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Contents Timetable ··································································································································· 2 Floor Plan ··································································································································· 3 Scientific Programs ···················································································································· 4 Plenary Lectures ······················································································································ 15 PL1 ······································································································································ 17 PL2 ······································································································································ 20 Symposia ································································································································· 21 S1 ········································································································································· 23 S2 ········································································································································· 34 S3 ········································································································································· 40 S4 ········································································································································· 49 S5 ········································································································································· 56 S6 ········································································································································· 60 S7 ········································································································································· 68 S8 ········································································································································· 76 S9 ········································································································································· 85 S10 ······································································································································· 92 S11 ······································································································································· 99 S12 ····································································································································· 106 S13 ····································································································································· 114 Colloquium ······························································································································· 123 Workshop ······························································································································· 129 Poster Sessions ····················································································································· 133 Author Index ··························································································································· 209 The Microbiological Society of Korea Rm. 810 (New Bldg.), The Korea Science & Technology Center 635-4, Yeoksam-dong, Gangnam-gu, Seoul 135-703, Korea Tel: +82-2-3453-3321/86 Fax: +82-2-3453-3322 Email: [email protected] Homepage: www.msk.or.kr This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government[NRF-2009-028-C00044]. 1 www.msk.or.kr Timetable 2 www.msk.or.kr Floor Plan 1st Floor Lobby Registration Desk Session Room Baegam Hall Secretariat Taebaek Hall 1st Basement Session Room Apricot Room MSK Lounge Orchid Room Lobby Poster Sesson & Exhibition 2nd Basement Session Room Art Hall Lobby Poster Session Exhibition 3 www.msk.or.kr Plenary Lectures PL1 Plenary Lecture 1 May 6 (Thu.), Art Hall (B2) Chair: Kye-Joon Lee, Seoul National University (Emeritus Professor) 17:30-18:15 Excellent DNA Polymerase from Thermococcus kodakaraensis KOD1 and Its Application for PCR Tadayuki Imanaka, Ritsumeikan University, Japan PL2 Plenary Lecture 2 May 7 (Fri.), Art Hall (B2) Chair: Doo-Hyun Nam, Yeungnam University 14:00-14:45 Post-transcriptional Regulation of Virulence Gene Expression in Pseudomonas aeruginosa Stephen Lory, Harvard Medical School, USA 4 www.msk.or.kr Symposia S1 Bacterial Communications to the Environments May 6 (Thu), Art Hall (B2) Chair: Hyung-Yeel Kahng, Sunchon National University S1-1 09:00-09:30 Exploration Untapped Halophilic Filamentous Actinomycetes: Opportunity and Challenge Wen-Jun Li, Yunnan University, China S1-2 09:30-10:00 Viral Metagenomics and Phage Genomics Jin-Woo Bae, Kyung Hee University S1-3 10:00-10:30 Diversity and Biogeographic View of Thermophilic Archaea Isolated from Terrestrial Hot Springs Takashi Itoh, RIKEN BioResource Center, Japan S1-4 10:30-11:00 Environmental Modulations and Progressive Selections in Dynamics of Genotypic and Ecotypic Microdiversities of Coastal Vibrio Populations Young-Gun Zo, Kyungsung University S1-5 11:00-11:30 Geobacter for Harvesting Electricity from Waste Organic Matter and Uranium Bioremediation Byoung-Chan Kim, Korea Research Institute of Bioscience and Biotechnology S2 Gene Expression and Growth Control May 6 (Thu), Apricot Room (B1) Chair: Sa-Ouk Kang, Seoul National University S2-1 09:00-09:30 Potassium Mediates Escherichia coli Enzyme IIANtr-Dependent Regulation of Sigma Factor Selectivity Yeong-Jae Seok, Seoul National University S2-2 09:30-10:00 Biochemical Characterization and Mutational Analysis of Fur-Family Proteins from Bacillus licheniformis Jin-Won Lee, Hanyang University 5 www.msk.or.kr S2-3 10:00-10:30 Delicate Control of Mitotic Exit by Bfa1 Asymmetry in Budding Yeast Saccaromyces cerevisiae Kiwon Song, Yonsei University S2-4 10:30-11:00 Chromatin Dynamics during Transcription Eun-Jung Cho, Sungkyunkwan University S2-5 11:00-11:30 Insertion of Transmembrane Helices into the Mitochondrial Inner Membrane : the Rules of the Game Joy (Hyun) Kim, Seoul National University S3 Pathogenesis of Clinically Important Bacterial Pathogens May 6 (Thu), Baegam Hall (1F) Chair: Myung-Je Cho, Gyeongsang National University S3-1 09:00-09:30 Stimulatory Effect of Cell Elongation on Biofilm Formation in Pseudomonas aeruginosa under Anaerobic Growth Condition Sang Sun Yoon, Yonsei University S3-2 09:30-10:00 Vibrio vulnificus Biofilm: Regulation and Roles of Extracellular Polysaccharides Kyu-Ho Lee, Hankuk University of Foreign Studies S3-3 10:00-10:30 Helicobacter pylori Infection in the Korean Population: An Epidemiological Link Between Toxin Polymorphism and Disease Douglas Scott Merrell, Uniformed Services University of the Health Sciences, USA S3-4 10:30-11:00 Entry Mechanism and Intracellular Localization of Orientia tsutsugamushi into Nonphagocytic Cell Myung Sik Choi, Seoul National University S3-5 11:00-11:30 Distinct Pathogenesis of Mycobacterium abscessus Isolated from Patients with Upper Lobe Fibrocavitary Form of Pulmonary Disease Sung Jae Shin, Chungnam National University 6 www.msk.or.kr S4 Dokdo's Biodiversity in the East Sea of Korea (GIMB Session 1) May 6 (Thu.), Art Hall (B2) Chair: Jung-Sook Lee, Korea Research Institute of Bioscience and Biotechnology S4-1 13:30-13:45 A Sustainable Research and Development on Dokdo Hyun Soo Rho, East Sea Research Institute, KORDI S4-2 13:45-14:00 The Study of Species Composition and Molecular Plant Geography in Dokdo Island Jae-Hong Pak, Kyungpook National University S4-3 14:00-14:15 Marine Invertebrate Fauna of Dokdo and Molecular Phylogeography of Sea Slaters and Chitons in Korea and Japan Ui Wook Hwang, Kyungpook National University S4-4 14:15-14:30 Exploring Microbial Functional Diversity of the Abyssal Seafloor in the East Sea Jong-Shik Kim, Gyeongbuk Institute for Marine Bio-Industry (GIMB) 14:30-15:00 Discussion and Q & A S5 Joint Symposium on Microorganism Resources Center May 6 (Thu.), Baegam Hall (1F) Organized by the Korea National Environmental Microorganisms Bank Chair: Sang Seob Lee, Kyonggi University S5-1 13:30-14:00 Practical Application of Environmental Microorganisms as Bioresources Sang Seob Lee, Kyonggi University S5-2 14:00-14:30 Current Status and Future Plan of KCTC Jung-Sook Lee, Korea Research Institute of Bioscience and Biotechnology S5-3 14:30-15:00 Management of Microbial Genetic Resources in Korean Agricultural Culture Collection In-Cheol Park, Rural Development Administration 7 www.msk.or.kr S6 Viruses and Immune Responses May 6 (Thu.), Art Hall (B2) Chair: Sin-Hyeog Im, Gwangju Institute of Science and Technology S6-1 15:00-15:30 Molecular Signature of DC Subsets during Acute vs. Chronic Virus Infection Sang-Jun Ha, Yonsei University S6-2 15:30-16:00 Options for Control of Pandemic Influenza: Active and Passive Immunization Huan Huu Nguyen, International Vaccine Institute S6-3 16:00-16:30 Chromatin Remodeling, HIV Reservoir, and Drug Development for Eradication of Chronic HIV Infection Kee-Jong Hong, Korea National Institute of Health S6-4 16:30-17:00 Automated HTS/HCS for Antivirals Using Visual HIV Full Replication Assays Peter Sommer, Institut Pasteur Korea S6-5 17:00-17:30 Probiotics as an Immune Modulator - Mechanism of Action and Therapeutic Applications Sin-Hyeog Im, Gwangju Institute of Science and Technology S7 Molecular Food Microbiology May 6 (Thu.), Apricot Room (B1) Chair: Jae-heon Kim, Dankook University S7-1 15:00-15:35 Adaptive Cellular Survival Response to Oxidative and Inflammatory Stresses Induced by Helicobacter pylori Young-Joon Surh, Seoul National University S7-2 15:35-16:10 Physiological Functionalities of Small Peptides Toshiro Matsui, Kyushu University, Japan S7-3 16:10-16:45 Anti-inflammatory Compounds from Medicinal Plants Jae-Ha Ryu, Sookmyung Women's University S7-4 16:45-17:20 Extraction of Bioactive Materials from Rice Hulls Seung-Cheol Lee, Kyungnam University 8 www.msk.or.kr S8 Response to Environmental Cues May 6 (Thu.), Baegam Hall (1F) Chair: Hyoung Tae Choi, Kangwon National University S8-1 15:00-15:30 Differential Roles of Yeast Glutamate Dehydrogenase Isozymes in Maintaining Resistance to Stress-Induced Apoptosis Pil Jae Maeng, Chungnam National University S8-2 15:30-16:00 Cadmium Regulates Copper Homeostasis by Inhibiting Mac1 Activity, a Transcriptional Activator of Copper Regulon, in Saccharomyces cerevisiae Cheol-Won Yun, Korea University Chair: Hyejoo Lee, Dong-A University S8-3 16:00-16:30 Membrane Potential Changes by Proton Pumping Microbial Rhodopsins Kwang-Hwan Jung, Sogang University S8-4 16:30-17:00 Different Role of the DosS and DosT Histidine Kinases in Mycobacterial Response to Hypoxic Conditions Jeong-Il Oh, Pusan National University S9 Design and Fabrication of Minimal Synthetic Cells May 7 (Fri.), Art Hall (B2) Chair: Ki-Sung Lee, Pai Chai University S9-1 10:00-10:30 Synthetic 5′-Untraslated Regions for Fine-Tunable and Predictable Gene Expression in Escherichia coli Gyoo Yeol Jung, Pohang University of Science and Technology S9-2 10:30-11:00 Rewriting Biology with Gene Synthesis Duhee Bang, Yonsei University S9-3 11:00-11:30 From Oligos to Organisms Mikkel Algire, J. Craig Venter Institute, USA S9-4 11:30-12:00 Artificial Assembly of a Minimal Cell Giovanni Murtas, Enrico Fermi Centre, Italy S9-5 12:00-12:30 Toward the Bacterial Minimal Chromosome Young-Chang Kim, Chungbuk National University 9 www.msk.or.kr S10 Omics Study on the Marine Hyperthermophilic Archaea (GIMB Session 2) May 7 (Fri.), Baegam Hall (1F) Sponsored by Marine & Extreme Genome Research Center, KORDI Chair: Sung-Jae Lee, Kyung Hee University S10-1 10:00-10:30 Novel Enzymes and Metabolic Pathways in Hyperthermophilic Archaea Haruyuki Atomi, Kyoto University, Japan S10-2 10:30-11:00 Global Proteome Analysis of Sulfur-Reducing Hyperthermophilic Archaeon Thermococcus onnurineus NA1 Jong-Soon Choi, Korea Basic Science Institute S10-3 11:00-11:30 Transcriptome Analysis on a Heat Shock Regulon in the Hyperthermophilic Archaeon, Thermococcus kodakaraensis Tamotsu Kanai, Kyoto University, Japan S10-4 11:30-12:00 Molecular Architecture and the Mechanics of Lon, a Protease-Chaperone Machine Sun-Shin Cha, Korea Ocean Research & Development Institute (KORDI) S10-5 12:00-12:30 Novel DNA Ligases with Broad Nucleotide Cofactor Specificity from the Hyperthermophilic Crenarchaeota Suk-Tae Kwon, Sungkyunkwan University 10 www.msk.or.kr S11 Pseudomonas Toxins and Pathogenicity May 7 (Fri.), Art Hall (B2) Chair: Joon-Hee Lee, Pusan National University S11-1 14:45-15:15 In Situ Fluorescent Imaging of Bacteriogenic Cyanide in Lungs of Live Mice Infected with Pseudomonas aerugionosa and Burkholderia cepacia Sungsu Park, Ewha Womans University S11-2 15:15-15:45 Iron Homeostasis Affects Antibiotic-Mediated Bacterial Cell Death Woojun Park, Korea University Chair: Woojun Park, Korea University S11-3 15:45-16:15 Pseudomonas Virulence by a Quorum-Dependently Secreted Exoprotease Joon-Hee Lee, Pusan National University S11-4 16:15-16:45 Apoptotic Elimination of Natural Killer Cells by Pseudomonas aeruginosa Jin Woong Chung, Dong-A University S12 Protein Structure and Function May 7 (Fri.), Apricot Room (B1) Chair: Chankyu Park, Korea Advanced Institute of Science and Technology S12-1 14:45-15:15 Structural Basis for Hypoxia Sensing by Histidine Kinases from Mycobacterium tuberculosis Beom Sik Kang, Kyungpook National University S12-2 15:15-15:45 Structural Studies of the Transmembrane Domain of Bacterial Histidine Kinase Young Ho Jeon, Korea Basic Science Institute S12-3 15:45-16:15 Functional Characterization of Native and Recombinant Antifreeze Protein of Arctic yeast, KOPRI-AY30 Hak Jun Kim, Korea Polar Research Institute Chair: Kwang-Hwan Jung, Sogang University S12-4 16:15-16:45 From Oxylipin to Volatile Dongsun Lee, Jeju National University S12-5 16:45-17:15 Spectroscopic Study of Rhodopsins in Action Hideki Kandori, Nagoya Institute of Technology, Japan 11 www.msk.or.kr S13 Marine Biotechnology (GIMB Session 3) May 7 (Fri.), Baegam Hall (1F) Chair: Kae Kyoung Kwon, Korea Ocean Research & Development Institute S13-1 14:45-15:15 Marine Brown Kelp Diversity Based on the Analyses of Multigenes from Plastid and Mitochondrion Ga Youn Cho, National Institute of Biological Resources S13-2 15:15-15:45 Functional Analysis in Escherichia coli of Biosynthesis Genes for Isoprenoids, Carotenoids, and Sesquiterpenes, and Their Production with Higher Plants Norihiko Misawa, Ishikawa Prefectural University, Japan S13-3 15:45-16:15 5-Hydroxyindole-Type Alkaloids from Marine Organisms Hyi-Seung Lee, Korea Ocean Research & Development Institute S13-4 16:15-16:45 Marine Microbial Water Quality off Hawaii Hans J. Krock, University of Hawai, USA (Emeritus Professor) S13-5 16:45-17:15 Plant Growth-Promoting Fungi from the Sand-Dune Plants in East Coast of Korea Jong-Guk Kim, Kyungpook National University 12 www.msk.or.kr Colloquium C1 Young Scientists Session 1 May 7 (Fri.), Apricot Room (B1) Chair: Jin-ho Lee, Kyungsung University C1-1 10:00-10:25 Pyrene Metabolism and Enzymes Involved in Novosphingobium pentaromativorans US6-1 Yuanrong Luo, Korea Ocean Research & Development Institute C1-2 10:25-10:50 Microarray Analysis of Knockout Mutant LuxS relevant to the Quorum Sensing Mechanism in Escherichia coli K-12 Su-Wan Son, Kyonggi University C1-3 10:50-11:15 Osmotic Stress Inhibits the Cell Growth of Listeria monocytogenes by the Downregulation of PTS Genes Dongryeoul Bae, Mississippi State University, USA C1-4 11:15-11:40 Direct Activation of Transcription of Fur-Positive Regulon by Iron-Free Form of Fur Protein Hyun-Jung Lee, Hankuk University of Foreign Studies 13 www.msk.or.kr Workshop W1 Workshop : Chunlab, Inc. May 7 (Fri.), Baegam Hall (1F) Chair: Yu-Seok Oh, Chunlab, Inc. 12:30-14:00 Microbial Community Analysis Using Pyrosequencing Jongsik Chun, CEO of Chunlab, Inc. 14 www.msk.or.kr 2010 International Meeting of the Microbiological Society of Korea Plenary Lectures 15 www.msk.or.kr 16 Plenary Lectures PL-1 Excellent DNA Polymerase from Thermococcus kodakaraensis KOD1 and Its Application for PCR Tadayuki Imanaka Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu, Shiga 525-8577, Japan E-mail: [email protected] We reflect on some studies on the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 and its enzymes. This strain can grow at temperatures up to the boiling point and also represents one of the simplest forms of life. In fact, we have determined the complete genome sequence, and found that around 2300 ORFs are enough to support the life. As expected, all enzymes displayed remarkable thermostability, and we have determined some of the basic principles that govern this feature. To our delight, many of the enzymes exhibited unique biochemical properties and novel structures not found in mesophilic proteins. Here, I focus on DNA polymerase that is useful in application. Hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 Novel and useful enzymes J. Biol. Chem., 281(15): 10533-10539 (2006) J. Bacteriol., 187(20): 7072-7080 (2005) J. Bacteriol., 185, 1705-1711 (2003) Appl. Environ. Microbiol., 68, 3925-3931 (2002) J. Biol. Chem., 277, 31656-31662 (2002) J. Bacteriol., 184, 3689-3698 (2002) J. Bacteriol., 184, 3305-3312 (2002) J. Bacteriol., 184, 777-784 (2002) Genome analysis Genome Res., 15(3): 352-363 (2005) Transcriptome analysis J. Bacteriol., 188(16):5915-5924 (2006) J. Biol. Chem. Published online (2007) Gene disruption system J. Bacteriol., 185, 210-220 (2003) Appl. Environ. Microbiol., 71(7):3889-3899 (2005) J. Bacteriol. Published online (2007) 17 www.msk.or.kr The KOD DNA polymerase is one of the most efficient thermostable PCR enzymes exhibiting higher accuracy and elongation velocity than any other commercially available DNA polymerases. The crystallographic studies were also done. However, even when KOD DNA polymerase was used for PCR, troubles with nonspecific DNA amplification and primer dimer formation still remain because of undesirable DNA polymerase activity during the first denaturing step of PCR. In order to inhibit this undesirable DNA polymerase activity (hot start PCR), two neutralizing monoclonal antibodies (mAbs), 3G8 and beta-G1, to KOD DNA polymerase were obtained. Exonulease activity measurement and epitope mapping revealed that the epitope for 3G8 is located in conserved regions among (family B) DNA polymerases (Region II), and the epitope for beta-G1 is located in the 3'-5' exonuclease domain. When hot start PCR with each of these mAbs was performed, the specificity of target gene amplification became much higher than in reactions without monoclonal antibody. Furthermore, this method can easily be applied to long distance PCR (>17.5 kb). <20 bp <20 bp Deep Vent Pfu Processivity (Bases/Reaction) 300 bp KOD 0 100 Taq 200 61 Deep Vent 23 Pfu 25 300 400 Extension efficiency (Bases/sec) 138 KOD 0 50 100 150 200 Taq Deep Vent 1.16 Pfu 7.39 0.63 Mutation frequency (%) 0.15 0.09 KOD KOD-Plus 0 2 4 6 8 In this symposium, development of PCR enzymes including (a) Characterization of a new PCR enzyme, KOD DNA polymerase, (b) Engineering of a new long and accurate (LA) PCR enzyme, (c) Improvement of PCR by neutralizing monoclonal antibodies, and (d) Structural analysis of KOD DNA polymerase will be presented. 18 Plenary Lectures References [1] Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR, M. Takagi, M. Nishioka, H. Kakihara, M. Kitabayashi, H. Inoue, B. Kawakami, M. Oka & T. Imanaka, Appl. Environ. Microbiol.,63, 4504-4510 (1997). [2] Characterization and application for hot start PCR of neutralizing monoclonal antibodies against KOD DNA polymerase, H. Mizuguchi, M. Nakatsuji, S. Fujiwara, M. Tagaki & T. Imanaka, J. Biochem., 126, 762-768 (1999). [3] Crystallographic studies on family B DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis strain KOD1, H. Hashimoto, T. Matsumoto, M. Nishioka, T. Yuasa, S. Takeuchi, T. Inoue, S. Fujiwara, M. Takagi, T. Imanaka & Y. Kai, J. Biochem., 125, 983-986 (1999). [4] Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease deficient mutant enzyme, M. Nishioka, H. Mizuguchi, S. Fujiwara, S. Komatsubara, M. Kitabayashi, H. Uemura, M. Takagi & T. Imanaka, J. Biotechnol., 88, 141-149, (2001). [5] Crystal structure of DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1, H. Hashimoto, M. Nishioka, S. Fujiwara, M. Takagi, T. Imanaka, T. Inoue, & Y. Kai. J. Mol. Biol., 306, 469-77 (2001). [6] Evolution of PCR Enzymes: Towards a better PCR system based on a KOD DNA polymerase, T. Imanaka & M. Takagi, J. Chin. Inst. Chem. Engrs., 32, 277-288, (2001). 19 www.msk.or.kr PL-2 Post-Transcriptional Regulation of Pseudomonas aeruginosa Virulence Gene Expression Stephen Lory Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Waren Alpert 363, Boston, MA 02115, USA Bacteria that are common inhabitants of soil or water reservoirs cause some of the most serious infections of humans. Moreover, the ability of these opportunistic pathogens to thrive in a wide range of environments often depends on the activities of specialized signal transduction pathways and complex regulatory networks. Pseudomonas aeruginosa can cause serious acute infections in immunocompromized individuals, or chronic infections in patients with cystic fibrosis. We have recently uncovered a regulatory network that functions as a molecular switch controlling the expression of hundreds of genes, including those encoding acute toxic proteins, and in a reciprocal mode, the formation of biofilm determinants important for chronic infections. The switch operates by controlling the reversible phosphorylation of GacS/GacA two component system, which regulates transcription of only two genes, the small RNAs (sRNA) RsmZ and RsmY. The global impact of the GacS/GacA system is therefore due to post-transcriptional activities of these sRNAs, acting by antagonizing the binding of the translational regulator RsmA to its target sites at the 5′ ends of transcripts. A number of gense that are positively and negatively regulated by the RsmA, RsmY and RsmZ system were uncovered. This post-translational regulatory mechanism allows bacteria to respond more rapidly to environmental changes than by utilization of transcriptional regulatory systems. 20 Plenary Lectures 2010 International Meeting of the Microbiological Society of Korea Symposia 21 www.msk.or.kr 22 Symposia S1-1 Geobacter for Harvesting Electricity from Waste Organic Matter and Uranium Bioremediation Byoung-Chan Kim Microbial Resource Center, Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology (KRIBB) Dissimilatory Fe(III)-reducing bacteria can obtain energy by coupling the oxidation of organic compounds to the reduction of Fe(III). Molecular analysis of the composition of microbial communities has shown that the Geobacteraceae, a family of dissimilatory Fe(III)-reducing bacteria in the delta subdivision of the class Proteobacteria, are the predominant Fe(III)-reducing microorganisms in a wide variety of sedimentary environments in which Fe(III) reduction is the principal terminal electron-accepting process. Geobacter species can use alternate electron acceptors, such as the radionuclide U(VI) [1] and insoluble graphite electrodes [2]. Therefore, Geobacter species are important for harvesting electricity from waste organic matter and removing uranium from groundwater. Geobacter species are also of interest because of their role in environmental restoration. For example, Geobacter can destroy petroleum contaminants in polluted groundwater by oxidizing these compounds to harmless carbon dioxide [3]. Recent studies have indicated that outer membrane c-type cytochromes and conductive nanowires (Geopilin) are important for the electron transfer to Fe(III) and electrode by Geobacter sulfurreducens [4]. Elucidating which cytochromes are involved in what type of reduction and electron transfer is not trivial because the genome of G. sulfurreducens contains genes for over 110 c-type cytochromes, at least 30 of which are predicted to be localized in the outer membrane where Fe(III) and electrode reduction is likely to take place. Notably, only five of these outer membrane cytochromes have been found to be directly involved in electron transfer from inside of cells to outside [5]. It also have been revealed that the outer surface cytochrome, OmcZ which is localized in extra cellular biomatrix and the conductive nanowires are critical and essential for high-dense electricity generation and important for the conductive biofilm formation by G. sulfurreducens [6]. Now there are several evolved G. sulfurreducens strains including KN400 strain (TIME’s one of the 50 best inventions of 2009) that have been adapted on electrode [7]. These newly evolved electricigens are under investigation in order to find what others are the key components for increasing efficiency of electron transfer by Geobacter species. References [1] Lovley DR Nature reviews. Microbiology 1, 35 (2003). [2] Lovley DR Nature reviews. Microbiology 4, 797 (2006). 23 www.msk.or.kr [3] Lovley DR Science 293, 1444 (2001). [4] Reguera G, McCarthy KD, Mehta T, Nicoll JS, Tuominen MT, and Lovley DR Nature 435, 1098 (2005). [5] Voordeckers JW, Kim BC, Izallalen M, and Lovley DR Applied and environmental microbiology, In press. [6] Nevin KP, Kim BC, Glaven RH, Johnson JP, Woodard TL, Methé BA, Didonato RJ, Covalla SF, Franks AE, Liu A, and Lovley DR PloS ONE 4(5), e5628 (2009). [7] Yi H, Nevin KP, Kim BC, Franks AE, Klimes A, Tender LM, and Lovley DR Biosensors & bioelectronics 24, 3498 (2009). 24 Symposia S1-2 Exploration Untapped Halophilic Filamentous Actinomycetes: Opportunity and Challenge Shu-Kun Tang1, Yun Wang2, Xiao-Yang Zhi1, Kai Lou2, Li-Li Zhang3, Li-Xin Zhao1, Cheng-Lin Jiang1, Li-Hua Xu1, Chang-Jin Kim4, and Wen-Jun Li1* 1 The Key Laboratory for Microbial Resources of the Ministry of Education, P. R. China, and Laboratory for Conservation and Utilization of Bio-Resources, Yunnan Institute of Microbiology, Yunnan University, Kunming, 650091, P. R. China 2 Xinjiang Institute of Microbiology, Xinjiang Academy of Agricultural Science, Urumqi, Xinjiang, 830091, P. R. China 3 Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Xinjiang Production & Construction Corps, Tarim University, Alar, Xinjiang 843300, P. R. China 4 Functional Metabolite Research Center KRIBB, Daejeon 305-806 *Corresponding author: Wen-Jun Li, E-mail: [email protected]; [email protected] Actinomycetes are still the most prolific producers of pharmacologically important compounds, accounting for about 70% of the naturally derived antibiotics that are currently in clinical use (Bérdy, 2005). However, most researches over the past decades were focusing on those actinomycetes in the general terrestrial soil environments, such as soils, and sediments of the rivers or lakes, and thus induced re-discovering the redundancy strains (special for members of the genera Streptomyces and Micromonospora) and well-known compounds. Actinomycetal resources under extreme environments (including extreme high and low temperature, extreme high or low pH, high salt concentration, etc) have received comparatively little attention from microbiologists. Actinomycetes are regarded as one kind of sideline microorganisms and those under extreme environments are better research materials for biological evolution and phylogenetic analysis. Moreover, these extremephilic actinomycetes may have new functional genes and can produce new secondary metabolites, which can be used for further new antibiotics screening. Since 1990s’, our research group has paid much attention to the research on extremophilic actinomycetal resources. Here, we just report our research results on the diversity of halophilic filamentous actinomycetes from three levels: species, functional genes and metabolites. Since the first halophilic actinomycete Actinopolyspora halophila (Gochnauer et al., 1975) was reported, people knew that some filamentous actinomycetes could grow and tolerate with high salt concentrations. Meanwhile, in the past three decades, only few halophilic actinomycetes were discovered and cultured, which included six species within three genera: Actinopolyspora halophila (Gochnauer et al., 1975), Actinopolyspora motivallis (Yoshida et al., 1991), Actinopolyspora iraqiensis (Ruan et al., 1994), Nocardiopsis halophila (Al-Tai and Ruan, 1994), Nocardiopsis kunsanensis (Chun et al., 2000) and Saccharomonospora halophila Al-Zarban et al., 2002). The big problem is that scientists don’t know too much about their nutrient requirements and ecological adaptive mechanisms, and they have no effective isolation methods, which are the 25 www.msk.or.kr main bottleneck to restrict the research development in this field. Since the late 1990s’, our research group initiated the study on halophilic filamentous actinomycetes from hypersaline environments, especially focusing on salt lakes in north-west China including Xinjiang and Qinghai provinces. Based on our extensive studies on the biological characteristics of halophilic filamentous actinomycetes (Tang et al., 2003), one efficient isolation medium (cellulose-casein multi-salts) (Tang et al., 2008) was designed and applied specially for the isolation of filamentous halophilic actinomycetes in hypersaline environments. By using this method, over 3000 halophilic filamentous actinomycete strains were isolated from hypersaline soils or sediments of salt lakes in Xinjiang Province, China, and most of them are truly halophilic, which have optimal NaCl growth concentrations, ranging from 5% to 15% (w/v), but not grow in absent of NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that isolated strains belong to fourteen genera within seven families. In addition to the above mentioned three genera, there are one new suborder Jiangellineae (Tang et al., 2010) with family Jiangellaceae (Tang et al., 2010), six new genera: Streptomonospora (Cui et al., 2001), Myceligenerans (Cui et al., 2004), Haloactinospora (Tang et al., 2008), Haloglycomyces (Guan et al., 2009), Haloechinothrix (Tang et al., 2010), Haloactinopolyspora (Tang et al., 2010), and some newly reported halophilic actinomycete species in four known genera, which had never halophilc strains discovered before: Prauserella halophila (Li et al., 2003, 2009); Amycolatopsis halophila (Tang et al., 2010), Saccharopolyspora halophila (Tang et al., 2009), Georgenia halophila (Tang et al., 2010). Our research results clearly indicated that there were so diverse halophilic filamentous actinomycetes, and the high density of new or unknown actinomycetal resources in these untapped hypersaline extreme environments totally overtook our expectation. Furthermore, our results also showed that the genera Nocardiopsis, Saccharomonospora and Streptomonospora were the predominant groups of culturable halophilic filamentous actinomycetes and they distributed widely in different saline environments, and until now none truly halophilic Streptomyces and Micromonospora isolates were discovered in hypersaline environments in Xinjiang. Thus, on the basis of our research results, we believe that microbial community in hypersaline environments is completely different from that constitute of microbial community in general terrestrial environments, which was generally predominated by genera Streptomyces and Micromonospora. It is undoubtedly that new species will have new functional genes and new secondary metabolites, and will certainly have new potential commercial use. Actinomycetes from hypersaline environments may be an important source for discovery of new drugs. Thus, the specific PCR-based was preformed for further screening from these isolated strains with PKS I, PKS II and NRPS functional genes. In addition, we also selected some positive halophilic strains for further sequencing their functional gene sequences to explore their diversity and difference with some other known from other environments. Moreover, large-scale batch fermentations were performed for some selected positive strains for further deriving their secondary metabolites. Our research results show that most halophilic filamentous actinomycete strains are positive for PKS I, PKS II, and NRPS genes, their protein coding functional gene sequences are quite different from those actinomycetes from other environments, and they also have quite different ecological and taxonomically characteristics, which clearly 26 Symposia indicates that halophilic filamentous actinomycetes have huge potential capacity for producing novel antibiotics. Our secondary metabolite research results can further confirm this conclusion. For example, Erythronolides H and I, novel congeners of the clinically important antibacterial drug erythromycin A, have been isolated from the new halophilic filamentous actinomycete strain, Actinopolyspora sp. YIM 90600. In addition to producing the new erythromycin congeners, Actinopolyspora sp. YIM 90600 produces erythromycin C in a high titer. The presence of the C-14 hydroxyl moiety and the C-6/C-18-epoxide in erythronolide H and the spiroketal moiety of erythronolide I sheds new insights into structural diversity of erythromycin analog libraries potentially accessible by combinatorial biosynthesis (Huang et al., 2009). In conclusion, our route to discovering novel actinomycetal resources from those environments that have been un- and under-explored extreme/neglected habitats is quite successful. Our research group not only established the isolation method on halophilic filamentous actinomycetes, realized them from unculturalbe to culturable, but also found their potential ability to produce new antibiotics. However, from the above inspiring research results, we should be aware in our mind that it is not only an opportunity, but also a challenge brought forward to us that how to make these extremophilic actinomycetal resources becoming practical antibiotic products like scientists did before. References [1] Al-Tai, A. M. and Ruan, J. S. 1994. Nocardiopsis halophila sp. nov., a new halophilic actinomycete isolated from soil. Int. J. Syst. Bacteriol. 44, 474-478. [2] Al-Zarban, S. S., Al-Musallam, A. A., Abbas, I., Stackebrandt, E. and Kroppenstedt. R. M. 2002. Saccharomonospora halophila sp. nov., a novel halophilic actinomycete isolated from marsh soil in Kuwait. Int. J. Syst. Evol. Microbiol. 52, 555-558. [3] Bérdy, J. 2005. Bioactive microbial metabolites. J. Antibiot. 58, 1-26. [4] Chun, J., Bae, K. S., Moon, E. Y., Jung, S. O., Lee, H. K. and Kim, S. J. 2000. Nocardiopsis kunsanensis sp. nov., a moderately halophilic actinomycete isolated from a saltern. Int. J. Syst. Evol. Microbiol. 50, 1909-1913. [5] Cui, X-L., Mao, P-H., Zeng, M., Li, W-J., Zhang, L-P., Xu, L-H and Jiang, C-L. 2001. Streptimonospora salina gen. nov., sp. nov., a new member of the family Nocardiopsaceae. Int. J. Syst. Evol. Microbiol. 51, 357-363. [6] Cui, X-L., Schumann, Stackebrandt, E., Kroppenstedt, R. M., Pukall, R., Xu, L-H., Rohde, M and Jiang, C.L. 2004. Myceligenerans xiligouense gen. nov., sp. nov., a novel hyphae-forming member of the family Promicromonosporaceae. Int. J. Syst. Evol. Microbiol. 54, 1287-1293. [7] Gochnauer, M. B., Leppard, G. G., Komaratat, M. K., Novitsky, T. and Kushner, D. 1975. Isolation and characterization of Actinopolyspora halophila, gen. et sp. nov., an extremely halophilic actinomycete. Can. J. Microbiol. 21, 1500-1511. [8] Guan, T-W., Tang, S-K., Wu, J-Y., Zhi X-Y., Li-Hua Xu, L. H., Zhang, L-L. and Li. W. J. 2009. Haloglycomyces albus gen. nov., sp. nov., a novel halophilic filamentous actinomycete of the family Glycomycetaceae. Int. J. Syst. Evol. Microbiol. 59, 1297-1301. 27 www.msk.or.kr [9] Huang, S-X., Zhao, L-X., Tang, S-K., Jiang, C-L., Duan, Y-W. and Shen, B. Erythronolides. 2009. H and I, new erythromycin congeners from a new halophilic actinomycete Actinopolyspora sp. YIM90600. Organic Letters 11, 1353-1356. [10] Li. W. J., Xu, P., Tang, S-K., Xu, L-H., Kroppenstedt, R.M., Stackebrandt, E and Jiang, C-L. 2003. Prauserella halophila sp.nov. and Prauserella alba sp. nov., two new moderately halophilic actinomycetes isolated from the saline soil. Int. J. Syst. Evol. Microbiol. 53, 1545-1549. [11] Li, Y., Tang S-K., Chen, Y-G., Wu, J-Y., Zhi X-Y., Zhang, Y-Q. and Li. W. J. 2009. Prauserella salsuginis sp. nov., Prauserella flava sp. nov., Prauserella aidingensis sp. nov. and Prauserella sediminis sp. nov., isolated from a salt lake. Int. J. Syst. Evol. Microbiol. 59, 2923-2928. [12] Ruan, J. S., Al-Tai, A. M., Zhou, Z. H., and Qu, L. H. 1994. Actinopolyspora iraquiensis sp. nov., a new halophilic actinomycete isolated from soil. Int. J. Syst. Bacteriol. 44, 759-763. [13] Tang, S-K., Li. W. J., Zhang, R-G., Wang Dong, Li-Hua Xu, and Cheng-Lin Jiang. 2003. Studies on biological characteristics of Some halophilic and halotolerant actinomycetes Isolated from saline and alkaline soils. Actinomycetologica 17, 6-10. [14] Tang, S-K., Tian, X-P., Zhi, X-Y., Cai, M., Wu, J-Y., Yang, L-L., Xu, LH. and Li. W-J. 2008. Haloactinospora alba gen. nov., sp. nov., a halophilic filamentous actinomycete of the family Nocardiopsaceae. Int. J. Syst. Evol. Microbiol. 58, 2075-2080. [15] Tang, S-K., Wang, Y., Cai, M., Zhi, X-Y., Lou, K., Xu, L. H., Jiang, C. L. and Li, W. J. 2009. Saccharopolyspora halophila sp. nov., a novel halophilic actinomycete isolated from a saline lake in China. Int. J. Syst. Evol. Microbiol. 59, 555-558. [16] Tang, S-K., Wang, Y., Guan, T-W., Lee, J-C., Kim, C-J. and Li, W-J. 2010. Amycolatopsis halophila sp. nov., a novel halophilic actinomycete isolated from a salt lake in China. Int. J. Syst. Evol. Microbiol. (In Press). [17] Tang, S-K., Wang, Y., Lee, J-C., Lou, K., Park, D-J., Kim, C-J. and Li, W-J. 2010. Georgenia halophila sp. nov., a novel halophilic actinobacterium isolated from a salt lake in China. Int. J. Syst. Evol. Microbiol. (In Press). [18] Tang, S-K., Wang, Y., Wu, J-Y., Cao, L-L., Lou, K., Xu, L-H., Jiang, C-L. and Li, W. J. 2009. Saccharopolyspora qijiaojingensis sp. nov., a novel halophilic actinomycete isolated from a salt lake in China. Int. J. Syst. Evol. Microbiol. 59, 2166-2170. [19] Tang, S-K., Wang, Y., Zhang, H., Lee, J-C., Lou, K., Kim, C-J. and Li, W-J. 2010. Haloechinothrix alba gen. nov., sp. nov., a novel halophilic filamentous actinomycete of the suborder Pseudonocardineae. Int. J. Syst. Evol. Microbiol. (In Press). [20] Tang, S-K., Zhi, X-Y., Wang, Y., Shi, R., Lou, K., Xu L-H. and Li, W-J. 2010 Haloactinopolyspora alba gen. nov. sp. nov., a novel halophilic filamentous actinomycete isolated from a salt lake in China, with proposal of Jiangellaceae fam. nov. and Jiangellineae subord. nov. Int. J. Syst. Evol. Microbiol. (In Press). [21] Yoshida, M., Matsubara, K., Kudo, T. and Horikoshi, K. 1991. Actinopolyspora mortivallis sp. nov., a moderately halophilic actinopmycete. Int. J. Syst. Bacteriol. 41, 15-20. 28 Symposia S1-3 Viral Metagenomics and Phage Genomics Jin-Woo Bae*, Eun-Jin Park, and Kyoung-Ho Kim Department of Life and Nanopharmaceutical Sciences and Department of Biology, Kyung Hee University, Seoul 130-701 Although viruses are known to be the most numerous biological entities in soil and seawater, little is known about their diversity in this environment. To investigate viruses in environment, we combined two kinds of approaches, the culture-independent and the culture-dependent method. We used the viral metagenomics and viral genomics to understand viral diversity and physiology. For the metagenomic approach, viruses were separated and concentrated with centrifugation and filtration from a soil, foods, feces, and a marine environment. Viral DNA was extracted and amplified with the multiple displacement amplification (MDA) method, a whole genome amplification method which uses the phi29 DNA polymerase and random hexamer to amplify DNA isothermally. The metagenomes amplified by MDA were sequenced and/or compared with sequences from metagenomes amplified by the linker amplified shotgun library method (LASL) which amplified only double strand DNA. The analysis of sequences showed that the MDA method amplify single stranded DNA viral genomes more preferentially than other (mostly double stranded) viral DNA. Changes of circular DNA and linear DNA amount during MDA were observed with quantitative real time PCR to confirm the preferential amplification of circular DNA. As a result, we detected that various kinds of unknown single stranded DNA viruses exit in soil and marine environment. We also could assemble several circular genomic compounds of unknown putative single stranded DNA viruses from metagenomic sequences retrieved from soil and marine environment. We also obtained the metagenomic viromes in order to identify and characterize viral diversity and community structure in fermented foods. Viral particles were purified and concentrated by sequential filtrations and ultracentrifugation. Extracted DNA was amplified by the linker amplified shotgun library (LASL) method and the amplified metagenomes were sequenced by 454 pyrosequencing. Moreover, in order to look more deeply the biological entities and correlation of viral with bacterial communities in the fermented foods, we also analyzed the bacterial community by pyrosequencing at the same time. Here, we report the first metagenomic analyses to investigate the unknown viral communities from three fermented foods, fermented shrimp, kimchi, and sauerkraut, fermented by the action of innate microorganisms. The story of metagenomic viromes gives us to better understand biological ecosystems in the fermented foods. References [1] M. Breitbart, F. Rohwer, Trends Microbiol, 13, 278, 2005. 29 www.msk.or.kr [2] J. A. Fuhrman, U. Campbell, Nature, 393, 410,1998. [3] F. Rohwer, R. Edwards, J Bacteriol, 184, 4529, 2002. [4] T. Allander et al., Proc Natl Acad Sci USA, 102, 12891, 2005. [5] A. F. Andersson et al., PLoS One, 3, e2836, 2008. [6] M. Breitbart et al., J Bacteriol, 185, 6220, 2003. [7] S. R. Gill et al., Science, 312, 1355, 2006. [8] A. Lopez-Bueno et al., Science, 326, 858, 2009. [9] S. G. Tringe et al., Science, 308, 554, 2005. [10] P. J. Turnbaugh et al., Nature, 457, 480, 2009. [11] P. J. Turnbaugh et al., Nature, 444, 1027, 2006. [12] J. C. Venter et al., Science, 304, 66, 2004. [13] T. Woyke et al., Nature, 443, 950, 2006. 30 Symposia S1-4 Diversity and Biogeographic View of Thermophilic Archaea Isolated from Terrestrial Hot Springs Takashi Itoh Japan Collection of Microorganisms, RIKEN BioResource Center, Wako-shi, Saitama 351-0198, Japan Thermophilic archaea would play important roles in promising areas of biosciences and biotechnologies: for example, understanding of the adaptive machineries and contribution to biogeochemical cycles in geothermally-heated environments, speculation of models to infer an early life form, and providing sources of heat-stable enzymes. In order to support researches in these fields, RIKEN BRC-JCM has been collecting, preserving, and distributing strains of the thermophilic archaea. Besides, we have isolated the thermophilic archaea extensively from terrestrial hot springs in Japan and the Philippines to incorporate novel thermophilic archaea into our collection. In this paper, I survey the phylogenetic positions of the archaeal isolates on the basis of the 16S rRNA gene sequences to discuss their taxonomic diversity. In addition, a comparison between the phylogenetic relationship and the geographic distribution of a certain group of the thermophilic archaea is made to shed light on the speciation process. Up to now, more than 190 archaeal strains were isolated from hot springs in Japan and the Philippines by using modified media of the “Sulfolobus” medium. The 16S rRNA gene sequence analyses revealed that they were distributed within five archaeal orders: Sulfolobales (131 isolates), Thermoproteales (36 isolates), Acidilobales (12 isolates), Desulfurococcales (1 isolate), and Thermoplasmatales (11 isolates). In addition to the genera published by ourselves (i.e., Thermocladium, Caldivirga, Vulcanisaeta, Caldisphaera, and Thermogymnomonas), there were three hitherto undescribed genera in the order Sulfolobales, and at least eight new species as predicted by the phylogenetic distance to known species (<98% similarity). Among these isolates, members of the genus Vulcanisaeta seemed to be distributed endemically in view of the 16S rRNA gene sequence comparisons. The genus Vulcanisaeta was created to encompass two rod-shaped, hyperthermophilic arachaeal species, V. distributa and V. souniana (Itoh et al., 2002). The V. distributa strains, which were all isolated from spouting hot spring or heated solfataric soil samples, are classified into the 16S rRNA gene-based subgroups, which are congruent with the geographic locations as well. On the other hand, the V. souniana strains, isolated from pipelined hot spring water, allocate in a different lineage. This may indicate that the geographic separation and the environmental difference promote the genetic diversification, leading to the speciation, among members of the genus Vulcanisaeta. In order to verify the hypothesis, we have conducted the phylogenetic analyses on radA and DEAD/DEAH box helicase-like genes. These results would underpin the usefulness of these Vulcanisaeta strains as model organisms to study the allopatric microbial speciation. Reference Itoh, T. et al., Int. J. Syst. Evol. Microbiol. 52: 1097-1104 (2002). 31 www.msk.or.kr S1-5 Environmental Modulations and Progressive Selections in Dynamics of Genotypic and Ecotypic Microdiversities of Coastal Vibrio Populations Young-Gun Zo1* and Nipa Chokesajjawatee2 1 Department of Biology, Kyungsung University, National Center for Genetic Engineering and Biotechnology, National Science and Technology Agency, Thailand 2 How bacterial populations respond to environmental fluctuations has been an intriguing question. There have been three kinds of approaches employed to address this question, namely [1] relating fine-scale genotypic variation of one or a few house-keeping genes to environmental variations using of a massive number of bacteria present at a given geographical location, [2] relating genome-wide genotypic information of a limited number of bacterial isolates to environmental variation, and [3] relating genotypic and phenotypic variations of a census-collection of bacteria to spatio-temporal variations of environmental conditions from which the collection was obtained. In this study, findings from these approaches were synthesized by regarding Vibrio populations in coastal environments as a model case. When massive collections of 16S rRNA gene sequences and gyrB were analyzed, the fine-scale diversity comprising natural bacterial assemblages, i.e., microdiversity structure, appeared to be derived from a neutral radiation of genetic traits and occasional selection. In this case, the dynamics of phylogenetic signals detected in the DNA sequences from natural environments did not corresponded to environmental parameters [1-2]. However, the correspondence was found when genome-wide genetic information of Vibrio cholerae was employed as the tool for genotyping [3-4]. Therefore, assessment of phenotypic correspondence from a large number of bacterial isolates warranted the proper resolution from this conflict. In this study, a set of V. cholerae isolates from a census of the species undertaken in the upper Chesapeake Bay was employed in an analysis of its 25 phenotypic and genotypic traits. Two distinct V. cholerae types were observed for every three isolates in the set. Results of time scale analysis indicated that turnover in the operational taxonomic units of V. cholerae populations occurred monthly, whereas prevalence of bacterial traits changed at three month intervals. Environmental variables, including pH, salinity and zooplankton composition showed significant correlation with variation in both genotypic and phenotypic characteristics. Progressive selective pressure on V. cholerae population structure in the natural environment is concluded to operate at the fine scale of the ecological niche. As evidence validating the progressive selection of fine-scale ecological niches, the unique energy-demanding accessory trait of bioluminescence was examined. Succession from non-luminescent to luminescent populations 32 Symposia of V. cholerae in coastal waters occurred during spring to mid-summer, along with a shift from neutral to alkaline (pH>8) pH vales. The occurrence of an intermediate luminescent population that had phylogenetic relatedness to non-luminescent populations peaked in the middle of the pH shift. It is concluded that each cluster of luminescent V. cholerae occupy a distinct ecological niche. In conclusion, active communication of bacterial cells with the environment appears to results in tight coupling between slight changes in the environment and gradual succession in bacterial community. The success is neither stochastic nor striking. These findings warrants that fine-scale variation in genotypic/ phenotypic characteristics of bacteria community is predictable based on some knowledge in environmental variations. References [1] Acinas S.G., Klepac-Ceraj V., Hunt D.E., Pharino C., Ceraj I., Distel D.L., and Polz M.F. Nature, 430, 551, 2004. [2] Thompson J.R., Pacocha S., Pharino C., Klepac-Ceraj V., Hunt D.E., Benoit J., Sarma-Rupavtarm R., Distel D.L., and Polz M.F. Science 307, 1311, 2005. [3] Keymer D. P., Miller M.C., Schoolnik G.K., and Boehm A.B. Appl. Environ. Microbiol. 73, 3705, 2007. [4] Miller M.C., Keymer D.P., Avelar A., Boehm A.B., and Schoolnik G.K. Appl. Environ. Microbiol. 73, 3695, 2007. 33 www.msk.or.kr S2-1 Potassium Mediates Escherichia coli Enzyme IIANtr-Dependent Regulation of Sigma Factor Selectivity Chang-Ro Lee1, Seung-Hyon Cho1, Hyun-Jin Kim1, Miri Kim1, Alan Peterkofsky2, and Yeong-Jae Seok1,3* 1 Laboratory of Macromolecular Interactions, Department of Biological Sciences and Institute of Microbiology, Seoul National University, Seoul 151-742 2 Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA 3 Department of Biophysics and Chemical Biology, Seoul National University, Seoul 151-742 While the intracellular concentration of Na+ does not exceed 20 mM, that of K+ is maintained at 300-500 mM in Escherichia coli (1). A high cytoplasmic K+ concentration is required for processes such as maintenance of cell turgor and adaptation of cells to osmotic conditions. Here we show that sigma factor selectivity is one of the important reasons for the accumulation of such high concentrations of K+ in E. coli cytosol. Recently, we have shown that an Escherichia coli K12 mutant devoid of enzyme IIANtr (EIIANtr) of the nitrogen PTS becomes extremely sensitive to leucine or leucine-containing peptides due to the defect in derepression of the ilvBN operon encoding acetohydroxy acid synthase I (2). The derepression mechanism was turned out to be due to the interaction of unphosphorylated EIIANtr with TrkA, an essential component of the Trk potassium transport system to decrease the intracellular potassium level (3). In this study, we report the mechanism for regulation of gene expression by the intracellular K+ level. We report here the mechanism for regulation of gene expression by the intracellular K+ level. The leucine hypersensitivity of a ptsN (encoding EIIANtr) mutant was suppressed by deleting the rpoS gene, encoding the stationary phase σ factor. Despite intracellular levels of sigma factors comparable to the wild-type strain, most of the genes down-regulated in a ptsN mutant are controlled by σ70, while all the up-regulated genes are controlled by σS, implying that the balance of sigma activities is modified by ptsN deletion. This change of sigma factor activity was found to be due to increased levels of K+. In vitro transcription assays showed that a σ70 controlled gene and a σS controlled gene were differentially affected by potassium concentration. Biochemical studies revealed that K+ is responsible for sigma factor competition by differentially influencing the binding of σ70 and σS to core RNA polymerase. Taken together, the data indicate that EIIANtr controls sigma factor selectivity by regulating the intracellular K+ level. References [1] Bossemeyer, D., Borchard, A., Dosch, D. C., Helmer, G. C., Epstein, W., Booth, I. R., and Bakker, E. P. (1989) J Biol Chem 264, 16403-16410. 34 Symposia [2] Lee, C.-R., Koo, B.-M., Cho, S.-H., Kim, Y.-J., Yoon, M.-J., Peterkofsky, A., and Seok, Y.-J. (2005) Mol Microbiol 58, 334-344. [3] Lee, C.-R., Cho, S.-H., Yoon, M.-J., Peterkofsky, A., and Seok, Y.-J. (2007) Proc Natl Acad Sci USA 104, 4124-4129. 35 www.msk.or.kr S2-2 Biochemical Characterization and Mutational Analysis of Fur-Family Proteins from Bacillus licheniformis Chang-Jun Ji, Jung-Hoon Kim, and Jin-Won Lee* Department of Life Science, Hanyang University, Seoul 133-791 The ferric uptake regulator (Fur) family proteins includes sensors of Fe (Fur), Zn (Zur), Mn (Mur), Ni (Nur), and peroxide (PerR). Bacillus subtilis, a model Gram-positive bacterium, contains three Fur homologues: Fur, Zur, and PerR. However, the genome sequence of Bacillus licheniformis indicates that there are two additional Fur homologues, BL00690 and BL00950, in addition to Fur, Zur, and PerR homologues. We confirmed that the fur, zur and perR homologous genes from B. licheniformis can complement the fur, zur and perR mutants respectively, and that PerR from B. licheniformis can sense H2O2 by oxidation of two histidine residues like PerR from B. subtilis. Although, untill now, no target genes have been identified for BL00690 and BL00950, our biochemical and mutational analyses strongly indicate that both BL00690 and BL00950 contain one Zn atom presumably coordinated by four cysteine residues and can sense H2O2 by histidine oxidation like PerR protein. 36 Symposia S2-3 Delicate Control of Mitotic Exit by Bfa1 Asymmetry in Budding Yeast Saccaromyces cerevisiae Kiwon Song Dept. of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 All eukaryotic cells become duplicated by a process called cell cycle. The key issue of cell cycle is to maintain genomic integrity while duplicating itself. Chromosomes are replicated once and equally segregated as daughter chromosomes at the anaphase of mitosis. Once chromosomes have segregated properly, a pathway called mitotic exit network (MEN) becomes activated to finish mitosis. Timing of the MEN activation should be tightly coordinated with proper segregation of the chromosomes to ensure genomic integrity. How the timing of mitotic exit is controlled is a current topic of interest. In budding yeast Saccharomyces cerevisiae that provides an excellent model to study mitotic exit, the Tem1 functions at the top of the MEN and Bfa1 negatively regulates Tem1. The polo kinase Cdc5 also activates MEN by directly phosphorylating and inhibiting Bfa1. The spindle pole body (SPB) acts as a platform for these MEN components. Bfa1/Bub2 complex localizes to SPBs and Tem1 association with SPBs depends on Bfa1/Bub2. As the spindle aligns along the mother-bud axis to segregate duplicated chromosomes in anaphase, Bfa1/Bub2 becomes exclusively present on the bud-oriented SPB. Conversely, when spindles are misaligned, Bfa1/Bub2 is present on both SPBs and mitotic exit is delayed, suggesting that the spatial distribution of Bfa1/Bub2 controls the timing of mitotic exit. However, the molecular mechanism and function of how asymmetric Bfa1 localization to the bud-directed spindle pole body (SPB) during anaphase controls mitotic exit are not well understood, particularly in unperturbed cells. Here, we identified novel Cdc5 target residues within the Bfa1 C-terminus, 452S, 453 S, 454S, and 559 S. A Bfa1 mutant in 4A which all of these residues have been changed simultaneously (called Bfa1 ) persisted on both SPBs at anaphase and was hypo-phosphorylated, despite retaining its GAP activity against Tem1. These observations demonstrate a tight link between localization and phosphorylation, and no direct connection between asymmetric localization and GAP activity of Bfa1. Consistent with this, in kinase-defective cdc5-2 cells, Bfa1 was unphosphorylated and localized to both SPBs. The BFA14A cells progressed through anaphase normally, but displayed a delayed mitotic exit in unperturbed cell cycles. Altogether, we suggest that Cdc5 modulates the asymmetric Bfa1 distribution to the bud-directed SPB independently of Bfa1 GAP activity at anaphase, and that Bfa1 asymmetry fine-tunes the timing of MEN activation. 37 www.msk.or.kr S2-4 Chromatin Dynamics during Transcription Eun-Jung Cho Sungkyunkwan University The eukaryotic genome is packed by formation of nucleosomes with histones H2A, H2B, H3, and H4. The bulk of the nucleosomes are assembled when DNA is replicated in the S phase via the replication coupled (RC) pathway, which is mediated by histone H3/H4 chaperones, CAF-1 and Asf1. Outside of the S phase, histones are deposited into the nucleosome by HIR (mammalian homolog of HIRA) and Asf1 through a replication independent (RI) pathway. Recently, considerable attention has been devoted to these deposition pathways because RC or RI pathways are considered to be the potential mechanistic tool to sort out various types of histones along the genome according to the biological function of chromatin. My lab is interested in the role of the H3/H4 chaperones in the chromatin dynamics and wishes to determine the chromatin homeostasis during transcription. We examined the role of histone chaperones and different histone forms during transcription. The potential consequence of histone deposition via the RI pathway will be discussed with the yeast model system. 38 Symposia S2-5 Insertion of Transmembrane Helices into the Mitochondrial Inner Membrane: the Rules of the Game Joy (Hyun) Kim Laboratory of Membrane Biology, School of Biological Sciences, Seoul National University, Seoul 151-747 While overall hydrophobicity is generally recognized as the main characteristic of transmembrane a-helices, the only membrane system for which we have detailed quantitative data on how different amino acids contribute to the overall efficiency of membrane insertion is the endoplasmic reticulum (ER) of eukaryotic cells [1, 2, 3]. Here, we provide data for TIM23-mediated membrane protein insertion into the inner mitochondrial membrane of yeast cells. We find that hydrophobicity and the location of polar and aromatic residues are strong determinants of membrane insertion. These results parallel what has been found previously for the ER. However, we see striking differences between the effects elicited by charged residues flanking the transmembrane segments when comparing the mitochondrial inner membrane and the ER, pointing to an unanticipated dissimilarity between the two insertion systems. References [1] Hessa, T. et al. Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature 433, 377-381, (2005). [2] Hessa, T. et al. Molecular code for transmembrane-helix recognition by the Sec61 translocon.Nature 450, 1026-1030, (2007). [3] Hessa, T., Reithinger, J. H., von Heijne, G. & Kim, H. Analysis of transmembrane helix integration in the endoplasmic reticulum in S. cerevisiae. J Mol Biol 386, 1222-1228, (2009). 39 www.msk.or.kr S3-1 Stimulatory Effect of Cell Elongation on Biofilm Formation in Pseudomonas aeruginosa under Anaerobic Growth Condition Sang Sun Yoon Department of Microbiology, Yonsei University College of Medicine, Seoul Pseudomonas aeruginosa, a Gram-negative bacterium of clinical importance, forms robust biofilm under anaerobic conditions that mimic the abnormally thickened mucus layer lining the airway of patients with cystic fibrosis (CF). The molecular basis behind this enhanced biofilm formation is unknown. We identified a morphological change naturally accompanied by anaerobic respiration in P. aeruginosa and investigated its effect on biofilm formation. A standard laboratory strain, PAO1, was elongated during anaerobic respiration compared to bacteria grown aerobically. The degree of cell elongation was dependent on the presence of nitrite reductase (NIR) that reduces nitrite (NO2-) to nitric oxide (NO) and was repressed in PAO1 in the presence of carboxy-PTIO, a NO antagonist. This demonstrated that cell elongation involves a process that responds to NO, a spontaneous byproduct of anaerobic respiration. The non-elongated NIR-deficient mutant failed to form biofilm, while the mutant of nitrate reductase (NAR) and PAO1, both of which were highly elongated, formed robust biofilm. Furthermore, decreased biofilm formation was also observed in PAO1 anaerobically grown in the presence of C-PTIO. Outer membrane protein, OprE, was highly upregulated during anaerobic respiration. An ΔoprE mutant was not as elongated as PAO1 and formed defective biofilm. Moreover, OprE synthesis was minimal in the non-elongated NIR-deficient mutant further supporting its vital role in cell elongation and biofilm formation under anaerobic condition. Taken together, our data suggest that anaerobiosis-induced cell elongation plays a critical role in biofilm formation. 40 Symposia S3-2 Vibrio vulnificus Biofilm: Regulation and Roles of Extracellular Polysaccharides Han-Suk Kim and Kyu-Ho Lee* Department of Environmental Science, Hankuk University of Foreign Studies To identify the genetic elements required for biofilm formation, we screened a pool of random Vibrio vulnificus mutants for their ability to form biofilms. One mutant displaying significantly decreased biofilm-forming activity was found to contain a transposon insertion in the ntrC gene encoding a well-known transcriptional activator. We examined how this regulator modulates a biofilm-forming process in V. vulnificus by searching for NtrC target gene(s). Comparison of the proteomes of ntrC mutant and wildtype strains grown under planktonic and biofilm stages revealed that synthesis of the protein homologous to GmhD (ADP-glycero-manno-heptose-6-epimerase) was elevated during the growth period for biofilm formation and was strongly influenced by NtrC. A luxAB-transcriptional fusion with the gmhD promoter region indicated that gmhD expression was positively regulated by NtrC. The function of the gmhD gene product in V. vulnificus was assessed by constructing and phenotypic analyses of an isogenic mutant. The gmhD mutant was defective in production of mature lipopolysaccharide (LPS) and demonstrated an attenuated ability to form a biofilm. These results suggest that NtrC acts as a key regulator of LPS biosyntheses and, thereby, modulates critical steps in biofilm development of V. vulnificus. In addition, the regulatory roles of NtrC in exopolysaccharide (EPS) biosynthesis were studied with three gene clusters for EPS biosyntheses. Transcriptions of the three clusters were positively controlled by NtrC and showed maximal expression at the early stage of biofilm development. Mutants deficient in one of the genes (VV1_2661, VV2_1579, and VV1_2305) in each cluster showed decreased production of EPS, attenuated ability to form biofilm, and lowered cytoadherence to human epithelial cells. However, mutations in VV2_1579 and VV1_2305 resulted in lower cytotoxicity to human cells and mortality to mice than the mutation in VV1_2661. These results demonstrate that NtrC-regulated EPS are crucial in biofilm formation of V. vulnificus, and some EPS components play important roles in interacting with hosts. References [1] Grau, B.L., Henk, M.C., Garrison, K.L., Olivier, B.J., Schulz, R.M., O’Reilly, K.L., and Pettis, G.S. (2008) Further characterization of Vibrio vulnificus rugose exopolysaccharide gene cluster. Infect Immun 76: 1485-1497. [2] Joseph, L.A., and Wright, A.C. (2004) Expression of Vibrio vulnificus capsular polysaccharide inhibits 41 www.msk.or.kr biofilm formation. J Bacteriol 186: 889-893. [3] Kim, H.-S., Lee, M.-A., Chun, S.-J., Park, S.-J., and Lee, K.-H. (2007) Role of NtrC in biofilm formation via controlling expression of the gene encoding an ADP-glycero-manno-heptose-6-epimerase in the pathogenic bacterium, Vibrio vulnificus. Mol Microbiol 63: 559-574. [4] Kim, H.-S., S.-J. Park, and K.-H. Lee. (2009) Role of NtrC-regulated exopolysaccharides in the biofilm formation and pathogenic interaction of Vibrio vulnificus. Molecular Microbiology. Mol Microbiol 74: 436-453. [5] Lee, J.H., Rho, J.-B., Park, K.-J., Kim, C.-B., Han, Y.-S., Choi, S.H., Lee, K.-H., and Park, S.-J. (2004). Role of flagellum and motility in pathogenesis of Vibrio vulnificus. Infect Immun 72: 4905-4910. [6] Park, N.Y., Lee, J.H., Kim, M.W., Jeong, H.G., Lee, B.C., Kim, T.S., and Choi, S.H. (2006) Identification of the Vibrio vulnificus wbpP gene and evaluation of its role in virulence. Infect Immun 74: 721-728. [7] Yildiz, F.H., and Visick, K.L. (2009) Vibrio biofilms: so much the same yet so different. Trends Microbiol 17: 109-118. 42 Symposia S3-3 Helicobacter pylori Infection in the Korean Population: An Epidemiological Link Between Toxin Polymorphism and Disease D. Scott Merrell, Ph.D. Associate Professor, Uniformed Services University, Bethesda, MD 20814, USA Helicobacter pylori is a medically important pathogen, and although infection rates vary geographically, globally this bacterium colonizes over 50% of the world’s population (1, 2). This spiral shaped, Gram-negative, microaerophilic bacterium chronically inhabits the unforgiving environment of the stomach, and causes subclinical gastritis in the majority of patients. However, in some individuals, H. pylori colonization results in peptic ulcer disease; 75% of gastric ulcers and 90% of duodenal ulcers are attributed to H. pylori infection (3). In its most severe sequelae, H. pylori infection can lead to the development of two forms of gastric cancer: adenocarcinoma and MALT lymphoma (4-7). The association of H. pylori with stomach cancer led the World Health Organization to classify it as a class I carcinogen in 1994 (8). It currently remains the only bacterium to obtain this perilous distinction. H. pylori strains express various toxins that enable the bacteria to cause host cell damage. Included among these toxins are the cytotoxin associated gene A (CagA) and the vacuolating cytotoxin (VacA) (9). CagA has emerged as a major contributor to disease severity, and there is a direct link between presence of CagA and increased cancer risk (10, 11). CagA induces various pathologic changes by modulating host cell signaling pathways, primarily after tyrosine phosphorylation at the EPIYA motif (12-19). The most common motifs have been designated as EPIYA-A, -B, -C, and -D (14), and are found in two distinct combinations by geographic location. VacA is another important toxin that is produced and secreted by all H. pylori strains (20, 21), and has been shown to have various modes of action (22-29). Like CagA, VacA has been shown to contain a number of polymorphisms. Currently, three polymorphic regions of vacA have been identified: signal (s), intermediate (i), and middle (m). Each of these polymorphic regions has two main types that divide them further into type 1 and type 2 (30, 31). The s region encodes the N-terminal signal sequence (32, 33), and polymorphisms in the s region affect anion channel-forming efficiency of the toxin (32). The s1 type has an increased ability to form membrane channels (32). Polymorphisms in the m region affect the cell tropism of the toxin (34); the m1 type of VacA shows toxicity to a broader range of cells than the m2 type (35, 36). The i region, located between the s and m regions, also displays two main polymorphisms (30). The i1 type of VacA has stronger vacuolating activity than the i2 type (30). Individually, the s1, i1, and m1 types have been shown to be associated with more severe forms of H. pylori induced disease (30, 37). Gastric cancer is the second most common cause of cancer death worldwide, and this fact could be reflective 43 www.msk.or.kr of the high incidence of H. pylori infection (38-41). Interestingly, geographic areas with the highest level of gastric cancer, which include most East Asian countries, also have the highest rate of H. pylori infection (39, 40, 42). Additionally, in East Asian countries, 90% of strains carry cagA. Indeed, South Korea, has one of the highest rates of H. pylori colonization (43) and one of the highest rates of gastric cancer in the world (11, 44). Recently, we conducted a large-scale molecular epidemiologic analysis of South Korean strains, and found a statistical link between the East Asian CagA, EPIYA-ABD genotype and development of gastric cancer. Statistical analysis showed that the proportion of ABD genotype varied significantly by diagnosis (P=0.022), and that this distribution was statistically different when compared to gastritis (P=0.004) or duodenal ulcer patients (P=0.014). Characterization of a subset of the Korean isolates showed that all strains from cancer patients expressed and delivered phosphorylateable CagA to host cells, whereas the presence of the cagA gene did not strictly correlate to expression and delivery of CagA in all non-cancer strains. Moreover, we genotyped the isolates for vacA, and then analyzed the data to determine if particular genotypes varied across disease state, sex, or cagA allele. Of these strains, 206 strains carried a s1/i1/m1 allele, 11 strains carried a s1/i1/m2 allele, and 8 strains carried a s1/i2/m2 allele. A statistical association between variation in the cagA and vacA alleles was identified (P=0.0007), and log linear modeling revealed that this variation affects severity of disease outcome (P=0.027). Additionally, we found evidence that variation within the middle (m) region of VacA contributes significantly to the distribution of vacA alleles across gender (P=0.008) as well as the association with disease outcome (P=0.011). In the Korean population, the majority of H. pylori strains carry the vacA s1/i1/m1 allele and the CagA EPIYA-ABD allele. These facts may contribute to the high incidence of gastric maladies including gastric cancer within this population. *Excerpts taken from Journal of Clinical Microbiology, 47(4):959-68, 2009 and Journal of Clinical Microbiology, 48(2):559–567, 2010. References [1] The EUROGAST Study Group, Gut 34, 1672 (Dec, 1993). [2] T. Matysiak-Budnik, F. Megraud, J Physiol Pharmacol 48 Suppl 4, 3 (Sep, 1997). [3] P. B. Ernst, B. D. Gold, Annu Rev Microbiol 54, 615 (2000). [4] M. J. Blaser, Bmj 316, 1507 (May 16, 1998). [5] J. Parsonnet et al., N Engl J Med 330, 1267 (May 5, 1994). [6] J. Parsonnet et al., N Engl J Med 325, 1127 (Oct 17, 1991). [7] N. J. Talley et al., J Natl Cancer Inst 83, 1734 (Dec 4, 1991). 44 Symposia [8] International Agency for Research on Cancer., in Monographs on the Evaluation of Carcinogenic Risks to Humans. (Lyon, 1994), vol. 61, pp. 177-240. [9] C. Montecucco, R. Rappuoli, Nat Rev Mol Cell Biol 2, 457 (Jun, 2001). [10] M. J. Blaser et al., Cancer Res 55, 2111 (May 15, 1995). [11] J. Gwack et al., Br J Cancer 95, 639 (Sep 4, 2006). [12] M. Hatakeyama, Oncogene 27, 7047 (Nov 24, 2008). [13] H. Higashi et al., Science 295, 683 (Jan 25, 2002). [14] H. Higashi et al., Proc Natl Acad Sci U S A 99, 14428 (Oct 29, 2002). [15] H. Higashi et al., J Biol Chem 279, 17205 (Apr 23, 2004). [16] B. G. Neel, H. Gu, L. Pao, Trends Biochem Sci 28, 284 (Jun, 2003). [17] K. Roovers, R. K. Assoian, Bioessays 22, 818 (Sep, 2000). [18] R. Tsutsumi, A. Takahashi, T. Azuma, H. Higashi, M. Hatakeyama, Mol Cell Biol 26, 261 (Jan, 2006). [19] M. Hatakeyama, Nat Rev Cancer 4, 688 (Sep, 2004). [20] T. L. Cover, S. R. Blanke, Nat Rev Microbiol 3, 320 (Apr, 2005). [21] J. C. Atherton et al., J Clin Microbiol 37, 2979 (Sep, 1999). [22] T. L. Cover, S. A. Halter, M. J. Blaser, Hum Pathol 23, 1004 (Sep, 1992). [23] B. Gebert, W. Fischer, E. Weiss, R. Hoffmann, R. Haas, Science 301, 1099 (Aug 22, 2003). [24] L. Manente et al., J Cell Physiol 214, 582 (Mar, 2008). [25] R. Pai, T. L. Cover, A. S. Tarnawski, Biochem Biophys Res Commun 262, 245 (Aug 19, 1999). [26] I. Szabo et al., EMBO J 18, 5517 (Oct 15, 1999). [27] M. R. Terebiznik et al., Autophagy 5, 370 (Apr, 2009). [28] V. J. Torres, S. E. VanCompernolle, M. S. Sundrud, D. Unutmaz, T. L. Cover, J Immunol 179, 5433 (Oct 15, 2007). [29] D. C. Willhite, S. R. Blanke, Cell Microbiol 6, 143 (Feb, 2004). [30] J. L. Rhead et al., Gastroenterology 133, 926 (Sep, 2007). [31] J. C. Atherton et al., J Biol Chem 270, 17771 (Jul 28, 1995). [32] M. S. McClain et al., J Bacteriol 183, 6499 (Nov, 2001). [33] A. P. Pugsley, Microbiol Rev 57, 50 (Mar, 1993). [34] X. Ji et al., Infect Immun 68, 3754 (Jun, 2000). [35] C. Pagliaccia et al., Proc Natl Acad Sci U S A 95, 10212 (Aug 18, 1998). [36] M. R. Amieva, E. M. El-Omar, Gastroenterology 134, 306 (Jan, 2008). [37] D. Basso et al., Gastroenterology 135, 91 (Jul, 2008). [38] A. I. Neugut, M. Hayek, G. Howe, Semin Oncol 23, 281 (Jun, 1996). [39] K. D. Crew, A. I. Neugut, World J Gastroenterol 12, 354 (Jan 21, 2006). [40] S. Yamamoto, Jpn J Clin Oncol 31, 471 (Sep, 2001). [41] D. M. Parkin, F. Bray, J. Ferlay, P. Pisani, CA Cancer J Clin 55, 74 (Mar-Apr, 2005). [42] Y. O. Ahn et al., J Korean Med Sci 6, 7 (Mar, 1991). [43] S. Tokudome et al., Asian Pac J Cancer Prev 8, 462 (Jul-Sep, 2007). [44] A. Shin et al., Br J Cancer 92, 1273 (Apr 11, 2005). 45 www.msk.or.kr S3-4 Entry Mechanism and Intracellular Localization of Orientia tsutsugamushi into Nonphagocytic Cell Myung Sik Choi*, Hyuk Chu, Sang Wook Kim, Nam Hyuk Cho, and Ik Sang Kim Department of Microbiology and Immunology, Seoul National University College of Medicine Orientia tsutsugamushi are obligate intracellular bacteria that grow within the cytoplams of the eukaryotic host cell. Therefore its capability of the attachment and entry into the cell surface and intracellular localization are critical steps in oriential pathogenesis. O. tsutsugamushi enters the host cell via unknown mechanism, this intracellular bacterium needs to exploit host endocytotic pathway. It has been reported that clathrin-mediated or caveolar-mediated endocytosis pathways are the main targets for many other intracellular bacteria such as Chlamydia, Listeria, and Rickettisa. To investigate the endocytotic pathway exploited by O. tsutsugamushi, we used several specific inhibitors to block, either clathrin-mediated or caveolar-mediated endocytotic pathway. The inhibitors blocking clathrin-mediated endocytotic pathway have clearly reduced the infectivity of O. tsutsugamushi into nonprofessional phagocytic cells. In contrast, the inhibitors blocking caveolar-mediated endocytotic pathway did not affect the infectivity. We further confirmed a localization of O. tsutsugamushi with clathrin or adaptor protein 2 (AP-2) in clathrin coated vesicles. In order to identify the endosomal escape of O. tsutsugamushi, colocalization of O. tsutsugamushi with molecules that are expressed either in early endosome or late endosome in early endosome antigen 1 (EEA1) and lysosomal-associated protein (LAMP2) was achieved. The result showed that orientia were colocalizaed with the late endosomal-lysosomal glycoprotein, LAMP2, during an early infection, but within 2 h this colocalization decreased significantly disappeared. This study suggested that O. tsutsugamushienter nonprofessional phagocytic cells such as endotheilal cells and fibroblast through clathrin-mediated endocytotic pathway and escapes from the endosome to the cytosol at phagosome/lysome state not ealry endosome. Next, we examined the mechanism of intracellular localization using ECV 304 cells, endothelial cell line. ECV 304 cells infected with O. tsutsugamushi recealed the collocation of microtubule organizing center (MTOC) and cytosolic orientiae by indirect immunoflurescence assay. Using immunofluorescence microscopy in presence and absence of MT depolymerizing agents (colchicine and nocodazole), it was shown that the cytosolic oriential movement was mediated by MTs. By transfection study (overexpression of p50/dynamitin, which is known to associate with dynein-dependent movement), the movement of O. tsutsugamushi to the MTOC was also mediated by dynein, the minus end directed MT-related motor. Although the significance of this movement in the cycle of O. tsutsugamushi was not proven, we propose that the cytosolic O. tsutsugamushi 46 Symposia uses MTs and dyneins to propel them from the cell periphery to the MTOC. This study is one of the few examples of the use of MT network by a nonmembrane bounded particle or microorganism. References [1] SW Kim, KS Ihn, SH Han, SY Seong, IS Kim, and MS Choi. Microtubule- and Dynein Mediated Movement of Orientia tsutsugamushi to the Microtubule Organizing Center Infection and immunity. 69, 494, 2001. [2] H Chu, JH Lee, SH Han, SY Kim, NH Cho,IS Kim, and MS Choi. Exploitation of the Endocytic Pathway by Orientia tsutsugamushi in Nonprofessional Phagocytes Infection and Immunity, 74, 4246, 2006. 47 www.msk.or.kr S3-5 Distinct Pathogenesis of Mycobacterium abscessus Isolated from Patients with Upper Lobe Fibrocavitary Form of Pulmonary Disease Sung Jae Shin*, Byung Soo Lee, Choul-Jae Won, Kwang-Wook Kim, Hyun Bae Kang, and Hosung Sohn Department of Microbiology and Infection Signaling Network Research Center, College of Medicine, Chungnam National University, Daejeon 301-747 Mycobacterium abscessus is a member of the group of rapidly growing mycobacteria (RGM), which cause a wide range of clinical diseases, including cutaneous disease, osteomyelitis, post-traumatic wound infection, and chronic lung disease [1-4]. M. abscessus has been identified as the causative organism in approximately 80% of cases of RGM pulmonary disease [1, 2]. In addition, M. abscessus is resistant to many antibiotics and thus is very difficult to treat [2, 5]. Pulmonary disease caused by nontuberculous mycobacteria (NTM), including M. abscessus, can be classified into two distinct types of clinical disease; the upper lobe fibrocavitary (UC) form and nodular bronchiectatic (NB) form [6]. In a very recent publication, our group reported the differential virulence between mycobacterial strains in accordance to the forms of the pulmonary M. abscessus diseases. In present study, we further to discuss the distinct pathogenesis of clinical strains based on the recent analysis of whole genome sequence , transposon mutants, and the pattern of macrophage cell death. References [1] Brown-Elliott BA and Wallace RJ Jr. Clinical and taxonomic status of pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria. Clin Microbiol Rev 2002;15:716-46. [2] Griffith DE, Girard WM, and Wallace RJ Jr. Clinical features of pulmonary disease caused by rapidly growing mycobacteria. An analysis of 154 patients. Am Rev Respir Dis 1993;147:1271-8. [3] Sanguinetti M, Ardito F, Fiscarelli E, La Sorda M, D'Argenio P, Ricciotti G, et al. Fatal pulmonary infection due to multidrug-resistant Mycobacterium abscessus in a patient with cystic fibrosis. J Clin Microbiol 2001;39:816-9. [4] Wallace RJ Jr., Swenson JM, Silcox VA, Good RC, Tschen JA, Stone MS. Spectrum of disease due to rapidly growing mycobacteria. Rev Infect Dis 1983;5:657-79. [5] Jeon K, Kwon OJ, Lee NY, Kim BJ, Kook YH, Lee SH, et al. Antibiotic treatment of Mycobacterium abscessus lung disease: a retrospective analysis of 65 patients. Am J Respir Crit Care Med 2009; doi:10.1164/rccm.200905-0704OC. [6] Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, et al. ATS Mycobacterial Diseases Subcommittee; American Thoracic Society; Infectious Disease Society of America. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 2007;175:367-416. 48 Symposia S4-1 A Sustainable Research and Development on Dokdo Hyun Soo Rho East Sea Research Institute (ESRI), Korea Ocean Research and Development Institute (KORDI) To further the dream of new, prosperous national marine system, the Dokdo Research Center has spearheaded the protection and governance of the Dokdo territory through integrated research and systematic result coordination based on the “Sustainable Use of Dokdo Act”. By compiling marine data collected through scientific investigations on Dokdo and surrounding waters, the program for “Sustainable Research and Development on Dokdo” has established efficient/sustainable use and control measures for this island. The data for Dokdo are being used to institute various strategies and a national policy for the environment of the islands and neighboring seas, including strategies for control and preservation of the ecosystem, resource securement, demarcation of the sea boundary between nearby countries, future spatial use, and strengthening of the protection and governance of Dokdo. The goals of this study were to characterize the topography and structural traits of Dokdo, conduct a field survey and empirical analysis for ecosystem and environmental monitoring of the surrounding waters, and coordinate data for Dokdo through the creation and operation of an integrated Dokdo database and web-site (www.dokdo.re.kr). These research results are important not only for the sustainable management and future use of this island but also for their protection and governance; they also serve to increase awareness of Dokdo both within and outside Korea through theses and conference presentations. In addition, for the systemic control and efficient use of a Dokdo archive, all data were compiled in a standardized database containing both previously collected data and data from our research team. All data on Dokdo are available to the public through the Dokdo website as a national authorized portal site. The website offers an abundance of information about Dokdo through a variety of activities, such as animations allowing the user to indirectly experience the marine survey, communication between users, and an informational communication that can both receive and offer professional advice. Because the East Sea Research Institute was built in Jukbyeon-myun, Uljin-Gun, Gyeongbuk, the closest city to Dokdo on the Korean mainland, we believe that the “Sustainable Research and Development of Dokdo” program will become widely known throughout Korea, thus fostering more comprehensive and systematic research endeavours. 49 www.msk.or.kr And, diverse scientific research into this island has allowed ‘Dokdo’ and the ‘East Sea’ to become internationally recognized, both at home and abroad. The interest and involvement of the Korean people will ensure that a national policy concerning Dokdo and the “Sustainable Research and Development of Dokdo” program will be legislated to help resolve the issues related to Dokdo that are important to Korea’s national security and national developmental potential. 50 Symposia S4-2 The Study of Species Composition and Molecular Plant Geography in Dokdo Island Jae-Hong Pak*, Woong Lee, Don-Hwa Lee, and Mi-Seon Kim Department of biology, College of natural sciences, Kyungpook National University, Daegu 702-701 Dokdo island belongs to the administrative district of Ulleung-gun, Gyeongsangbuk-do, Korea. It is Korean easternmost island, situated in the middle of the East Sea, at a latitude of N 37°14′26.8″ and a longitude of E 131° 52′ 10.4″. Location is 87.4 km away from the island of Ulleungdo in the East Sea, and 216.8 km from Jukbyeon, Gyeongsangbuk-do Province, and 157.5km away from the Japanese island of Oki. It is composed of two main islands, Seodo (“West island”: 88,639m2) and Dongdo (“East island”: 73,297m2), as well as 89 tiny rocks and reefs (25,517m2). Dokdo island is an oceanic island that created in 2~4.5 million years ago. It is Boreal Kingdom, Sino-Japanese region, Korea and South Japan, Ulleungdo province in floral region so that has independent characteristics different from the Japanese islands and Russia. The flora of vascular plants in Dokdo island were confirmed to be consisted of 31 families, 56 genera, 57 species, 1 subspecies, 4 varieties totaling 62 taxa growing spontaneously. Woody plants were 8 taxa and herbaceous plants were 54 taxa. Fern was 1 taxa, dicotyledons were 45 taxa and monocotyledons were 16 taxa. Based on The specific plant species for environmental assessment by the Ministry of the environment in the republic of Korea, a total of 13 taxa were identified including Orobanche coerulescens Stephan for the floristic degree V. The dispersion type of plant migration in Dokdo island was investigated by classifying anemochore(39 taxa, 62.9%), zoochore (12 taxa, 19.4%), hydrochore (2 taxa, 3.2%), artificial means. To research the molecular distributional pattern of Dokdo island’s plants, we used 2 endemic taxa of Sedum takesimense Nakai and Lonicera insularis Nakai in Ulleungdo and Dokdo island. L. insularis, grows on the seaside in Ulleungdo and Dokdo island, is deciduous shrub. It has 2 hypotheses about its origin: 1) Japanese Lonicera morowii A.Gray, which arrived Ulleungdo island by way of Dokdo island, is a progenitor of L. insularis in Dokdo island according to Ulleungdo & Dokdo island’s characteristics of historical geology. 2) L. insularis in Dokdo island planted by Ulleungdo island’s individual. To improve these 2 hypothesis, we investigated cpDNA non-coding region trnL-trnF, trnS-trnG, petN-psbM, psbM-trnD, matK gene of L. insularis. As a result, 4 region of trnL-trnF, trnS-trnG, psbM-trnD, matK gene corresponded with L. morowii’s it completely but petN-psbM region has 2 different types. L. insularis has type 1 of Ulleungdo island, Dongdo of Dokdo island, and type 2 of Ulleungdo island, Seodo of Dokdo island. L. morowii has only type 2. Accordingly, this result supported both of 2 hypothesis. Because there were no records about L. insularis’s distiribution in Dokdo island until L. insularis in Dokdo island planted by Ulleungdo island’s individual, the odds are against 51 www.msk.or.kr hypothesis 2. S. takesimense is particularly found only in Ulleung and Dokdo islands. This is an endemic species first reported by Nakai, that is the major species distributed in the Ulleungdo island. S. takesimense was classified family Crassulaceae, genus Sedum, subgenus Aizoon, but different various opinions about their classification were existed. So necessity of intensive studying was raised until the present. The comparative study of Chloroplast DNA trnL/F intergenic region among the 32 materials in Ulleungdo and Dokdo island results 2 distinctive types of cpDNA haplotype. Type 1 showed in Albong region of Ulleungdo island and Dokdo island, but Type 2 worked in Ulleungdo island only. Therefore, we got the result of two cpDNA lineages which has separate molecular distributional pattern. This result suppose that S. takesimense originated from multiple progenitors, also long-distance dispersal. References [1] Stuessy T.F. and Mikio Ono Evolution and speciation of island plants, 1-357, 1998. [2] Pfosser M., Jakubowsky G., Schluter P.M., Fer T., Kato H., Stuessy T.F. and Sun B.Y. Pl. Syst. Evol., 256, 159-170, 2006. 52 Symposia S4-3 Marine Invertebrate Fauna of Dokdo and Molecular Phylogeography of Sea Slaters and Chitons in Korea and Japan Ui Wook Hwang Department of Biology, Teachers College & Institute for Phylogenomics and Evolution, Kyungpook National University, Daegu 702-701 Benthic fauna were studied on the intertidal and subtidal rocky shores of Dokdo (island), Korea, between August 2007 and June 2008. A total of 98 marine invertebrate species in 57 families was identified throughout the study period, and 21 of those species are newly recorded including Porifera (n=1), Cnidaria (n=1), Platyhelminthes (n=1), Mollusca (n=3), Annelida (n=4), Arthropoda (n=9), and Echinodermata (n=2). A topographical description and ecological comment are given for each major species. Among the seven phyla presented, Mollusca and Arthropoda predominated, accounting for >70-100% of the total taxa at each station. In general, the faunal composition and distribution of invertebrates considered by habitat type (viz. attached, sessile or mobile species) seemed to be closely related to the topographical characteristics of each site. Altogether, the total of 403 species (172 families and 10 phyla) recorded in the present study and 13 earlier ones indicate that the marine invertebrates inhabiting rocky bottoms in the Dokdo ecosystem show high and dynamic biodiversity. A sea slater Ligia exotica is a cosmopolitan species, widely distributed throughout all over the world. In South Korea, it has been known there is only one species (L. exotica). Genetic diversity and phylogenetic relationships among populations of L. exotica were examined based on the partial 16S rDNA (429bp) and cytochrome C oxidase subunit I gene (COI, 539bp) of 310 individuals: 246 from various sites in South Korea and 64 from Japan. Comparative analyses of the nucleotide sequences of 16S rDNA and COI revealed that L. exotica consist of two distinct genetic lineages, A and B types. The sequence dissimilarity between the two types is about 11.4 %. In total, 51 haplotypes (21 in A and 30 in B) were identified from the 310 individuals examined in this study. In common, both the types A and B were found mixed in most collecting sites. However, in the eastern coastline of the Korean Peninsula, the A type is more dominant, whereas the B type is more dominant in in the western coastline vice versa. On the other hand, the results suggested that, in the four islands (Ulleungdo, Dokdo, Jejudo and Tsushima islands), there exists only one type of the two exclusively and there are shown absolute dominance of a type compared to the others. Through the AMOVA test, statistical significance of genetic differences was examined between the two types of L. exotica and among individuals within each of the two types. Consequently, the present data indicated that a large population of L. exotica in South Korea and Japan consists of the two distinct mitochondrial genetic lineages. 53 www.msk.or.kr Liolophura japonica (Lischke, 1873) is one of the chitons distributed widely in the Korean Japanese coasts. In this study, genetic diversity among Korean and Japanese L. japonica populations was analyzed, focusing on those of Dokdo and Ulleungdo islands, Korea. Nucleotide sequence analyses of COI, 16S rDNA, and ITS revealed that genetic diversity of L. japonica populations of Dokdo and Ullengdo islands was twice higher than those of other examined regions. In total, 76 and 23 haplotypes were identified in COI and 16S rDNA, respectively. Statistical analyses of the two mitochondrial genes showed that L. japonica comprises two genetically distinct groups (types A and B). Type A has star-like structure indicative of a recent population expansion, whereas type B has a more complicated genealogical pattern. The present study elucidated that type A is widely distributed in the Korean and Japanese rocky coasts whereas type B is distributed below latitude of 35°N to 36°N. The two types of L. japonica are inhabitant below about 36°N with significant genetic difference between them. In addition, phylogenetic analyses consistently showed that the two types were divided into two separate clades. Comparative analyses of molecular, morphological, and ecological data of the two types A and B strongly suggested that L. japonica may be divided into two independent species. References [1] Kim SH, and Kim YT Marine invertebrates II of Dokdo, Ministry of Environment, p. 32, 2006. [2] Choe BL, Park JK and Lee JR Marine Molluscs of Ulrung and Dokdo Islands, Korean National Council for Conservation of Nature, 353, 1996. 54 Symposia S4-4 Exploring Microbial Functional Diversity of the Abyssal Seafloor in the East Sea Jong-Shik Kim Research and Development Department, Gyeongbuk Institute for Marine Bioindustry (GIMB) Marine microbial communities play roles in the functioning of abyssal seafloor ecosystems and are believed to be major contributors to the biogeochemical cycles. Despite their importance, the vast majority of microorganisms are uncultivated, thus their roles in the ecosystems are poorly understood. Recently, however, the study of microbial diversity in the ocean has been advanced as presented by recent papers [1, 2, 3]. These papers all provide invaluable methods and knowledge. Ocean samples, collected across Northwest Atlantic through Eastern Pacific [1], at a depth of 4,000 m [2] and from ocean’s surface to near-sea floor depths [3], have been studied for understanding microbial diversity. In order to understand microbial functional diversity of the abyssal seafloor in East Sea, we are here investigating the microbial community organization and specific functional adaptations from the level of the entire microbial communities to the individual isolates. Our goal is to advance the understanding of structure-function relationships in microbial communities addressing the followings: 1) diversity of nonculturable microorganisms in the abyssal seafloor in the East Sea, 2) diversity of cultured microorganisms adapted to the deep-sea floor in the East Sea. ACKNOWLEDGEMENTS This work was supported by the Gyeongsanbuk-Do and Uljin-Gun’s research support program. References [1] Rusch D.B., A.L. Halpern, G. Sutton, K.B. Heidelberg, S. Williamson, S. Yooseph, D. Wu, J.A. Eisen, J.M. Hoffman, K. Remington, K. Beeson, B. Tran, H. Smith, H. Baden-Tillson, C. Stewart, J. Thorpe, J. Freeman, C. Andrews-Pfannkoch, J. E. Venter, K. Li, S. Kravitz, J. F. Heidelberg, T. Utterback, Y.-H. Rogers, L.I. Falcon, V. Souza, G. Bonilla-Rosso, L.E. Eguiarte, D.M. Karl, S. Sathyendranath, T. Platt, E. Bermingham, V. Gallardo, G. Tamayo-Castillo, M.R. Ferrari, R.L. Strausberg, K. Nealson, R. Friedman, M. Frazier, and J.C. Venter. 2007. The Sorcerer II global ocean sampling expedition: northwest Atlantic through eastern tropical Pacific. PLoS Biol. 5(3): e77. [2] Konstantinidis K.T., J. Braff, D.M. Karl, and E.F. DeLong. 2009. Comparative metagenomic analysis of a microbial community residing at a depth of 4,000 meters at station ALOHA in the North Pacific subtropical gyre. Appl. Environ. Microbiol. 75(16): 5345-5355. [3] DeLong E.F., C.M. Preston, T Mincer, V. Rich, S.J. Hallam, N.U. Frigaard, A. Martinez, M.B. Sullivan, R. Edwards, B.R. Brito, S.W. Chisholm, and D.M. Karl. 2006. Community genomics among stratified microbial assemblages in the ocean’s interior. Science 311: 496-502. 55 www.msk.or.kr S5-1 Practical Application of Environmental Microorganisms as Bioresources Sang Seob Lee Dept. Biological Science, School of Natural Science, Kyonggi University Environmental microorganisms can be used to help clean up pollution created by human activities, a process called bioremediation. Various microorganisms can be used to consume spilled oil, solvents, pesticides, and other environmentally toxic pollutants, The great diversity of microorganisms on Earth contains vast genetic resources to mediate solutions for cleaning up the environment, and much research in this area is taking place at present. In this paper, recent progress in practical application of environmental microorganisms are briefly presented with emphasis on their potential for bioresources. Development of Biological Treatment System of Water Pollutants The enhanced nutrients removal system for wastewater was developed using phototrophic purple non-sulfur bacteria isolated from the streams in Kyonggi area, Korea using selective media, cultivated anaerobically under the light (2,000 Lux). A 15-liter reactor modified from the A2/O process was employed for the cultivation and the light apparatus was used for the dominant growth of photosynthetic bacteria. Experiments were performed into two Phases and the results were compared: the synthetic wastewater was tested for the removal efficiency of nutrients and organics during Phase 1 and the real wastewater during Phase 2. For both Phases, the F/M ratio was maintained at 0.14-0.18 mg BOD5/mg MLVSS.day. Results showed that 97-99% of organics were removed during Phase 1 and 96-99% during Phase 2. Nutrients (nitrogen and phosphorus) were also removed efficiently: 85-91% removal of T-N and 78-92% removal of T-P were achieved for Phase 1, and 76-89% removal of T-N and 73-88% removal of T-P for Phase 2. Development of Biodegradation System of Oil Contaminated Soil and Ground Water In a oil contaminated site in In-chun, Korea, 43 strains of benzene degraders were isolated from soil by growing on mineral medium with Benzene as a sole carbon source. Among them, 12 isolates grew up quickly. 12 strains were used for benzene removal screen test, and 7 strains for toluene removal were tested, respectively. Consiquently, BJ10 which were identified as Pseudomonas sp. by 16S rDNA showed the highest removal efficiency for benzene and toluene. In basal medium with 56 μmol benzene, BJ10 could degrade benzene over 95% and showed good biomass growth after 3 h of incubation(incubation condition : temperature; 30°C, cell concentration; 1 g/L, pH8). With 47 μmol of toluene, BJ10 removed over 98% of toluene on liquid medium with 56 Symposia same condition. Also, It can degrade toluene, ethylbenzene and o-Xylene without other nutrients. Development of Biodegradation System of Explosives Contaminated Soil Total 235 bacterial strains were screened for TNT (2.4.6-trinitrotoluene), 2,4 and 2,6-DNT (2,4 and 2,6-dinitrotoluene) removal, respectively. KT22 was identified as Serratia sp., KD4 was identified as Pseudomonas sp., and KD6 was identified also as Pseudomonas sp., showed the highest removal efficiency for TNT, 2,4-DNT, and 2,6-DNT, respectively. Optimal conditions were shown cell concentration 1.0 g/L, pH 7.0, and temperature 25-30°C. The growth rate was lower for 2,6-DNT, compared to TNT and 2,4-DNT. Mixed culture of KT22 and Bacillussp. showed increased TNT removal efficiency. In LB medium with 100 mg/L TNT, KT22 could degrade TNT over 97.7% and showed good biomass growth after 6 h of incubation. The growth rate was lower for 2,6-DNT, compared to TNT and 2,4-DNT. KD4 and KD6 could degrade 2,4 /2,6-DNT over 99.0% and 92.66% after 12 h and 18 h of incubation, respectively. In Stanier’s basal mineral medium, the growth rate(μ) of KT22 was 0.253 h-1, KD4 and KD6 were 0.212 h-1, 0.027 h-1. Mixed culture of KT22 and Bacillus sp. showed increased TNT removal efficiency. 57 www.msk.or.kr S5-2 Current Status and Future Plan of KCTC Jung-Sook Lee Korean Collection for Type Cultures (KCTC), Biological Resource Center (BRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), 111 Gwahangno, Yusong-gu, Daejeon 305-806 Tel: +82-42-860-4600, Fax: +82-42-860-4677, E-mail: [email protected] The largest and oldest culture collection in Korea, Korean Collection for Type Cultures (KCTC) has three functions as follows. 1) We carry out the collection and preservation of core biological resources from home and abroad, offering public support by distributing biological resources to academia, industry, and research institutions, and organizing patent strain deposit. Especially, we were designated as an International Depositary Authority (IDA) under Budapest Treaty by World Intellectual Property Organization (WIPO). Now we can manage about 17 kinds of biological resources including bacteria, yeast, fungi, algae, animal and plant viruses, embryos, animal and plant cell cultures, seeds, RNA and etc. We manage more than 18,000 biological resources including patent strains. Every year over 1,700 strains are newly added to KCTC. We also distribute more than 5,200 strains every year to home and abroad. 2) We develop the platform technology for the screening, identification and preservation of useful biological resources. We publish more than 60 papers concerning biological resources. Since 1997, we reported more than 160 new species. Last year, KCTC was cited by over 280 papers in PubMed search. 3) We are trying to construct on local and international network for biological resources and related information, and support workshops, conferences, and consultation, etc. As a national bio-infrastructure for biological resources, we perform the role of a biotechnology think-tank in the field of bio R&D. KCTC plays an important part in networking among the collections in domestic, and participates actively in international network among the collections in the world. We concentrate our efforts to increase KCTC to international biological resource center. 58 Symposia S5-3 Management of Microbial Genetic Resources in Korean Agricultural Culture Collection (KACC) In-Cheol Park*, Hang-Yeon Weon, Seung-Beom Hong, Soon-Ja Seok, Soo-Jin Kim, Yi-Seul Kim, and Soon-Wo Kwon Korean Agricultural Culture Collection (KACC), National Agrobiodiversity Center, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-853 Korean Agricultural Culture Collection (KACC) was established in 1995, in order to conserve microbial diversity in agro-environment and food, providing related services to professional microbial research societies. KACC classifies and preserves diverse Korean microbial resources including bacteria, actinomycetes, yeasts, filamentous fungi and mushrooms. Moreover, in collaboration with Centraalbureau voor Schimmelcultures (CBS), Deutsche Smmlung von Microorganismen und Zellkulturen GembH (DSMZ), NITE Biological Resources Center, and other international collections, type strains from overseas were kept in KACC. A total number of microbial strains registered in the KACC was up to 12,924 by end of the year, 2009. The 17,800 herbarium specimens of mushrooms are preserved in Herbarium Conservation Center of National Academy of Agricultural Science (HCCN). Registered microbial strains were preserved and maintained by various methods such as lyophilization, cryopreservation by liquid nitrogen and ultra-low temperature storage below -80°C, water storage, mineral oil storage and other methodologies according to their preservation properties. The registered strains were filed up to database in the KACC homepage (http://www.genebank.go.kr) to facilitate the efficient utilization of microbial resources and to make them easily accessible through internet. In 2009, the number of distributed isolates scored up to 3,992 strains including 1,973 strains of bacteria and 2,019 strains of fungi. The distributed strains were used in microbial research and development at laboratory of universities or institutes. It is estimated that import substitution corresponds to eighty million won through distributions by KACC. 59 www.msk.or.kr S6-1 Molecular Signature of DC Subsets during Acute vs. Chronic Virus Infection Sang-Jun Ha*, Yun-Hee Jeong, Young-Ho Bahn, Joon-Seok Park, Chan-Hee Park, and Hyo-Jin Park Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 Today, chronic viral infections such as HIV, HBV, and HCV, became a major issue to threaten public health. Chronically infected individuals not only act as a reservoir for viral spread, but also chronic infection in general increases the risk of subsequent diseases and secondary infections with other pathogens. In the face of continuously spreading viruses, there is an urgent need to develop vaccines that can be used therapeutically. Many chronic viral infections are marked by pathogen persistence and a generalized immunosuppression. The exact mechanisms by which this occurs are still unknown. The strategy comparing immune responses after acute vs. chronic viral infection might be one of the ways to uncover the mechanism of immunosuppression caused by chronic viral infection. We analyzed the function and phenotype of both T cells and dendritic cells (DCs) after acute vs. chronic infection. By comparing antigen-specific acute and chronic T cells, we found that co-inhibitory molecules including Programmed death 1 (PD-1) are highly expressed on T cells, suppress their function, and finally leads to a progressive exhaustion. We also observed that conversion of DC subtypes priming T cells (from lymphoid DCs to myeloid or plasmacytoid DCs) occurred during chronic infection but not acute infection. Especially, myeloid DCs showed high level expression of co-inhibitory molecules and low level expression of co-stimulatory molecules during chronic virus infection. Therefore, the strategy both blocking inhibitory signals on T cells/DCs and targeting antigen to proper DCs might overcome immunosuppression and subsequently enhance the clearance of persistent virus. References [1] Ha SJ, West EE, Araki K, Smith KA, and Ahmed R Immunological Reviews 223, 317 (2008). [2] Ha SJ, Mueller SN, Wherry EJ, Barber DL, Aubert RD, Sharpe AH, Freeman GJ, and Ahmed R Journal of Experimental Medicine 205, 543 (2008). [3] Wherry EJ, Ha SJ, Kaech SM, Haining WN, Sarkar S, Kalia V, Subramaniam S, Blattman JN, Barber DL, and Ahmed R Immunity 27, 670 (2007). [4] Blattman JN, Wherry EJ, Ha SJ, van der Most RG, and Ahmed R Journal of Virology 83, 4386 (2009). [5] Brooks DG, Ha SJ, Elsaesser H, Sharpe AH, Freeman GJ, and Oldstone MBA Proceedings of the National Academy of Sciences USA 105, 20428 (2008). [6] Li Q, Skinner PJ, Ha SJ, Duan L, Mattila TL, Hage A, White C, Barber DL, O’Mara L, Southern PJ, Reilly 60 Symposia CS, Carlis JV, Miller CJ, Ahmed R, and Hasse AT Science 323, 1726 (2009). [7] Slifka MK, Homann D, Tishon A, Pagarigan R, and Oldstone MBA Journal of Clinical Investigation 111, 805 (2003). [8] Zuniga EI, Liou LY, Mack L, Mendoza M, and Oldstone MBA Cell Host & Microbe 4, 374 (2008). 61 www.msk.or.kr S6-2 Options for Control of Pandemic Influenza: Active and Passive Immunization Huan H. Nguyen International Vaccine Institute, Seoul, Korea and Department of Microbiology, University of Alabama at Birmingham, USA Pandemic influenza poses a serious threat to global health and the world economy. Highly pathogenic avian influenza A virus (HPAIV) of the H5N1 subtype that has emerged since 2004, resulted in more than 430 cases of laboratory-confirmed human infection in 15 countries with a death rate of more than 50% and remains a global threat because of its continued transmission among domestic poultry and wild birds. Options for control of pandemic influenza are vaccination in combination with novel immunization routes and therapeutic anti-virals including virus-specific antibodies (Abs). The current influenza vaccines designed to inducing antibody (Ab) responses against viral surface antigens (i.e., hemagglutinin [HA] and neuraminidase [NA]) is limited by the ability of the virus to mutate these major antigenic glycoproteins. Vaccines that target determinants conserved among influenza A viruses (IAV) to generate broad protection against infection with different influenza A subtypes (i.e., heterosubtypic immunity [HSI]) remain elusive. We have currently developed a recombinant adenovirus (Ad) vector co-encoding HA (H5 subtype) and a conserved ectodomain of matrix protein 2 (M2e) (AdH5/M2e) for induction of protective immunity to H5N1 and other subtypes. Another approach in influenza vaccine development includes new generation of live-attenuated vaccine based on genetic deletion of the nonstructural NS1 protein of IAV. Since NS1 enables the virus to disarm the host cell type 1 IFN defense system, mutation or deletion of the NS1 gene leads to attenuation of the viruses and enhances host antiviral response. Therefore, NS1 deleted viruses (DelNS1) can serve as a candidate live-attenuated influenza vaccine that provides better protection than inactivated vaccines and could induce HSI to infection with different influenza virus A subtypes. Sub-lingual immunization has been found to be a safe and effective route for induction of protective specific immune responses in systemic and mucosal compartments including respiratory tract. We found that sublingual immunization with either AdH5/M2e or DelNS1 induces broad protective immunity to H5 viruses and other influenza virus A subtypes including H1N1. Passive immunization (the transfer of specific immunoglobulins/Abs to a previously non-immune recipient host) could offer an alternative strategy to prevent and treat influenza virus infection and an additional therapeutic option to antiviral drugs that are limited by widespread drug resistance among influenza virus strains. Even after targeted vaccines become available, passive immunization could still have prophylactic effects and 62 Symposia provides an additional countermeasure against influenza. Attempts to develop monoclonal Abs (mAbs) have been made. However, passive immunization based on mAbs may require a cocktail of mAbs with broader specificity in order to provide full protection since mAbs are generally specific for single epitopes. We found that H5N1-specific immunoglobulins (IgY) prepared from eggs laid by H5N1-vaccinated hens protect against infections with HPAIV H5N1 and related H5N2 strains. When administered intranasally before or after lethal infection, IgY prevent disease or significantly reduce viral replication resulting in complete recovery from the disease, respectively. We further generated H1N1 virus-specific IgY by immunization of hens with inactivated H1N1 A/PR/8/34 as a model virus for current pandemic H1N1/09 and found that such H1N1-specific IgY protect mice from lethal influenza virus infection. These results underscore the usefulness of recombinant Ad vectors encoding surface glycoprotein (HA) and conserved protein (M2e) and NS1 deleted viruses (DelNS1) as vaccine candidates for control of pre-pandemic H5N1 and newly emerging subtypes. In addition, our data on antiviral efficacy of IgY provide a proof-of-concept for the approach using virus-specific IgY as affordable, safe, and effective alternative for the control of influenza outbreaks, including the potential H5N1 and current H1N1 pandemic. 63 www.msk.or.kr S6-3 Chromatin Remodeling, HIV Reservoir, and Drug Development for Eradication of Chronic HIV Infection Kee-Jong Hong Center for Infectious Diseases, Korea National Institute of Health Chronic infection of HIV in CD4+ T cell reservoir is considered as a major barrier to eradicate the infected viruses from AIDS patients. During chronic reservoir development after integration of HIV, chromatin remodeling through acetylation and deacetylation may have a key role for latent replication of proviral DNA. We established four new latently HIV-infected cell lines showing unique characteristics about latency; four Clicj cells from jurkat T lymphocytes and three NCHA cells from A3.01 T lymphocytes. Those cell lines were different from another already developed latent HIV cell lines J1.1 and ACH2 in basic characteristics; receptor distribution, p24 induction and chemokine production. Our microarray experiments using latent cells showed higher expression of histone deacetlyase 4 and H2B compared to parent T cells (A3.01 and Jurkat). p24 production by HIV replication in latent HIV cells was increased by treatment of the novel synthetic HDAC inhibitors (CG05 and CG06), also the acetylation level of all latent cell lines was decreased. These two novel HDAC inhibitors may support the new strategy to break HIV reservoir when used with HAART or other inhibitors. This kind of approach will provide us helpful information to develop new advanced drug candidate for HIV treatment, also can support new strategy for virus eradication from HIV infected patients. 64 Symposia S6-4 Automated HTS/HCS for Antivirals Using Visual HIV Full Replication Assays Peter Sommer Cell Biology of Retroviruses Unit, Institut Pasteur Korea, Gyeonggi-do E-mail: [email protected] There are currently 25 drugs belonging to 6 different inhibitor classes approved for the treatment of human immunodeficiency virus (HIV) infection. However, new anti-HIV agents and treatment strategies are still needed to confront the emergence of drug resistance and various adverse effects associated with long-term use of antiretroviral therapy and the inability to cure infected individuals. We developed visual, HIV full replication assays and implemented them in high-throughput compound (n=200.000) and genome-wide siRNA screens, which allowed the identification of a few thousand novel small molecules with potent anti-retroviral activity and a few hundred host factors required for HIV infection, respectively. The identified compounds and host factors are opening unexplored avenues to novel antiviral drug and target discovery and validation, and should feed the drug development pipeline in the near future. 65 www.msk.or.kr S6-5 Probiotics as an Immune Modulator - Mechanism of Action and Therapeutic Applications Ho-Keun Kwon1, Choong-Gu Lee1, Jae-Seon So1, Chang-Suk Chae1, Ji-Sun Hwang1, Anupama Sahoo1, Jong Hee Nam2, Joon Haeng Rhee2, and Sin-Hyeog Im1,2* 1 Department of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712 2 Chonnam National University Medical School, Gwangju 501-749 Introduction Gastrointestinal microorganisms affect host physiology through diverse mechanisms, including modulation of the host immune system. Probiotics are nonpathogenic microorganisms that confer a number of beneficial effects on the health of the host (1). Among them, species of Lactobacilli and Bifidobacteria are prominent probiotics with anti-inflammatory properties (2). For example, administration of Lactobacillus casei suppresses pro-inflammatory responses by increasing IL-10 levels (3, 4), while Lactobacillus acidophilus increases Th1 type cytokines (5). However, many questions remain unanswered, such as which probiotic strains are the most effective in modulation of specific immune disorders and how orally administrated probiotics affects systemic immune system (6). In this study, we sought to identify which probiotics, or mixture of probiotics, could confer potent anti-inflammatory effects by increasing CD4+Foxp3+ regulatory T cells (Tregs). The forkhead family protein Foxp3 is a transcription factor highly expressed in CD4+ Tregs. It is a regulator of T cell tolerance, and is necessary for the development and function of Tregs (7). Methods Following in vitro and in vivo functional studies on Treg generation, we developed a probiotics mixture that exhibits potent anti-inflammatory properties and investigated its modulation of diverse immune disorders. The probiotics mixture, designated IRT5, consists of a combination of five probiotic strains. The effect of IRT5 administration on host immune system was investigated. Results and conclusion Oral administration of IRT5 induced T and B cell hypo-responsiveness without inducing apoptosis. IRT5 administration increased the level of CD4+Foxp3+ Tregs in mesenteric lymph nodes (MLN) by augmenting Foxp3+ levels in CD4+CD25- T cells. Conversion of T cells into Foxp3+ Tregs is directly mediated by regulatory dendritic cells (rDCs) that express increased levels of IL-10, TGF-β, Cox2 and indoleamine 2,3-dioxygenase (iDO). Administration of IRT5 suppressed the progression of experimental inflammatory bowel disease (IBD), 66 Symposia atopic dermatitis (AD) and rheumatoid arthritis (RA). In addition, migration of Tregs to inflammatory regions in response to chemokines (CCL1 and CCL22) and their receptors (CCR4 and CCR8) mediated disease suppression. Our results present the first evidence of generation of CD4+CD25-Foxp3+ Tregs in response to probiotics, an effect that may be therapeutically useful for the modulation of inflammatory immune disorders. Acknowledgements This research was supported by grants from the Regional Technology Innovation Program of the MOCIE (RT105-01-01) and the BioGreen 21 Program (20070501034009; PJ007054) in Rural Development Administration and an Agricultural R&D Promotion Center, Republic of Korea. References [1] Marteau P (2006) Gut 55(12):1692-1693. [2] Braat H, et al. (2004) Am J Clin Nutr 80(6):1618-1625. [3] So J-S, et al. (2008) Mol Immunol 46(1):172. [4] So J-S, et al. (2008) Mol Immunol 45(9):2690. [5] Sudo N (2002) Clinical & Experimental Allergy 32(7):1112-1116. [6] Shanahan F (2003) Scand J Gastroenterol Supplement (237):34-36. [7] Ziegler SF (2006) FOXP3 Annu Rev Immunol 24(1):209-226. 67 www.msk.or.kr S7-1 Adaptive Cellular Survival Response to Oxidative and Inflammatory Stresses Induced by Helicobacter pylori Young-Joon Surh College of Pharmacy, Seoul National University, Seoul 151-742 Accumulating evidence from preclinical and clinical studies suggest that chronic infection and inflammation contribute the carcinogenesis. Helicobacter pylori infection has been implicated in gastritis and peptic ulcer, which can eventually progress to gastric cancer [1]. However, in a long-term study conducted in a wild-type mouse model, H. pylori infection itself did not induce gastric cancer. To elucidate the mechanisms underlying this discrepancy, we investigated the molecular mechanisms responsible for H. pylori-induced inflammatory response in human gastric cancer (AGS) cells and genetically engineered animal models. Infection of AGS cells with H. pylori up-regulated the levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase, enhanced secretion of interleukin-8, and increased phosphorylation of ERK. The nuclear translocation and subsequent DNA binding of NF-kappaB, which is known to regulate pro-inflammatory gene expression, were augmented after H. pylori treatment. The DNA binding of AP-1 and the expression of c-Jun and phospho-c-Jun were also induced by H. pylori infection. A wide array of antioxidant enzymes and other cytoprotective proteins constitute a fundamental cellular defense system against oxidative and proinflammatory insults. Treatment of AGS cells with H. pylori resulted in elevated expression of heme oxygenase-1 (HO-1), a representative stress-responsive enzyme, and its mRNA transcript. The immunofluorescence staining, using an FITC-conjugated HO-1 antibody, verified the elevated cytoplasmic localization of HO-1 after H. pylori treatment. The H. pylori treatment enhanced antioxidant response element (ARE) binding of Nrf2, a redox-sensitive transcription factor responsible for regulating expression of many antioxidant and cytoprotective genes, including ho-1. Chromatin-immunoprecipitation and ARE-luciferase reporter gene assays revealed that H. pylori treatment induced Nrf2 binding to the ARE site and that the binding site is located at -0.5 kB of human ho-1 promoter region. After repeated H. pylori inoculation (x 3), mouse stomach tissues were collected at 16 weeks. Following inoculation at 1, 4, 8 and 16th week of H. pylori inflammation was evident in gastric mucosa, and there was the Nrf2 activation in mouse stomach as monitored by utilizing ARE transgenic mice harbouring the human placenta alkaline phosphatase reporter gene. Analysis of stomach tissues collected from Nrf-2-/-, Nrf-2+/- and Nrf-2+/+ mice infected with H. pylori for 16 weeks revealed that H. pylori-induced inflammation as well as oxidative stress was more pronounced in Nrf2 knock-out mice than in hetero and wild-type mice. Taken together, H. pylori infection can trigger the proinflammatory response through up-regulation of COX-2, which provokes adaptive cellular defense response through Nrf2-mediated 68 Symposia upregulation of antioxidant enzyme, such as HO-1, thereby protecting cells and tissues against subsequent prooxidative and proinflammatory insults. Some phytochemicals, by stumulating Nrf2-driven cytoprotective gene expression, may exert chemoprotective and chemopreventive eddects against H. pylori-induced gastritis and gastric carcinogenesis [2]. Figure 1. Role of Nrf2 in adaptive survival response to H. pylori-induced oxidative and inflammatory gastric damage. Acknowledgements This work was supported by the National Research Foundation (NRF) grant (20100001707) through the Ministry of Education, Science and Technology (MEST), Republic of Korea References [1] M. Miyamoto, K. Haruma, M. Yoshihara, T. Hiyama, M. Sumioka, T. Nishisaka, S. Tanaka, and K. Chayama Dig. Dis. Sci., 48, 9.68-975, 2003. [2] Y.-J. Surh Nature Rev. Cancer, 3, 768-780, 2003. 69 www.msk.or.kr S7-2 Physiological Functionalities of Small Peptides Toshiro Matsui, PhD Faculty of Agriculture, Graduate School of Kyushu University, Fukuoka, 812-8581, Japan Tel/Fax: +81-92-642-3012 E-mail address: [email protected] Bioactive peptides derived from natural proteins by enzymatic hydrolysis or fermentation have been examined for the years whether they can prevent life-style related diseases, including hypertension, diabetes, atherosclerosis and renal dysfunction. The well-recognized physiological functionality of small peptides is a preventive potential against elevated blood pressure in mild hypertensive subjects. For the effect, much interest has been focused on their inhibitory potential of angiotensin I-converting enzyme (ACE) that is closely associated with the production of pressor hormone, angiotensin (Ang) II from Ang I or the onset of hypertension. More than 400 ACE inhibitory peptides have been reported so far. It was evidently proven that some ACE inhibitory peptides had a significant blood pressure (BP) lowering effect in mild hypertensive subjects. A good example is a di-peptide-induced anti-hypertensive effect, in which Val-Tyr isolated from sardine muscle hydrolysate showed a ca. 10 mmHg BP reduction in human study for a 1-month protocol. Val-Tyr also claimed another physiological potential, since it suppressed tissue renin-angiotensin systems in aorta and kidney, not in blood system in transgenic Tsukuba-hypertensive mice bearing the human renin-angiotensin system. To clarify the effect of small peptides on vessel function(s), ex vivo vascular contractive experiments were conducted in a rat aorta-Magnus force measurement. As a result, we obtained an interesting finding that Val-Tyr evoked vascular relaxation on thoracic aorta from SHR in endothelium- and ACE inhibition-independent manners, although it has already been reported that longer or oligo-peptides such as Arg-Ala-Asp-His-Pro (-Phe) could relax contractive tension of aorta in an endothelium-dependent manner through a promotion of NO/cGMP vasorelaxation pathways. It indicates that small peptides may play a role in the regulation of vascular response in vascular smooth muscle layer by alternative vasorelaxation mechanism(s). In a screening study of vasoactive small peptides, basic amino acid-containing peptides such as Trp-His (EC50: 3.4 mmol/L) and His-Arg-Trp (EC50: 1.2 mmol/L) were found to preferably relax the contractive aorta in an endotheliumindependent manner. In vascular smooth muscle cell (VSMC) experiments both peptides showed unique anti-proliferative action, in which they inhibited a WST-8 incorporation in Ang II-stimulated VSMCs, and a non-selective AT-related receptors’ antagonist did not alter the effect. Moreover, elevated incorporation by Bay K8644 stimulation was also inhibited by small peptides, which suggested that they might be associated with Ca2+ influx control. To 70 Symposia make clear the possibility, further contractive experiments of small peptides in rat aorta were performed in combination with L-type Ca2+ channel blockers. Although nifedipine, a dihydropyridine (DHP) type Ca2+ channel blocker, did not affect the Trp-His-induced relaxation; Verapamil, a phenylalkyl amine (PAA) type Ca2+ channel blocker, reduced its relaxation power. It suggested that Trp-His as well as His-Arg-Trp would be partly involved in the regulation of intracellular Ca2+ concentration ([Ca2+]i) in VSMC via an L-type Ca2+ channel. In a direct fluorescence measurement of [Ca2+]i by Fura-2 they significantly reduced the increase in [Ca2+]i by Bay K 8644 stimulation to VSMC, suggesting their binding to the channel protein. On the other hand, it was noted that no reduction of [Ca2+]i by their corresponding amino acids was observed, provided useful information that any peptide skeleton must be essential for exerting vascular relaxation via [Ca2+]i suppression. A marked reduction of [Ca2+]i by both vasoactive peptides in Ang II stimulation-VSMC study compared with Bay K 8644 also revealed their alternative action toward [Ca2+]i regulation systems. Our interest on peptide functionality has been then moved to preventive effect on vascular diseases, because of their potent vascular relaxation peptides via inhibition of [Ca2+]i influx. Atherosclerosis is one of typical vascular diseases related to VSMC migration or proliferation. In our challenging animal study using apo E-deficient mice a 9-week-successive administration of Trp-His at a dose of 10 or 100 mg/kg/day resulted in a significant reduction of atherosclerotic lesion area on aortic tree, while no difference in growth parameters and lipid profiles was observed. The first finding that vasoactive di-peptide could ameliorate atherosclerosis without any change in lipid profile may provide us a new physiological potential of small peptides. Much interest has been focused on the elucidation of peptide functionalities. Recent studies by other researchers also demonstrated some new findings regarding the improvement of renal dysfunction via Ca2+-ATPase activation by Val-Tyr in renal proximal cells and hyperglycemia via post-transcriptional down-regulation of SGLT1 expression by Gln-Cys-Pro or Gln-Ser-Pro. Given these diverse functionalities of peptides, the intake of bioactive small peptides would be of great benefit for maintaining our homeostasis. 71 www.msk.or.kr S7-3 Anti-inflammatory Compounds from Medicinal Plants Jae-Ha Ryu College of Pharmacy, Sookmyung Women’s University, Seoul 140-742 Natural products have served as an important source of drugs since ancient times and about half of the useful drugs today are derived from natural products. Contributing to this world-wide attention towards formulations based on natural products are their low or absent toxicity, their complete biodegradability, their availability from renewable sources, and, in most cases, their low-cost if compared with those of compounds obtained by total chemical synthesis. Chemodiversity in nature, e.g. in plants, microorganisms and marine organisms, still offer a valuable source for novel lead discovery for the drug development. In fact, individual plant species may contain over one thousand chemical substances and only a minor fraction of 300,000 plant species has been studied for medical application. But rapid identification of the bioactive compounds of natural product mixtures remains critical factor to ensure that this tool of drug discovery can compete with recent developed techniques such as chemical compound libraries and high-throughput screening of combinatorial synthetic efforts. Recently, owing to the renewed attention to pharmaceuticals, agrochemicals and nutraceuticals obtained from natural sources, the study of bioactive secondary metabolites, traditionally carried out by chemists, has increasingly attracted the attention of pharmacologists, biologists, botanists, agronomists, etc., stimulating cooperative work. So, we need to reinforce the interdisciplinary approach to the study of bioactive natural products for the successful drug development. In order to find new ant-inflammatory compounds from medicinal plants, we have screened bioactive compounds by using the activated macrophage culture system. Activated macrophages produce proinflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1 and 6, and nitric oxide (NO). The overproduction of NO by the oxidation of L-arginine by inducible nitric oxide synthase (i-NOS) activates the cyclooxygenase-2 (COX-2) resulting in markedly increased release of pro-inflammatory prostaglandins (PGs). In the presence of superoxide anion (O2-) NO can be converted into peroxynitrite (ONOO-) that is a highly reactive molecule capable of oxidizing proteins, lipids, and DNA. We have purified compounds that inhibited the production of NO in LPS-activated macrophages by activity-guided purification, and indentified their structures by using spectroscopic methods. We identified sesquiterpenes, polyacetylenes, diarylheptanoids, butanolides, flavonoids, lignans as active compounds from Allium sativum, Alpinia officinarum, Angelica gigas, Artemisia iwayomogi, Atremisia princeps, Broussonetia kazinoki, Carpesium macrocephalum, Curcuma zedoaria, Machilus thunbergii, Magnolia fargesii, Magnolia obovata, Opuntia ficus, Panax ginseng, Perilla frutescens, Psoralea corylifolia, Saussurea lappa, Siegesbeckia 72 Symposia glabrescens, Torilis japonica, Tussilago farfara, Xanthium strumarium. They showed their activity through the suppression of i-NOS/COX-2 expression via the modulation of NF-κB activity and I-κB degradation and also through the scavenging of peroxynitrite. I will discuss the purification, structural determination, action mechanism of active principles from medicinal plants. Fig. 1. Chemical structures of compounds 1-4 (A) and HMBC correlations of 4 from Magnolia fargesii (B). The effects of 4 on I-κB degradation, nuclear translocation of p65 (C), production of PGE2 (D) and expression of i-NOS and COX-2 (C), (D) in LPS-activated macrophages. 73 www.msk.or.kr S7-4 Extraction of Bioactive Materials from Rice Hulls Seung-Cheol Lee Department of Food Science and Biotechnology, Kyungnam University Rice is the principal cereal in Asia, some countries in Africa, and Latin America. More than one million tons of rice hulls (RHs) have been produced annually in South Korea after processing of rice. However, RHs are wasted or destined to undervalued uses. Currently, agricultural and industrial residues are attractive sources of natural antioxidants. The extraction of antioxidant compounds from residue materials such as hulls, seed coats, peels, grape seeds, olive rape and cocoa byproduct has been reported. In general, seed coat plays an important role in protecting seeds from oxidative damage because seed coat possesses large quantity of endogenous antioxidant such as phenolic compounds. Far-infrared (FIR) rays are defined as electromagnetic waves having a wavelength of longer than 4 μm but shorter than microwave (λ>0.1 cm). FIR rays are biologically active and transfer heat to the center of materials evenly without degrading the constituent molecules of surface. FIR, however, may have capability to cleave covalent bonds and liberate antioxidants such as flavonoids, carotene, tannin, ascorbate, flavoprotein or polyphenols from repeating polymers. After irradiation of far-infrared (FIR) onto RHs, methanolic extract was prepared for the determination of antioxidant ability. After 30 min of FIR treatment, the radical scavenging activity (RSA) and total phenol contents (TPC) of RH extracts increased from 47.74% to 79.63% and from 0.12 mM to 0.19 mM, respectively, compared to control. The inhibition of lipid peroxidation in extracts was also increased from 41.07% to 47.96%. According to the GC-MS analysis, more phenolic compounds (p-coumaric acid, 3-vinyl-1-oxy benzene, p-hydroxy benzaldehyde, vanillin, p-hydroxy benzoic acid, and 4,7-dihydroxy vanillic acid) were detected in FIR-irradiated RH extract. These results indicated that FIR radiation onto RH could liberate and activate covalently bound phenolic compounds that have antioxidant activities. Subcritical water (SCW) is hot water under pressure sufficient to maintain water in the liquid state. Antioxidant characteristics of SCW extracts from RHs was also evaluated. The TPC in SCW extracts of RHs significantly increased with increasing temperature and treatment period. The TPC of RHs extract increased from 165.1 μM of extraction at 25°C for 10 min to 270.2 μM of 200°C for 60 min. Antioxidant activity was evaluated by determining RSA. The RSA of SCW extracts of RHs was also significantly increased with increasing temperature and treatment period. The RSA of RH extract increased from 8.40% of extraction at 25°C for 10 min to 79.33% of 200°C for 60 min. The effect of SCW extraction temperatures and treatment periods on TPC and RSA showed almost same trends. The cytotoxic and antitumor activity of methanolic extract of rice hulls (MERH) were also evaluated by the 74 Symposia MTT-dye reduction assay against human colon cancer cells, and the colonic aberrant crypt foci (ACF) assay in 1,2-dimethylhydrazine (DMH)-injected F344 male rats, respectively. MERH was found to be highly cytotoxic, with IC50 values of 0.5 μg/ml in vitro. Forty weeks of MERH supplementation (50 mg/kg body weight/day) reduced colonic pre-neoplastic ACF formation by 35% (p<0.01). An active compound, momilactone B, was isolated from MERH by silica gel chromatography, Sephadex LH-20 chromatography, and HPLC. The cytotoxic activity of momilactone B was evaluated by the MTT-dye reduction, lactate dehydrogenase (LDH) and colony-forming ability assays in human colon cancer HT-29 and SW620 cells. The results indicated that FIR irradiation or SCW extraction could be an excellent method for recovering antioxidant materials from RHs, and momilactone B from RHs might be a new candidate for chemotherapeutic agent against human colon cancer. References [1] Lee, S.C., Kim, J.H., Jeong, S.M., Kim, D.R., Ha, J.U., Nam, K.C., and Ahn, D.U. Journal of Agriculture and Food Chemistry, 51, 4400, 2003. [2] Kim, S.J., Park, E., Park, H.R., and Lee, S.C. Journal of Agriculture and Food Chemistry, 55, 1702, 2007. [3] Park, S.M. and Lee, S.C. Food Science and Biotechnology, 18, 1435, 2009. 75 www.msk.or.kr S8-1 Differential Roles of Yeast Glutamate Dehydrogenase Isozymes in Maintaining Resistance to Stress-Induced Apoptosis Pil Jae Maeng*, Kyung Jin Kim, and Yong Joo Lee Department of Microbiology and Molecular Biology, Chungnam National University, Daejeon 305-764 Glutamate is synthesized by glutamate dehydrogenases (GDHs). The yeast Saccharomyces cerevisiae has three isoforms of GDH encoded by three distinct genes, GDH1, GDH2, and GDH3. Gdh1 and Gdh3 catalyze the NADPH-dependent synthesis of glutamate from α-ketoglutarate, while Gdh2 catalyzes NAD+-dependent reconversion of glutamate to ammonia and 2-ketoglutarate. Here, we report differential roles of the two NADPH-dependent GDHs in stress-induced apoptosis. Cells with ∆gdh1 mutation exhibited significantly decreased survival rate when exposed to either heat or oxidative stress in pre-stationary phase culture, however, showed little difference compared to wild-type cells in stationary phase culture. On the other hand, ∆gdh3 cells showed dramatically increased sensitivity to heat and oxidative stress in stationary culture compared to wild-type cells, but moderately higher sensitivity in pre-stationary culture. The death of ∆gdh3 cells caused by oxidative stress was shown to be associated with typical apoptotic hallmarks, i.e., ROS accumulation, nuclear fragmentation, DNA breakage, and phosphatidylserine translocation [1, 2]. Upon long-term cultivation, ∆gdh3 cells showed increased potentials for both aging-induced apoptosis and adaptive regrowth. In addition, ∆gdh3 cells showed higher tendency to glutathione (GSH) depletion and subsequent ROS accumulation than the wild-type, which was rescued by exogenous GSH, glutamate, or glutathione disulfide (GSSG). It is thus suggested that GSH deficiency in ∆gdh3 cells is caused by an insufficient supply of glutamate necessary for biosynthesis of GSH rather than the depletion of reducing power required for reduction of GSSG to GSH. We also found that deletion of GDH2 suppresses the stress-induced apoptosis caused by the shortage of glutamate supply required for the biosynthesis of GSH. Despite the insignificance of GDH1 for the resistance to stressed-induced apoptosis in stationary phase cells, the level of GDH1 transcription remained consistently high throughout the 3-d culture period. However, the level of Gdh1 protein sharply decreased in stationary phase cells. On the other hand, transcription of GDH3 was low during pre-stationary phase, but significantly high during stationary phase. In addition, the level of Gdh3 protein changed similarly. This result suggests that the differential roles of GDH1and GDH3 in maintaining resistance to stress-induced apoptosis might be mediated by differential protein degradation, but not by their transcription. References [1] Lee, Y. J., K. L. Hoe, and P. J. Maeng. Mol. Biol. Cell 18, 3556 (2007). [2] Madeo, F., E. Frohlich, and K. U. Frohlich. J. Cell Biol. 139, 729 (1997). 76 Symposia S8-2 Cadmium Regulates Copper Homeostasis by Inhibiting Mac1 Activity, a Transcriptional Activator of Copper Regulon, in Saccharomyces cerevisiae Dong-Hyuk Heo, In-Joon Baek, and Cheol-Won Yun* School of Life Sciences and Biotechnology, Korea University, Seoul Physiological processes in virtually all organisms require metal ions as cofactors, and cellular depletion of particular metals can cause defects in numerous biological functions. On the other hand, excessive accumulation of certain metal ions induces the production of reactive oxygen species (ROS) and ultimately leads to cell death [1, 2]. Living organisms rely on delicate mechanisms to maintain intracellular metal homeostasis. Certain metal ions, such as cadmium, arsenate, lead, and mercury, are harmful to organisms. Thus, cells have unique mechanisms to protect themselves from such deleterious metal ions, such as exporting them from the cell [3]. Cadmium is among the better-known toxic metals that affect living organisms. Cadmium toxicity has been studied using various model systems, ranging from bacteria to humans. The sources of cadmium are numerous, and the diseases that are caused by cadmium are diverse and include conditions such as gastroenteritis [4], Itai-Itai disease, osteoporosis [5], renal dysfunction [6], and bone pain [7]. Cadmium directly damages the cell membrane and induces apoptosis [8], mitochondrial dysfunction [9], cancer [10, 11], and ultimately cell death. Interestingly, cadmium cannot directly form ROS, but it can induce their formation by inhibiting the scavenging activities of superoxide dismutase and catalase [11]. Furthermore, cadmium induces mutations both directly by damaging DNA and indirectly by inhibiting the activity of repair systems [12], although the mechanism of this inhibition is not completely understood. The mechanism of cadmium toxicity has recently been studied at the molecular level, and a link to sulfur metabolism was identified. Interestingly, sulfur compounds such as methionine, cysteine, glutathione, and their complexes are involved in the detoxification of cadmium [13, 14]. For example, cDNA microarray data show that cadmium up-regulates the expression levels of genes that are involved in sulfur metabolism in S. cerevisiae [14]. In addition, cadmium inhibits the SCFMet30 pathway, preventing this pathway from degrading Met4 [13]. Met4 is a primary transcription factor for the genes that are involved in sulfur metabolism. When cells are exposed to cadmium, Met4 is activated, and it up-regulates the biosynthesis of molecules that are involved in sulfur metabolism, such as glutathione. These molecules then scavenge the cadmium. These results suggest that sulfur metabolism is a key detoxification pathway in S. cerevisiae. Another aspect of cadmium toxicity comes from the metal ion’s ability to arrest the cell cycle, especially through the Rad53 pathway [15]. Cadmium activates the Mec1/Rad53 pathway and ribonucleotide reductase, 77 www.msk.or.kr increases the copy number of mtDNA, and ultimately induces cell death. Cadmium competes with physiologically important metal ions in the uptake pathway, and this competition has lethal effects on organisms. This process is one of the principal mechanisms of cadmium toxicity. Furthermore, the P-type ATPase Pca1 pumps Cd2+ out of the cell and into the extracellular environment, and this protein thereby contributes to Cd2+ tolerance [16]. In S. cerevisiae, the intracellular copper concentration is strictly regulated because copper is an essential metal for enzyme activity [17], defense against oxidative stress [18], and the electron transport system [19, 20]. Copper is taken up from the environment by two copper transporters, Ctr1 and Ctr3, which are localized to the plasma membrane [21, 22] and regulated by copper sensing transcription factor, Mac1 [21]. Ctr1 and Ctr3 confer a high-affinity copper binding activity. Another copper transporter, Ctr2, is localized to the vacuole membrane and confers a low-affinity copper binding activity [23]. Interestingly, CTR3 is not transcribed in laboratory S. cerevisiae strains, due to the insertion of transposable elements in the promoter region [23]. Thus, Ctr1 is the only high-affinity copper transporter protein in S. cerevisiae. The copper taken up by transporters is delivered to target organelles by intracellular delivery systems, such as Atx1, Ccs, and Cox17 [17-20]. These proteins deliver copper to the Golgi, Sod1, and the mitochondria, respectively. The Mac1 protein [21] is a transcriptional activator that regulates the expression of the copper metabolism genes mentioned above. High concentrations of copper bind to Mac1 and then inhibit the DNA-binding activity of Mac1 [24], thereby inhibiting transcription of its target genes. Multiple cysteine-rich domains are found in the C-terminus of the Mac1 protein [25], and these domains may confer copper-binding activity. Interestingly, copper metabolism is regulated in a concerted manner with iron metabolism[26]. For example, copper can affect iron metabolism through the regulation of the ferroxidase Fet3 protein. The Fet3/Ftr1 complex comprises the reductive iron uptake system of S. cerevisiae, in which the ferrous form of iron is oxidized by surface ferroxidase Fet3 and then taken up by iron permease Ftr1. The enzymatic activity of Fet3/Ftr1 depends largely upon cellular copper utilization. In this paper, we have identified a novel mechanism of cadmium toxicity in which cadmium inhibits Mac1 activity and, therefore, reduces CTR1 transcription. In addition, cadmium interferes with iron use by inhibiting Fet3 and this leads to changes in iron and copper homeostasis in S. cerevisiae. References [1] Halliwell, B. and Gutteridge, J. M. (1984) Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J. 219, 1-14. [2] Lefebvre, V. and Buc-Calderon, P. (1995) Desferal prevents against cell lysis induced by hydrogen peroxide to hypoxic hepatocytes: a role for free iron in hypoxia-mediated cellular injury. Chem Biol Interact. 94, 37-48. [3] Kim, D. Y., Bovet, L., Maeshima, M., Martinoia, E. and Lee, Y. (2007) The ABC transporter AtPDR8 is a cadmium extrusion pump conferring heavy metal resistance. Plant J. 50, 207-218. [4] Andersen, O., Nielsen, J. B. and Svendsen, P. (1988) Oral cadmium chloride intoxication in mice: effects of dose on tissue damage, intestinal absorption and relative organ distribution. Toxicology. 48, 225-236. 78 Symposia [5] Bhattacharyya, M. H., Whelton, B. D., Stern, P. H. and Peterson, D. P. (1988) Cadmium accelerates bone loss in ovariectomized mice and fetal rat limb bones in culture. Proc Natl Acad Sci U S A. 85, 8761-8765. [6] Itokawa, Y., Nishino, K., Takashima, M., Nakata, T., Kaito, H., Okamoto, E., Daijo, K. and Kawamura, J. (1978) Renal and skeletal lesions in experimental cadmium poisoning of rats. Histology and renal function. Environ Res. 15, 206-217. [7] Wilson, A. K. and Bhattacharyya, M. H. (1997) Effects of cadmium on bone: an in vivo model for the early response. Toxicol Appl Pharmacol. 145, 68-73. [8] Coonse, K. G., Coonts, A. J., Morrison, E. V. and Heggland, S. J. (2007) Cadmium induces apoptosis in the human osteoblast-like cell line Saos-2. J Toxicol Environ Health A. 70, 575-581. [9] Muller, L. (1986) Consequences of cadmium toxicity in rat hepatocytes: mitochondrial dysfunction and lipid peroxidation. Toxicology. 40, 285-295. [10] Gunn, S. A., Gould, T. C. and Anderson, W. A. (1963) Cadmium-Induced Interstitial Cell Tumors in Rats and Mice and Their Prevention by Zinc. J Natl Cancer Inst. 31, 745-759. [11] Huang, Y. H., Shih, C. M., Huang, C. J., Lin, C. M., Chou, C. M., Tsai, M. L., Liu, T. P., Chiu, J. F. and Chen, C. T. (2006) Effects of cadmium on structure and enzymatic activity of Cu,Zn-SOD and oxidative status in neural cells. J Cell Biochem. 98, 577-589. [12] Potts, R. J., Bespalov, I. A., Wallace, S. S., Melamede, R. J. and Hart, B. A. (2001) Inhibition of oxidative DNA repair in cadmium-adapted alveolar epithelial cells and the potential involvement of metallothionein. Toxicology. 161, 25-38. [13] Barbey, R., Baudouin-Cornu, P., Lee, T. A., Rouillon, A., Zarzov, P., Tyers, M. and Thomas, D. (2005) Inducible dissociation of SCF(Met30) ubiquitin ligase mediates a rapid transcriptional response to cadmium. Embo J. 24, 521-532. [14] Momose, Y. and Iwahashi, H. (2001) Bioassay of cadmium using a DNA microarray: genome-wide expression patterns of Saccharomyces cerevisiae response to cadmium. Environ Toxicol Chem. 20, 2353-2360. [15] Yen, J. L., Su, N. Y. and Kaiser, P. (2005) The yeast ubiquitin ligase SCFMet30 regulates heavy metal response. Mol Biol Cell. 16, 1872-1882. [16] Adle, D. J., Sinani, D., Kim, H. and Lee, J. (2007) A cadmium-transporting P1B-type ATPase in yeast Saccharomyces cerevisiae. J Biol Chem. 282, 947-955. [17] Lin, S. J. and Culotta, V. C. (1995) The ATX1 gene of Saccharomyces cerevisiae encodes a small metal homeostasis factor that protects cells against reactive oxygen toxicity. Proc Natl Acad Sci U S A. 92, 3784-3788. [18] Culotta, V. C., Klomp, L. W., Strain, J., Casareno, R. L., Krems, B. and Gitlin, J. D. (1997) The copper chaperone for superoxide dismutase. J Biol Chem. 272, 23469-23472. [19] Beers, J., Glerum, D. M. and Tzagoloff, A. (1997) Purification, characterization, and localization of yeast Cox17p, a mitochondrial copper shuttle. J Biol Chem. 272, 33191-33196. [20] Glerum, D. M., Shtanko, A. and Tzagoloff, A. (1996) Characterization of COX17, a yeast gene involved in 79 www.msk.or.kr copper metabolism and assembly of cytochrome oxidase. J Biol Chem. 271, 14504-14509. [21] Dancis, A., Haile, D., Yuan, D. S. and Klausner, R. D. (1994) The Saccharomyces cerevisiae copper transport protein (Ctr1p). Biochemical characterization, regulation by copper, and physiologic role in copper uptake. J Biol Chem. 269, 25660-25667. [22] Knight, S. A., Labbe, S., Kwon, L. F., Kosman, D. J. and Thiele, D. J. (1996) A widespread transposable element masks expression of a yeast copper transport gene. Genes Dev. 10, 1917-1929. [23] Rees, E. M., Lee, J. and Thiele, D. J. (2004) Mobilization of intracellular copper stores by the ctr2 vacuolar copper transporter. J Biol Chem. 279, 54221-54229. [24] Keller, G., Bird, A. and Winge, D. R. (2005) Independent metalloregulation of Ace1 and Mac1 in Saccharomyces cerevisiae. Eukaryot Cell. 4, 1863-1871. [25] Keller, G., Gross, C., Kelleher, M. and Winge, D. R. (2000) Functional independence of the two cysteine-rich activation domains in the yeast Mac1 transcription factor. J Biol Chem. 275, 29193-29199. [26] Dancis, A., Yuan, D. S., Haile, D., Askwith, C., Eide, D., Moehle, C., Kaplan, J. and Klausner, R. D. (1994) Molecular characterization of a copper transport protein in S. cerevisiae: an unexpected role for copper in iron transport. Cell. 76, 393-402. 80 Symposia S8-3 Membrane Potential Changes by Proton Pumping Microbial Rhodopsins Kwang-Hwan Jung Department of Life Science and Institute for Biological Interface, Sogang University, Seoul 121-742 ([email protected]) Microbial rhodopsins are retinal-binding proteins which function as ion pumps and photosensory receptors. A variety of rhodopsins was discovered in many habitats since proteorhodopsin, which functions as a light-driven proton pump, was found in uncultured marine bacteria of the SAR86 group. Recently, it was revealed that PR-like proteins exist in genomes of flavobacteria. It has been characterized the photochemical features of rhodopsins which were isolated from Korea East Sea and the surface of North & South pole. All of them have the proton acceptor (D85 in BR) and donor (D96 in BR) which mediate proton translocation from intracellular region to extracellular region when it receives light energy, and their actual proton pumping activity in vitro has been demonstrated. Additionally, flavorhodopsin from IMCC 1997 cells is very unstable compared to proteorhodopsin, which may be explained by the primary amino acid sequence differences. In order to measure the stability of FR, we used the method of heat endurance at 70°C, and some FR mutants showed that their endurance time against heat was increased. We showed that the Helix E is important for stability of flavorhodopsin. We also applied several rhodopsins to various environmental conditions such as E. coli membranes state, membrane gel state, DDM solubilized state, OG solubilized state, and DOPC reconstituted state. According to the light-induced difference spectroscopy, rhodopsins solubilized in 0.02% DDM clearly showed photointermediates like M, and O states which respond to the different wavelengths, respectively. The laser-induced difference spectra ATP synthase showed the fast formation and decay rate of photointermediates in the solubilized samples which contain DDM. For the application, we developed a bio-ATP-synthesis system using ATP synthase of E. coli PR as a biocatalyst and a microbial rhodopsin as light energy converter. GR was overexpressed in E. coli and made inverted membrane vesicle so that it could contain both of GR and ATP synthase. Gloeobacter rhodopsin (GR) produce a proton gradient which can be used in 81 www.msk.or.kr driving force of synthesizing ATP by ATP synthase in this vesicle. (This work was supported by grant from the 21C Frontier Microbial Genomics and Application Program) References [1] Lee KA, Jung KH. 2010. ATP regeneration system using E. coli ATP synthase and Gloeobacter rhodopsin and its stability. J. Nanosci. Nanotechno. Accepted. [2] Miranda MRM, Choi AR, Shi L, Bezerra AG, Jung KH, Brown LS. 2009. Feb 18. The Photocycle and Proton Translocation Pathway in a Cyanobacterial Ion-Pumping Rhodopsin. Biophys J. 96(4):1471-1481. [3] Jung JY, Choi AR, Lee YK, Lee HK, Jung KH. 2008. May 28. Spectroscopic and photochemical analysis of proteorhodopsin variants from the surface of the Arctic Ocean. FEBS Letters-Biophysics 582(12):1679-1684. [4] Brown LS, Jung KH. 2006. June. Bacteriorhodopsin-like proteins of eubacteria and fungi: extent of conservation of the haloarchaeal proton-pumping mechanism. Photochem. Photobiol. Sci. 5(6):538-546. 82 Symposia S8-4 Different Role of the DosS and DosT Histidine Kinases in Mycobacterial Response to Hypoxic Conditions Jin-Mok Lee, Kwang-Jin Park, Min-Ju Kim, and Jeong-Il Oh* Department of Microbiology, Pusan National University, Busan 609-735 The DosS (DevS) and DosT histidine kinases in mycobacteria are involved in their hypoxic adaptation and entry into the latent state. Although exhibiting high sequence similarities in their primary structures, DosS and DosT differ in redox-sensing mechanism: DosS senses the environmental redox state, while DosT recognizes the presence of O2 [1, 2]. The DevS histidine kinase of Mycobacterium smegmatis contains a tandem of the GAF domains (GAF-A and GAF-B) in its N-terminal sensory domain. The DevS protein is a hemoprotein, and a b-type heme is coordinated by its first GAF domain (GAF-A) with His-150 providing a proximal axial ligand to the heme [3]. The heme iron of DevS was in the ferrous (Fe2+) state when purified and was resistant to autooxidation from ferrous to ferric (Fe3+) state in the presence of O2. The binding of O2 to the deoxy-ferrous heme led to a decrease in DevS autokinase activity, whereas NO-binding did not [3]. Phylogenetic analysis of the GAF-A domains of DevS homologues revealed that the primary structure of GAF-A domain of M. smegmatis DevS is closely related to that of DosT in M. tuberculosis (MTB) rather than DosS in MTB. In good agreement with the result of the phylogenetic analysis, DosT of MTB complemented the DevS-phenotype of M. smegmatis DevS mutant strain as judged by the hypoxic induction of hspX, a gene belonging to the Dev regulon, whereas DosS did not. We also found through this complement test that DosT and DevS play a crucial role in the hypoxic induction of the Dev regulon during the transition phase from aerobic to hypoxic conditions in contrast to DosS. Multiple alignment of the amino acid sequences of GAF-A domains of DevS homologues showed that seven amino acids are conserved differently between DosS and DosT subclades. The role of these amino acids in different redox-sensing mechanism between DosS and DosT was investigated. It was demonstrated that five amino acids (E87, D90, H97, L118, and T169 in DosS of MTB) might be responsible for the DosS- and DosT-specific redox sensing mechanism. The three-dimensional structure of the GAF-A domain of DosS revealed that it has a GAF-folding structure with a b-type heme which is similar to the structure of the known PAS domain [4]. References [1] Kumar A, Toeldo JC, Patel RP, Lancaster JR, and Steyn JC. Proc. Nat. Acad. Sci. USA 104, 11568, 2007. 83 www.msk.or.kr [2] Sousa EHS, Tuckerman JR, Gonzalez G, and Gilles-Gonzalez MA. Protein Sci. 16, 1, 2007. [3] Lee JM, Cho HY, Cho HJ, Ko IJ, Park SW, Baik HS, Oh JH, Eom CY, Kim YM, and Oh JI. J. Bacteriol. 190, 6795, 2008. [4] Cho HY, Cho HJ, Kim YM, Oh JI, and Kang BS. J. Biol. Chem. 284, 13057, 2009. 84 Symposia S9-1 Synthetic 5′-Untraslated Regions for Fine-Tunable and Predictable Gene Expression in Escherichia coli Sang Woo Seo1, Jae-Seong Yang2, Inhae Kim3, Sanguk Kim2,3, and Gyoo Yeol Jung1,2* 1 Department of Chemical Engineering, 2School of Interdisciplinary Bioscience and Bioengineering, and Division of Molecular and Life Science, Pohang University of Science and Technology, Gyeongbuk 790-784 3 A large number of natural and unnatural products from microorganisms such as biologically active compounds and commodity chemicals have attracted huge attention in the various industries including pharmaceutical and chemical industries. However, functional improvement of microorganisms meeting with commercial need is limited by the biological constraints developed during natural evolutionary process. “Metabolic Engineering” aims the purposeful redesign of the biological systems and requires the accurate information of the cellular metabolic networks and proper tools for the reconstruction of the biological systems. Numerous regulatory elements such as promoter libraries and RBS calculator can be applied for the modulation of gene expression. However, without carefully considering the structural information of 5′-unstranslated region (5′-UTR) sequence, it is insufficient to precisely design and modulate the gene expression level. To address this issue, in this study, we randomized 5′-UTRs maintaining ribosome binding affinity using superfolder GFP as a reporting system in Escherichia coli. A mathematical model was constructed by mapping between the secondary structures of 5′-UTRs and the expression level of superfolder GFP. Examples using the other genetic contexts will show the potentials of this model to predict the precise expression level based on the structural information and consequently will provide a valuable tool for “Pathway Synthesis” for the production of biofuels and commodity chemicals including butanol, hydrogen, and amino acids. 85 www.msk.or.kr S9-2 Rewriting Biology with Gene Synthesis Duhee Bang Department of Chemistry, Yonsei University, Seoul 120-749 Advances in the chemical synthesis of DNA have facilitated our ability to synthesize genes at unprecedented error rates. These synthetic capabilities along with emerging genomic technologies are now making it possible to tackle the challenge of constructing new biological systems to address major problems in biology, medicine and energy. I will present efforts to scale up DNA synthesis to millions base pairs at an affordable cost [1-3]. A resulting revolution in the DNA synthesis will enable us to test our information-driven hypothesis by reconstructing DNA sequences in a highly controlled manner. The development of an ultra-low cost synthetic genomics platform will generate unprecedented opportunities in an emerging field of Synthetic Biology. References [1] D Bang, and GM Church, Nature Methods, 2008, 5, 37-39. [2] HB Kim, HJ Han, and D. Bang, submitted. [3] J Tian, H Gong, N Sheng, X Zhou, E Gulari, X Gao, and G Church. Nature, 2004, 432, 1050-1054. 86 Symposia S9-3 From Oligos to Organisms Mikkel Algire Synthetic Biology and Bioenergy Group, J. Craig Venter Institute The Synthetic Biology group has been trying to develop a minimal cell both to understand the fundamentals of biology and to build a new cell and organism with optimized functions. In order to construct a synthetic cell we need to synthesize and assemble a whole genome and activate or ‘boot up’ this synthetic genome. As a first step toward the construction of minimal synthetic cell and to demonstrate that a bacterial genome could be activated, we completely replaced the genome of a bacterial cell with one from another species by transplanting a whole genome as naked DNA. Intact genomic DNA from Mycoplasma mycoides subsp. capri was transplanted into Mycoplasma capricolum cells by polyethylene glycol-mediated transformation. These cells that result from genome transplantation are identical to the M. mycoides donor strain. We have also developed whole genome assembly techniques to allow us to have nucleotide level control over the synthetic genome. Using these tools assembled and cloned the synthetic Mycoplasma genitalium JCVI-1.0 genome in the yeast Saccharomyces cerevisiae by recombination to produce a 592-kb circle. We further extended this approach by assembling the synthetic genome from 25 overlapping fragments in a single step. The use of yeast recombination greatly simplifies the assembly of large DNA molecules from both synthetic and natural fragments. In order to produce a synthetic cell, the genome assembly and genome transplant steps must be combined. Following the chemical synthesis, assembly, and cloning of a bacterial genome in yeast the genome must be transferred from yeast to a receptive cytoplasm. To demonstrate the ability to transplant a cloned bacterial genome from yeast we cloned a M. mycoides genome as a yeast centromeric plasmid and then transplanted it into M. capricolum to produce a viable M. mycoides cell. While in yeast, the genome was altered by using yeast genetic systems and then transplanted to produce a new strain of M. mycoides. 87 www.msk.or.kr S9-4 Artificial Assembly of a Minimal Cell Giovanni Murtas Centro Enrico Fermi, Piazza del Viminale 1, 00184 - Rome - Italy Synthetic Biology approaches can assemble and/or reconstruct cell parts in synthetic compartments. A minimal cell as a model for early living cells can be artificially constructed in the laboratory resuming the main properties of a basic cell living system, a synthetic cell compartment or liposome to host a minimal metabolism based on protein synthesis, and a shell and core reproduction mechanism. Starting from the minimal cell assembly approach it is becoming realistic to construct artificial cells, and deliver cell-like bioreactors to synthesize pure proteins/enzymes or isolate single pathways. These artificial cell-like systems could perform different tasks in antimicrobial drug development, drug delivery and diagnostic applications With a bottom up approach we are aiming to construct a minimal cell using a minimal set of extant enzymes and/or genes to create basic bio-mechanisms in simple lipid compartments know as liposomes, to develop a cell-system alive that means implementing in a liposome compartment a basic metabolism, based on protein synthesis, and a self-replication mechanism to insure cell reproduction.1,2 These should be the essential bio-mechanisms for a cell system to be defined alive and to qualify for the lowest degree of complexity compatible with cellular life. A suitable cell-compartment to build a cell model in the lab is represented by the liposome, a closed spherical bilayer, considered the most likely precursor of early living cells. In order to establish a basic mechanism of protein synthesis for a minimal metabolism, a set of 36 pure enzymes with purified ribosomes, energy and nutrients has been implemented in the cell compartment known as the PURESYSTEM (PS).3 A second and fundamental milestone to achieve in this model is represented by the property, present in any cell or living system, known as the ability to self-reproduce itself. This is to be distinguished into cell-compartment or vesicle self-reproduction and core or genome reproduction. On this purpose we can foresee a system where enzymes for lipid membrane synthesis are introduced within liposomes and drive the catalysis required for the boundary layer reproduction. A simple biochemical mechanism can be proposed and this is to introduce into vesicles a Fatty Acid Synthetase (FAS) enzyme for fatty acid synthesis. The FAS enzyme with fatty acid catalysis could induce vesicle growth and shell self-reproduction by fatty acid incorporation into the lipid bilayer. In particular the FAS-B type I from Brevibacterium ammoniagenes has been already characterised and expressed in the heterologous system E. coli.4 FAS-B catalyze mainly the synthesis of palmitate. Preliminary experiments have already shown that the FAS-B enzyme, as pure enzyme, can control endogenous lipid synthesis within liposomes proving lipid pattern change of membrane vesicles due to fatty acids synthesis 88 Symposia and incorporation5. For a complete and autonomous shell self-reproduction process the genetic components must be introduced within the model system, therefore the FAS-B gene together with the PS should be encapsulated into vesicles. This is a system that would sustain the FAS-B expression and Fatty acid catalysis allowing a shell selfreproduction, from mother to daughter vesicles, for a few generations although the system would be exhausted soon after. The main problem is connected with the limited amount of any PS enzyme available within each vesicle that would diminish dramatically in a few generations. To overcome the limiting condition of this protocell a set of genes encoding the PS components such as all the aminoacyl-tRNA synthetases and translation factors, will be integrated into vesicles. In addition the genes for the ribosomal proteins and rRNA required to assemble minimal ribosome will be synthesised using the T7 RNA polymerase and the PS. The tRNA genes will be transcribed by a T7 RNA polymerase to complete the genetic composition of the PS. Again, although the PS genome insertion into vesicle will help to sustain the vesicle reproduction process, this will not stop the limitation caused from death by dilution of all the elements included. The real self reproduction of biological cells is based on the formation of identical copies, containing each time the full apparatus. Therefore conditions are required under which the ribosomal system and all enzymes assisting transcription-translation for protein synthesis self-replicate. This is in principle possible by introducing some of the enzymes/genes responsible for DNA replication such as the DNA polymerase III, a DNA primase, helicase, ligase and gyrase, plus a gene for single-stranded DNA binding protein. At this stage, to keep the cell model within a frame of low complexity and remain in the realm of minimal living, nutrients and energy such as molecules of ATP, amino acids, nucleotides and other low molecular weight components will be provided and not synthesized autonomously by the system. Finally the cell model system should be tested in the laboratory to prove that it is able to evolve in a changing environment. 1,2,6 Synthetic Biology is now offering new technologies and approaches for artificial cell assembly. Artificial cells are now prospects for biotechnical and biomedical applications7. A recent work on a cell-like bioreactor has provided interesting suggestions to build new bioreactors for protein/enzyme synthesis within lipid vesicles8. The artificial cell assembly exercise may shed light on the origin of living cells9 but at the same time can provide the basic tools and lay the foundation for a new generation of artificial cell bioreactor systems for biotechnology. A new cell-like structure can be developed using a simple lipid vesicle compartment, as described for the EGFP synthesis in liposome10, with the same or similar channel pore exchange system as introduced by Noireaux et al., but replacing the E. coli extract for protein synthesis with the pure set of enzymes (PS) to control protein synthesis. More in details the PS system together with the α-hemolysin gene and any gene of interest can be easily entrapped into lipid compartments or liposomes with the option to add post-translational modification components or membrane proteins to specialise this vesicle bioreactors for biotechnological applications. These ready formed liposomes can be fed in water solution with a mix of nutrients and energy to sustain the internal reactions for a few days. This would allow a long lasting, high yield, synthesis and catalysis with an high degree of pure products and in the absence of inhibitors. This system would be suitable for testing artificial genomes, any single and/or 89 www.msk.or.kr artificial metabolic pathway or new pharmaceutical molecule isolated from the whole and complex metabolic contest. The same artificial cell-like structure could be assemble to combat chronic infection known as biofilms by synthesizing and delivering bactericides in situ.11 A similar technology could be promising in medical diagnostic where a liposome could entrap an artificial metabolism and convert relevant molecular compounds associated or released from any pathology into detectable fluorescent signals. In conclusion spin-off projects are possible from the artificial minimal cell assembly approach where a pure and minimal metabolism is encapsulated within liposomes this would be a cell-like device without capability of self- replicate, and therefore a non bio-hazardous system. These artificial cell-like structures could be engineered for specific applications to allow testing new artificial metabolisms, most promising in pharmacology, medical diagnostic and drug delivery. References [1] P.L. Luisi, F. Ferri, P. Stano, Approaches to semi-synthetic minimal cells: a review, Naturwissenschaften, 2006, 93, 1-13, Review. [2] G. Murtas, Construction of a semi-synthetic minimal cell: a model for early living cells, Orig Life Evol. Biosph., 2007, 37, 419-22. [3] Y. Shimizu, A. Inoue, Y. Tomari, T. Suzuki, T. Yokogawa, K. Nishikawa, T. Ueda, Cell-freetranslation reconstituted with purified components. Nat. Biotechnol., 2001, 19, 751-5. [4] H.P. Stuible, G. Meurer, E. Schweizer, Heterologous expression and biochemical characterization of two functionally different type I fatty acid synthases from Brevibacterium ammoniagenes, Eur. J. Biochem. 1997, 247, 268-73. [5] G. Murtas, Internal lipid synthesis and vesicle growth as a step toward self-reproduction of the minimal cell. Systems and Synthetic Biology (DOI: 10.1007/s11693-009-90481) [6] P.L. Luisi, in The emergence of life, 2006, eds. Cambridge University Press [7] A. Pohorille, D. Deamer, Artificial cells: prospects for biotechnology, Trends Biotech, 2002, 20, 123-8. [8] V. Noireaux, A. Libchaber, A vesicle bioreactor as a step towards an artificial cell assembly, Proc. Natl. Acad. Sci U.S.A., 2004, 101, 17669-176747 [9] J.W. Szostak, D.P. Bartel & P. L Luisi, Synthesizing life, Nature, 409, 387-390 [10] G. Murtas, Y. Kuruma, P. Bianchini, A. Diaspro, P.L. Luisi, Protein Synthesis in liposomes with a minimal set of enzymes. Biochem. Biophys. Res. Commun. 2007, 363(1), 12-7 [11] M.N. Jones. Use of liposomes to deliver bactericides to bacterial biofilms, Methods Enzymol., 2005, 391, 211-28. 90 Symposia S9-5 Toward the Bacterial Minimal Chromosome Young-Chang Kim1,2,3*, Mun-Sik Shin 1, Kyung-Bae Min2, Woo-Yeong Jang2, Ji-Eun Yu2, Hye-Jin Bae 2, Joo-Han Gwak2, and Soon-Gee Cheong3 1 Department of Synthetic Biology, 2Department of Microbiology, 3Biotechnology Research Institute, Chungbuk National University, Cheongju, 361-763 The minimal genome is the smallest possible group of genes that would be sufficient to sustain a cellular life form under the most favorable conditions imaginable [1]. There are two ways to synthesize a minimal genome, which are top-down and bottom-up approaches. We are interested in the bottom-up approach, in which the major hurdle is how to design the minimal genome. We don’t know the principle of the organization of the genome. Moreover to verify the design experimentally, we should have to deal with some 200 or more essential genes. So, we use the divide-and-conquer strategy to solve the problems in designing the minimal genome [2]. We made E. coli having two chromosomes. One is a normal chromosome and the other is a model minimal chromosome, in which the replication and segregation processes and their regulation are occurred independently. Studies on the minimal chromosome will provide us useful information about designing the minimal genome. References [1] Wegrzyn, G. The minimal genome paradox. J Appl Genet. (42), 385-392, 2001. [2] Pal, D., Venkova-Canova, T., Srivastava, P., and Chattoraj, D. K. Multipartite regulation of rctb, the replication initiator gene of vibrio cholerae chromosome ii. J Bacteriol. (187), 7167-7175, 2005. 91 www.msk.or.kr S10-1 Novel Enzymes and Metabolic Pathways in Hyperthermophilic Archaea Haruyuki Atomi1*, Takaaki Sato1, Yuusuke Yokooji1, Ryo Takemasa1, Tamotsu Kanai1, and Tadayuki Imanaka2 1 Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan 2 Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu 525-8577, Japan E-mail: [email protected] Thermococcus kodakaraensis is a hyperthermophilic archaeon isolated from Kodakara Island, Kagoshima, Japan. The organism is an obligate anaerobe and heterotroph, and displays an optimal growth temperature of 85°C. We have previously determined the entire genome sequence of T. kodakaraensis, and have estimated the presence of 2,306 genes. Assignment of gene function based on primary sequence analysis is possible for approximately half of the genes, and this enables us to predict the presence or absence of particular metabolic pathways in this archaeon. Intriguingly, in many cases, although biochemical data suggest otherwise, there are pathways that are incomplete judging from the genome data. One of the main objectives of our research is to identify the enzymes or pathways that functionally replace these missing enzymes. Classical biochemical methods, combined with a comparative genomics approach, have proven to be effective [1-3]. Once identified, the function of the protein/gene in vivo is confirmed genetically using the gene disruption system developed for T. kodakaraensis. We will introduce several examples of novel metabolic enzymes and pathways discovered in T. kodakaraensis, focusing on our results concerning pentose metabolism and the coenzyme A biosynthesis pathway. We will also describe recent improvements in the gene manipulation system developed in T. kodakaraensis. The system now allows, in addition to multiple gene disruption, gene insertion (reporter genes/heterologous protein production), promoter exchange (over-expression), tag insertion (purification), and signal peptide insertion (protein secretion). Using these gene manipulation techniques along with the complete genome sequence data of this organism, we are now capable of utilizing T. kodakaraensis as host cells for protein production and cell engineering. References [1] Targeted gene disruption as a tool for establishing gene function in hyperthermophilic archaea. H. Atomi and T. Imanaka. Ch. 13, pp. 213-223, In Thermophiles, CRC Press, London, 2008. [2] Archaeal type III RuBisCOs function in a pathway for AMP metabolism. T. Sato, H. Atomi and T. Imanaka. Science 315, 1003-1006, 2007. [3] Pantoate kinase and phosphopantothenate synthetase, two novel enzymes necessary for CoA biosynthesis in the Archaea. Y. Yokooji, H. Tomita, H. Atomi and T. Imanaka. J. Biol. Chem. 284, 28137-28145, 2009. 92 Symposia S10-2 Global Proteome Analysis of Sulfur-Reducing Hyperthermophilic Archaeon Thermococcus onnurineus NA1 Jong-Soon Choi*, Seung Il Kim, and Young-Ho Chung Division of Life Science, Korea Basic Science Institute, Daejeon 305-333 Thermococcus onnurineus NA1 is a representative of sulfur-reducing hyperthermophilic archaeon, Thermococcus sp. T. onnurineus NA1 was isolated from a deep-sea hydrothermal vent area. The strain requires elemental sulfur as a terminal electron and organic compounds such as peptides, amino acids and sugar as carbon source. This strain shows the optimum growth at 80°C and pH 8.5. Recently, the genome sequence of T. onnurineus NA1 was completed, in which its genome retains the metabolic pathway not only for organotrophic growth but also for carboxydotrophic growth [1]. In the present study, we attempted (i) to analyze global proteome of T. onnurineus NA1 at organotrophic and carboxydotrophic culture condition and (ii) to identify hyperthermostable proteins by the enrichment of in vitro heat treatment. Furthermore, (iii) the recombinant target thermostable proteins were characterized for the biophysical properties such as enzyme activity, substrate specificity, and cation requirement for growth. For the proteomic profiling, T. onnurineus NA1 cells grown in each culture conditions (yeast-peptone-sulfur medium for organotrophic growth; carbon monoxide as a sole carbon for carboxydotrophic growth) were harvested and lysed by boiling. The soluble proteins were prepared by centrifugation and separated on 12% SDS-PAGE. The gel was fractionated according to molecular size and the sliced gel bands were subjected to in-gel trypsin digestion. The resulting peptides were subjected to LC-MSMS analysis with Mascot v2.2 database for protein identification and relative quantification. For studying thermostable proteins, organotrophic growth medium-grown T. onnurineus NA1 cells were harvested and cytosolic proteins were extracted by the previous method [2]. The cytosolic fraction containing 1 mg protein was exposed to heat treatment for 10 min to 24 h at 60°C, 80°C, and 100°C. Denatured and aggregated proteins were removed by centrifugation and the resultant soluble fraction was performed by gel-based and shotgun proteomic analyses. In the proteomic profiling study, we identified and quantified the proteomes of T. onnurineus NA1 cultured at these two different cultures by SDS-PAGE/FT-LC-MS analysis; 1,268 and 1,572 proteins were identified from YPS- and CO-culture medium, respectively. A total of 1,612 proteins were identified from T. onnurineus NA1, in which the identification rate corresponds to ~82% of genome-the top score among the archaeon proteome data for our best knowledge. By the analysis of functional categories of identified proteome, the relative percentage of proteins belonging to energy production in the group of metabolism was 2-fold increased in CO-culture condition compared to that in YPS-culture one. In particular, hydrogenase gene clusters responsible 93 www.msk.or.kr for biohydrogen production were prevalently increased in the carboxydotrophic growth. The altered metabolism is probably due to the feasible proteomes of T. onnurineus NA1 for high biohydrogen production. In the proteomic analysis of heat-stable proteins, 13 and 149 proteins were identified as intact ones after heat treatment at 100°C for 2 h by 2DE/MALDI and SDS-PAGE/LC-MS analysis, respectively. Representative thermostable ezymes like deblocking aminopeptidase and fructose 1,6-bisphosphatase were over-expressed in E. coli system. Thermostability of these proteins was confirmed by enzyme activities and in silico TargetStar analyses [3]. In conclusion, the global proteome analysis suggests that T. onnurineus NA1 can give a possibility of the industrial application for the production of biohydrogen and thermostable enzymes. References [1] Lee HS, Kang SG, Bae SS, Lim JK, Cho Y, Kim YJ, Jeon JH, Cha SS, Kwon KK, Kim HT, Park CJ, Lee HW, Kim SI, Chun J, Colwell RR, Kim SJ, Lee JH, J Bacteriol, 190, 7491, 2008. [2] Kwon SO, Kang SG, Park SH, Kim YH, Choi JS, Kim SI, Extremophiles 13, 379, 2009. [3] Kim HJ, Moon EJ, Moon S, Jung HJ, Yang YL, Park YH, Heo M, Cheon M, Chang I, Han DS, Int J Modern Physics C 18, 1513, 2007. 94 Symposia S10-3 Transcriptome Analysis on a Heat Shock Regulon in the Hyperthermophilic Archaeon, Thermococcus kodakaraensis Tamotsu Kanai1*, Shogo Takedomi1, Tomoyuki Kamashita1, Masahiro Nakanosho1, Shinsuke Fujiwara2, Haruyuki Atomi1, and Tadayuki Imanaka3 1 Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan, 2 Department of Bioscience, School of Science and Technology, Kwansei-Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan, 3 Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan The genome of the hyperthermophilic archaeon Thermococcus kodakaraensis (Topt = 85°C) contains a gene (TK2291) encoding a putative transcriptional regulator orthologous to the Pyrococcus furiosus Phr (Pf-Phr). Pf-Phr represses genes encoding the small heat shock protein, an AAA+ ATPase, and Phr itself under normal growth temperatures [1,2]. To examine the physiological roles of the Phr ortholog (Tk-Phr) and its contribution to heat shock response, its deletion mutant (KHR1) was constructed. Growth measurements indicated that the KHR1 cells showed similar specific growth rates with those of the wild-type strain, but an increase in cell yield in the stationary phase was observed. To determine the genes that were regulated by TK2291, a whole genome microarray analysis was performed between KHR1 and wild-type cells grown at 80˚C. Transcript levels of more than 20 genes were significantly higher in KHR1 cells than in wild-type cells. The majority of these genes contained a sequence motif virtually identical to that of Pf-Phr in their 5’-flanking regions. The TK2291 regulon includes genes encoding the small heat shock protein (TK1155), an AAA+ ATPase (TK1157), prefoldin (TK1121-TK1122), ATPase of the RecA superfamily (TK1590), and Tip49 (TK1199). On the other hand, more than half of the members in the regulon encode either conserved or hypothetical proteins, raising the possibility that these proteins participate in as-yet unidentified processes of the heat shock response in T. kodakaraensis [3]. Genetic and biochemical analyses of Tk-Phr are also discussed. References [1] Vierke, G., Engelmann, A., Hebbeln, C. and Thomm, M. (2003) J. Biol. Chem. Vol. 278, p. 18-26 (2003). [2] Liu, W., Vierke, G., Wenke, A. K., Thomm, M. and Ladenstein, R. J. Mol. Biol. Vol. 369, p. 474-488 (2007). [3] Kanai, T., Takedomi, S., Fujiwara, S., Atomi, H. and Imanaka, T., J. Biochem., Vol. 143, p. 361-370 (2010). 95 www.msk.or.kr S10-4 Molecular Architecture and the Mechanics of Lon, a Protease-Chaperone Machine Sun-Shin Cha Marine Biotechnology Research Center, Korea Ocean Research and Development Institute, Ansan 426-744 The ATP-dependent Lon protease, which has orthologs distributed in all kingdoms of life [1-3], is essential in bacteria and other microorganisms under stress conditions and is needed for survival of mammalian cells subjected to oxidative damage. Lon consists of a molecular chaperone belonging to the AAA+ family and a protease with a serine-lysine catalytic dyad encoded in tandem in a single polypeptide. Here, we report the 2.0 Å resolution crystal structure of Lon from Thermococcus onnurineus NA1 [4] (TonLon). Six subunits of TonLon assemble into a cylindrical structure with a sequestered internal chamber harboring the proteolytic active sites accessible only through restricted axial channels. Alternating subunits exist in two different nucleotide states displaying different domain orientations and intersubunit contacts indicative of the ATP hydrolysis-coupled motions driving protein unfolding and translocation. References [1] Rotanova TV. Bioorg Khim 25, 883 (1999). [2] Wang N, Maurizi MR, Emmert-Buck L and Gottesman MM J Biol Chem 269, 29308 (1994). [3] Wang N, Gottesman S, Willingham MC, Gottesman MM and Maurizi, MR Proc Natl Acad Sci USA 90, 11247 (1993). [4] Lee HS, Kang SG, Bae SS, Lim JK, Cho Y, Kim YJ, Jeon JH, Cha SS, Kwon KK, Kim HT, Park CJ, Lee HW, Kim SI, Chun J, Colwell RR, Kim SJ and Lee JH J. Bacteriol 190, 7491 (2008). 96 Symposia S10-5 Novel DNA Ligases with Broad Nucleotide Cofactor Specificity from the Hyperthermophilic Crenarchaeota Suk-Tae Kwon Department of Genetic Engineering, Sungkyunkwan University DNA ligases fall into two groups on the basis of the required cofactor for activity; ATP-dependent DNA ligase and NAD+-dependent DNA ligase. The ATP-dependent DNA ligase are widely distributed in eukaryotes and they are also found in eukaryotic DNA viruses, bacteriophages of the T series and archaebacteria. In contrast, the NAD+-dependent DNA ligase are found exclusively in eubacteria. In the past few years, information concerning amino acid sequences and biochemical properties of archaeal DNA ligase has accumulated. In contrast to the conventional idea that archaea possess only ATP-dependent DNA ligases, reports of dual specificity DNA ligase from some archaea raised evolutionary issues of cofactor determinations of DNA ligases. We assumed that some crenarchaeotal DNA ligases may utilize ADP instead of ATP, while certain euryarchaeotal DNA ligases may exploit NAD+, those DNA ligases especially belong to the order Thermococcales. After that, we have reported that DNA ligases from Thermococcus onnurineus NA1 and Staphylothermus marinus also demonstrated dual cofactor specificity (ATP/NAD+ and ATP/ADP, respectively). These facts once again prompted us to investigate another crenarchaeotal DNA ligase, Sulfophobococcus zilligii and Hyperthermus butylicus to analyze and to detect the evolutionary correlation of cofactor requirements of archaeal DNA ligases. Unlike other ATP-dependent DNA ligases, and even more distinct from previously reported dual specificity DNA ligases, the Szi DNA ligase and Hbu DNA ligase exploited ADP, and GTP in comparison to ATP. As we have confirmed broad cofactor specificity (ATP/ADP/GTP) of the Szi DNA ligase and Hbu DNA ligase, a study focusing on the evolutionary relationships of DNA ligases on cofactor perspectives was carried out. Similar to categorization of organisms using 16S rRNA, DNA ligase amino acid sequence alignment also classified organisms into four distinctive groups; crenarchaeota, euryarchaeota, eukarya, and bacteria. Then, we layered biochemical properties concerned with respective cofactor requirements of DNA ligases over the tree topology. Consequently, interesting relationships between cofactor requirements and organism classification was observed. Eukaryal DNA ligases were more phylogenetically proximate to crenarchaeotal DNA ligase than euryarchaeotal DNA ligase, while bacterial DNA ligases were relatively closer to euryarchaeotal DNA ligases than crenarchaeotal DNA ligases in the phylogenetic tree. In conclusion, it is likely that multiple cofactor specificity crenarchaeotal DNA ligases existed ahead of other 97 www.msk.or.kr DNA ligases. Initially, those ancestral DNA ligases utilized ADP, a low free energy source, as a nucleotide cofactor. So as to seek higher energy sources such as ATP or NAD+, protein structures that interact with those sources were acquired during the evolutionary process and diverged to eukaryal DNA ligases and euryarchaeotal DNA ligase. After the emergence of dual specificity ATP/NAD+ DNA ligases in the euryarchaeota, bacterial DNA ligases obtained NAD+-utilizing ability and subsequently evolved to contemporary NAD+-dependent bacterial DNA ligase. Broad cofactor specificity of archaeal DNA ligases seems to have vanished through the evolution and development of organisms in relatively moderate environments, and are now affixed to either ATP or NAD+-dependent DNA ligase. So far, Szi DNA ligase and Hbu DNA ligase seem to be the earliest archetype of DNA ligases, since it has most variant cofactor specificity than any other DNA ligases. Moreover, Szi DNA ligase and Hbu DNA ligase may exemplify the remnant of an evolution, or the transition phase of cofactor exploitation by showing its preference to utilize ATP rather than ADP, GTP. This unique property proves its value as evidence of the evolution of DNA ligase from the perspective of cofactor specificity. References [1] Seo MS, Kim YJ, Choi JJ, Lee MS, Kim JH, Lee J-H, and Kwon S-T. J. Biotechnol. 128, 519 (2007). [2] Sun Y, Seo MS, Kim JH, Kim YJ, Kim GA, Lee JI, Lee J-H, and Kwon S-T. Environ. Microbiol. 10, 3212 (2008). 98 Symposia S11-1 In Situ Fluorescent Imaging of Bacteriogenic Cyanide in Lungs of Live Mice Infected with Pseudomonas aerugionosa and Burkholderia cepacia Sungsu Park1*, Seong-Won Nam1, Xiaoqiang Chen1, You-Hee Cho2, and Juyoung Yoon1 1 Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750 2 Department of Life Science, Sogang University, Seoul 121-742 Cystic fibrosis (CF) patients often suffer from chronic respiratory infections caused by the opportunistic pathogens Pseudomonas aeruginosa (PA) and Burkholderia cepacia complex (Bcc), which often produce cyanide (CN- or HCN). Cyanide is a toxin that inhibits cellular respiration. The CF lung exhibits a microaerobic condition where the cyanogenic pathogens survive through anaerobic respiration and enhance their cyanide production. A previous report [1] showed that up to 130 µM cyanide was detected from the sputa of CF and non-CF bronchiectasis patients infected with PA, whereas cyanide was not detected in the sputum of patients without the PA infection. The presence of cyanide in the sputa from the patients infected with PA can be interpreted as an indication that cyanide is present in the infected lungs as well. However, the extent of the cyanide production and the pathogenic role of cyanide in the lungs are mostly unknown because of the lack of a proper determination method for the in situ concentration of cyanide in the lungs. In vivo imaging offers noninvasive insight into living organisms and provides spatial and temporal information of the disease-related changes in the body. Recently, we synthesized a chemosensor which enhances its fluorescence intensity upon binding CN- and were able to fluorescently visualize the bioaccumulation of cyanide in the nematode Caenorhabditis elegans using the chemosensor [2]. In this study, the amounts of bacteriogenic cyanide in the lungs of mice infected with the cyanogenic PA or B. cepacia strains were visualized using the cyanide chemosensor with a whole animal imaging system. For the chronic infection of PA or B. cepacia in the mouse lungs, BALB/c nude mice were infected through the intratracheal introduction of agar beads containing these bacteria. For the in vivo imaging, the infected mice were anesthetized and the cyanide sensor was then injected into their lungs. Using this in vivo imaging method, we were able to detect high concentrations (1.8 to 4 mM) of cyanide in the lungs of the infected mice, suggesting that the cyanide production was enhanced in the lungs. Since the in vivo imaging method did not require the experimental animals to be sacrificed, the long term monitoring of cyanide in the mouse lung was enabled. The PA and B. cepacia strains continuously produced cyanide in the lungs for at least 9 days and the cyanide concentration in the lungs was clearly related to the respective bacterial loads during these days. The in vivo imaging method was used to test the in vivo efficacy of the antibiotics against PA or B. cepacia. PA14 was 99 www.msk.or.kr susceptible to the β-lactam (e.g., ceftazidime) and fluoroquinolone (ciprofloxacin) antibiotics, while B. cepacia was resistant to as these antibiotics. These results suggest that the biogenic cyanide should be considered as an important virulence factor in the CF lung. In the future, the CFTR (Cystic fibrosis transmembrane conductance regulator)-deficient mice model should be used to elucidate the pathological role of the bacteriogenic cyanide against CF patients. References [1] B. Ryall, J.C. Davies, R. Wilson, A. Shoemark and H.D. Williams, Pseudomonas aeruginosa, cyanide accumulation and lung function in CF and non-CF bronchiectasis patients. Eur. Respir. J. 32, 740 (2008). [2] S.-Y. Chung, S.-W. Nam, J. Lim, S. Park and J. Yoon. A highly selective cyanide sensing in water via fluorescence change and its application to in vivo imaging. Chem. Commun. 2866 (2009). 100 Symposia S11-2 Iron Homeostasis Affects Antibiotic-Mediated Bacterial Cell Death Woojun Park* and Jinki Yeom Division of Environmental Science and Ecological Engineering, Korea University Pseudomonas species perform key roles in the environment, including the degradation of natural and man-made chemicals and the establishment of important interactions with plants and animals [1, 2]. One pseudomonad, Pseudomonas aeruginosa, is a focus of particular study because it is the predominant opportunistic pathogen of cystic fibrosis patients [1, 2]. Clearance is difficult because of its unusually low susceptibility to antibiotic treatment [2, 3]. Thus both because many pseudomonads inhabit natural environments in which antibiotic exposure is possible, and because P. aeruginosa in particular comprises a public health hazard, Pseudomonas species are model strains for studies of the development of antibiotic resistance. Until recently bactericidal antibiotics were believed to kill cells by several well-established mechanisms, typically involving the disruption of cell wall biosynthesis, the interruption of DNA replication, or the overwhelming inhibition of protein synthesis [4, 5]. However, a systems-analysis investigation conducted upon E. coli by Kohanski, Collins, and co-workers raised the possibility that in this organism bacteriocidal antibiotic might additionally create oxidative stress, and that a part of their toxicity in aerobic habitats might be due to this effect [4-6]. We previously discovered that ferredoxin-NADP+ reductase (FprB) is a ferric reductase in P. putida [7, 8]. Thus we hypothesized that if antibiotic exposure creates H2O2 stress, FprB might promote Fenton chemistry and contribute to cell death. An fprB mutant will be more resistant to antibiotics than was its wild-type parent. Conversely, a strain engineered to overproduce FprB will be more sensitive. In this study, northern blot analysis demonstrated that ahpC, gor, and recA were abundantly induced when cells were treated with antibiotics (Fig. 1). We also demonstrated directly that iron availability is important to the action of antibiotics and the ferric reductases of P. putida and P. aeruginosa could accelerate antibioticmediated cell death by promoting the Fenton reaction. The modulation of NADH levels and iron chelation affected the actions of antibiotics. Interestingly, the deletion of the ferric reductase gene confers more antibiotic resistance upon cells, and its overexpression accelerates antibiotic-mediated cell death (Fig. 2). The results of transcriptome analysis showed that both Pseudomonas species induce many oxidative stress genes under antibiotic conditions, which could not be observed in ferric reductase mutants. Our results indicate that iron homeostasis is crucial for bacterial cell survival under antibiotics, and should constitute a significant target for boosting the action of antibiotics. 101 www.msk.or.kr Figure 1. Antibiotics coupled with 100 μM ferric-citrate can Figure 2. The sensitivity tests were conducted under increase oxidative stress and DNA repair-related antibiotic condition using the wild-type, the gene expression. fprB mutant strain (∆fprB) and the fprB References [1] Gómez MI, Prince A. Curr. Opin. Pharmacol. 7(3), 244-251, 2007. [2] Gooderham WJ, Hancock RE. FEMS Microbiol. Rev. 33(2), 279-294, 2009. [3] Page MG, Heim J. Curr. Opin. Pharmacol. 9(5), 558-565, 2009. [4] Dwyer DJ, Kohanski MA, Collins JJ. Curr. Opin. Microbiol. 12(5), 482-489, 2009. [5] Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ. Cell 130(5), 797-810, 2007. [6] Kohanski MA, Dwyer DJ, Wierzbowski J, Cottarel G, Collins JJ. Cell 135(4), 679-690, 2008. [7] Yeom J, Jeon CO, Madsen EL, Park W. J. Bacteriol. 191(5), 1472-1479, 2009. [8] Yeom J, Jeon CO, Madsen EL, Park W. J. Biochem. 145(4), 481-491, 2009. 102 Symposia S11-3 Pseudomonas Virulence by a Quorum-Dependently Secreted Exoprotease Joon-Hee Lee Lab of Microbiology, Department of Pharmacy, College of Pharmacy, Pusan National University, Busan 609-735 Pathogenic bacteria use various virulence factors to achieve the invasive infection and hosts defend themselves against the pathogens by using effective immune systems. Invertebrates mainly depend on innate immunity that includes the expression of antibacterial peptides and the activation of prophenoloxidase cascades in hemolymph (insect blood). Generally, these systems are initiated from the recognition of bacteria-specific materials, so called as PAMPs (pathogen-associated molecular pattern) such as peptidoglycan, lipopolysaccharide, lipoteichoic acid, and glucan by the cognate specific recognition proteins (pattern recognition receptors). In insects, signals from the PAMP-recognition are amplified by a proteolytic cascade similar to the vertebrate complement system, which activates the Toll-mediated intracellular signaling pathway to produce antibacterial peptides, and the prophenoloxidase system that leads to melanization on the infected site to block further infection process. While these major innate defense mechanisms of invertebrates are specifically cascaded by host protease system, bacteria also produce many proteases during infection process, which are known as virulence factors. A multi-host pathogen, Pseudomonas aeruginosa produces many extracellular proteases involved in pathogenecity, including elastase (pseudolysin), alkaline protease (aeruginolysin), LasA (staphylolysin), LasD (staphylolysin), and Protease IV (lysyl endopeptidase). In this study, we investigated the possibility that bacterial proteases could lead the activation or interrupt of the insect innate immune system by cleaving the host proteases. When the cell-free culture supernatant (CFCS) of P. aeruginosa was injected into Tenebrio moliter, the melanization was induced. However, the small molecules passed through the 10 kDa cut-off membrane or the heat-inactivated CFCS failed to induce the melanization. Interestingly, CFCS from quorum mutant (lasI-, rhlI-) also failed to induce the melanization and it was restored by the supplement of synthetic quorum signals during the culture. This suggested that certain quorum-dependently secreted protein factor(s) may snatch the innate immune response, skipping the initial recognition step. When we mixed the CFCS with the purified protease cascade components of the T. moliter innate immune system in vitro, some of them were significantly cleaved into their active forms. In this study, we explored the bacterial proteases involved in this process and addressed the role of quorum sensing in the Pseudomonas infection and virulence. 103 www.msk.or.kr S11-4 Apoptotic Elimination of Natural Killer Cells by Pseudomonas aeruginosa Jin Woong Chung Department of Biological Science, Dong-A University Infectious complications are one of the major causes of morbidity and mortality in immunocompromised patients, despite recent advances in therapeutic approaches and supportive care. Among the infectious agents, the increasing incidence of Pseudomonas aeruginosa (PA) is a worldwide problem, particularly in patients with leukemia and in hematopoietic stem cell transplantation recipients. PA is a multi-drug resistant, Gram-negative, opportunistic pathogen and is associated with significant morbidity and mortality. PA constitutes the major cause of prolonged hospitalization, severe illness, death, and increased cost for immunocompromised patients. A high mortality rate occurs in patients with underlying disease, such as cystic fibrosis and cancer. PA pathogenesis involves the production of a variety of toxic products, including alkaline protease (AP), elastase, and several Type III system-dependent exotoxins that include Exo A, Exo T, and Exo U. AP and elastase have previously been implicated in the inhibition of NK cell activity, and the exotoxins have been reported to induce apoptosis of phagocytes, such as dendritic cells, macrophages, and neutrophils. Apoptosis and shedding of the infected, apoptotic cells may be beneficial to the survival of the host organism. However, apoptosis of lymphocytes by bacterial infection has detrimental effects on host survival. NK cells are lymphocytes that mature from hematopoietic stem cells (HSC) in the bone marrow (BM). Upon activation, they can eliminate leukemic cells, as well as pathogen-infected or transformed cells, either directly or indirectly through the release of cytokines and chemokines. Previous studies indicate that upon infection with PA, NK cells can produce interferon-γ that may assist in clearing the bacteria. However, a negative role of NK cells in the regulation of PA infection has also been reported. Furthermore, NKG2D and substance P have been shown to be important in host defense against PA infection, supporting the involvement of NK cells in resistance to such infections. However, little is known about the exact mechanisms or interactions between NK cells and PA during infection. In this report, we show that PA invades natural killer (NK) cells and induces phagocytosis-induced cell death (PICD) of lymphocytes. In vivo tumor metastasis was augmented by PA infection, with a significant reduction in NK cell number. Adoptive transfer of NK cells mitigated PA-induced metastasis. Internalization of PA into NK cells was observed by transmission electron microscopy. In addition, PA invaded NK cells via phosphoinositide 3-kinase (PI3K) activation, and the phagocytic event led to caspase 9-dependent apoptosis of NK cells. PA-mediated NK cell apoptosis was dependent on activation of 104 Symposia mitogen-activated protein (MAP) kinase and the generation of reactive oxygen species (ROS). These data suggest that the phagocytosis of PA by NK cells is a critical event that affects the relapse of diseases in immunocompromised patients, such as those with cancer, and provides important insights into the interactions between PA and NK cells. 105 www.msk.or.kr S12-1 Structural Basis for Hypoxia Sensing by Histidine Kinases from Mycobacterium tuberculosis Beom Sik Kang School of Life Sciences and Biotechnology, Kyungpook National University Mycobacterium tuberculosis is still one of the most dreaded pathogens in existence, and one of the reasons for its success as a pathogen lies in its ability to persist for years within its host. One-third of the world’s population is estimated to carry M. tuberculosis in the dormant form [1], and while in this state, the pathogen is insensitive to most available chemotherapy. M. tuberculosis has been shown to undergo a metabolic transformation to its non-replicating persistence state under the influence of environmental stimuli such as hypoxia or nitric oxide [2]. This transformation is thought to be mediated via two sensor histidine kinases, DosS and DosT [3, 4], each of which consists of a sensor core and a kinase core [5]. The sensor core contains two GAF domains that are responsible for detecting oxygen tension. The first GAF domain (GAF-A) contains a heme [6] while the second GAF domain (GAF-B) is not suitable to bind a small ligand such as cyclic nucleotides [7]. In the crystal structure of the first GAF domain (GAF-A) of DosS, a b-type heme was embedded in a hydrophobic cavity of the GAF-A domain and was roughly perpendicular to the β-sheet of the GAF domain. The heme iron was liganded by H149 at the proximal heme axial position. The iron, in the oxidized form, was six-coordinated with a water molecule at the distal position. Upon reduction, the iron, in ferrous form, was five-coordinated and when the GAF domain was exposed to atmospheric O2, the ferrous form was oxidized to generate the met form rather than a ferrous O2-bound form. Since the heme is isolated inside the GAF domain, its accessibility is restricted. However, a defined hydrogen bond network found at the heme site could accelerate the electron transferability and would explain why DosS was unable to bind O2. Flavin nucleotides were shown to reduce the heme iron of DosS while NADH was unable to do so. These results suggest that DosS is a redox sensor and detects hypoxic conditions by its reduction. The kinase core of histidine kinases contains an ATP-binding domain following a histidine kinase phosphor-acceptor (HisKA) domain. The ATP-binding domains have been known to contain conserved motifs, N, F, and three G boxes [8]. However, sequence alignment analysis revealed that DosS and DosT do not have F and G2 boxes, which play a role for ATP lid. Crystal structures of ATP-binding domains from DosS and DosT show a closed ATP-binding pocket with a short putative ATP-lid. The crystal structures suggest that the ATP-binding domain should undergo a conformation change, which opens the ATP binding site, probably by the sensor domain, GAF-A. 106 Symposia References [1] Parrish NM, Dick JD, and Bishai WR. Trends Microbiol 6, 107 (1998). [2] Wayne LG and Sohasjey CD. Annu Rev Microbiol 55, 139 (2001). [3] Dasgupta N, Kapur V, Singh KK, Das TK, Sachdeva S, Jyothisri K, and Tyagi JS. Tuber Lung Dis 80, 141 (2000). [4] Roberts DM, Liao RP, Wisedchaisri G, Hol WGJ, and Sherman DR. J Biol Chem 279, 23082 (2004). [5] Sardiwal S, Kendall SL, Movahedzadeh F, Rison SCG, Stoker NG, and Djordjevic S. J Mol Biol 353, 929 (2005). [6] Cho HY, Cho HJ, Kim YM, Oh JI, and Kang BS. J Biol Chem 284, 13057 (2009). [7] Lee J, Cho HY, Cho HJ, Ko I, Park SW, Baik H, Oh J, Eom C, Kim YM, Kang BS, and Oh J. J Bacteriol 190, 6795 (2008). [8] Nowak E, Panjikar Santosh, Morth JP, Jordanova R, Svergun DI, and Tucker PA. Structure, 14, 275 (2006). 107 www.msk.or.kr S12-2 Structural Studies of the Transmembrane Domain of Bacterial Histidine Kinase Eunha Hwang1, Young-Pil Kim1, Kwonjoo Yeo1, Innokentiy V. Maslennikov2, Christian Klammt2, Georgia Kefala2, Chaejoon Cheong1, Senyon Choe2, and Young Ho Jeon1* 1 Division of Magnetic Resonance, Korea Basic Science Institute, Ochang 363-883 2 Structural Biology Lab., Salk Institute, USA Histidine kinase plays an important role in signal response to the external stimuli in bacteria. Currently, the structural studies of the transmembrane domain of histidine kinase are deficient because of the difficulties of the sample preparation. The main difficulties include the low expression of the membrane protein and the inhomogeniety of the sample state. Here, we show the successful expression of transmembrane domain (PTD) of bacterial histidine kinases, with fusion of Vitreoscilla hemoglobin (VHb), and the sample preparation of selected proteins in mg quantities for nuclear magnetic resonance (NMR) studies. The PTDs of the histidine kinases were then screened with various conditions to obtain homogeneous NMR spectra with well-resolved resonances. We achieved the near-complete backbone assignments (>90%) of the two intergral membrane proteins of DPC micelles and LMPG micelles. These results may provide a valuable opportunity and information for the structural study by NMR. Fig. 1. Possible topology models of membrane proteins in detergent micelles. 108 Symposia References [1] Kim YP, Yeo KJ, Kim MH, Kim YC*, Jeon YH* (2010) Structural characterization of the intra-membrane histidine kinase YbdK from Bacillus subtilis in DPC micelles, Biochem Biophys Res Commun. 391(3):1506-11. [2] Kim HJ, Howell SC, Van Horn WD, Jeon YH, Sanders CR (2009) Recent Advances in the Application of Solution NMR Spectroscopy to Multi-Span Integral Membrane Proteins, Prog Nucl Magn Reson Spectrosc. 55(4):335-360. 109 www.msk.or.kr S12-3 Functional Characterization of Native and Recombinant Antifreeze Protein of Arctic Yeast, KOPRI-AY30 Kyung Sun Park1,2, Jong Kyu Lee1, Jong Soo Kim1, and Hak Jun Kim1,2* 1 Division of Polar Biology and Ocean Sciences, Korea Polar Research Institute, Incheon 406-840 2 Department of Polar Sciences, University of Science and Technology, Incehon 406-840 Antifreeze proteins (AFPs) are proteins that depress the freezing point but not the melting point of aqueous solutions by inhibiting the growth of ice crystals. This phenomenon is described as thermal hysteresis (TH), a difference between the freezing and melting points and provides organisms in subzero environments with one of cold adaption mechanisms. These characteristics make AFPs a potential biomedical, food, agricultural, and industrial applications. Recently, we have isolated AFPs from an Arctic psychrophilic yeast Leucosporidium sp. using ice-affinity chromatography, which showed antifreeze activity as well as ice recrystallization inhibition activity. To further characterize the protein, recombinant AFP was expressed and purified. However, the native AFP was observed to be N-glycosylated. To see the activity of the glycosylated AFP in relation to their structure, we conducted several experiments, such as, dynamic light scattering, circular dichroism, chemical cross-linking, and heat stability. The secondary structural analysis by CD spectra of native AFP showed minima around 200 nm, a typical characteristic of random coil protein while, recombinant AFP showed increased a-helicity. In Glutaraldehyde cross –linking experiment, recombinant AFP was prone to form oligomers esp. dimer but native AFP was not. Heat stability of the both proteins showed little difference although recombinant AFP seemed more stable. Effect of glycosylation on behavior and function of AFPs seemed to be negligible but yeast AFP seemed not to be case. Here, we will present the effect of glycosylation of yeast AFP in terms of antifreeze activity. 110 Symposia S12-4 From Oxylipin to Volatile Dongsun Lee Jeju National University The oxylipin pathway generates not only prostaglandin-like jasmonates but also green leaf volatiles (GLVs), which confer characteristic aromas to fruits and vegetables. Although allene oxide synthase (AOS) and hydroperoxide lyase are atypical cytochrome P450 family members involved in the synthesis of jasmonates and GLVs, respectively, it is unknown how these enzymes rearrange their hydroperoxide substrates into different products. Here we present the crystal structures of Arabidopsis thaliana AOS, free and in complex with substrate or intermediate analogues. The structures reveal an unusual active site poised to control the reactivity of an epoxyallylic radical and its cation by means of interactions with an aromatic p-system. Replacing the amino acid involved in these steps by a non-polar residue markedly reduces AOS activity and, unexpectedly, is both necessary and sufficient for converting AOS into a GLV biosynthetic enzyme. Furthermore, by combining our structural data with bioinformatic and biochemical analyses, we have discovered previously unknown hydroperoxide lyase in plant growth-promoting rhizobacteria, AOS in coral, and epoxyalcohol synthase in amphioxus. These results indicate that oxylipin biosynthetic genes were present in the last common ancestor of plants and animals, but were subsequently lost in all metazoan lineages except Placozoa, Cnidaria and Cephalochordata. 111 www.msk.or.kr S12-5 Spectroscopic Study of Rhodopsins in Action Hideki Kandori Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan We have studied structure-function relationship in visual and microbial rhodopsins by means of various spectroscopic techniques. In particular, light-induced difference Fourier-transform infrared (FTIR) spectroscopy is a promising method for this aim [1]. In the symposium, I will present on the microbial rhodopsins that function as light-driven ion pumps. (1) Mechanism of light-driven ion pumps Proton transport is essential for our life, because ATP is synthesized by an enzyme that utilizes proton motive force, and thus nature creates various proton pumps. Proton pump proteins can translocate protons even when the proton concentration is higher at the other side of the membrane. Proton pathways are necessary for pumps, but proton pathways cannot be fully connected between the two sides of the membrane, because the proton gradient formed will be easily collapsed. This is an important aspect in distinguishing pumps from channels. The former needs a "switch", which ensures the vectoriality of the pumping. As the heart of pumps, the switching machinery is interesting, but little is known about it. Halophilic bacteria possess two light-driven pumps; bacteriorhodopsin (BR) and halorhodopsin (HR) pump protons and chloride ions, respectively. BR is the best understood ion pump, whose structure and structural changes have been extensively investigated. Recently, we succeeded in visualizing the real-time motion of BR using high-speed Atomic Force Microscope (AFM) [2]. Outward proton transport is achieved by 5 proton transfers, and each proton transfer is spatially and temporally well controlled (Figure). By means of low-temperature FTIR spectroscopy, we revealed the presence of internal water molecules of BR and their important role in function before the X-ray structural determination [3]. In the active center of BR, we found strongly hydrogen-bonded water molecules (water O-D stretch in D2O at <2400 cm-1), and comprehensive study of various rhodopsins (>15 original papers including the latest one on Gloeobacter rhodopsin [4]) revealed that such water molecules 112 Symposia were found only in the proteins exhibiting proton-pumping activity [5]. We conclude that a single hydrogen bond of water is possibly the determinant of the proton-pump function in rhodopsins. (2) Functional conversion of light-driven ion pumps Functional conversion is important for both basic and application studies. We previously converted BR into an inward chloride-ion pump [6], but interestingly, HR has never been converted into proton pump. This also suggests the importance of hydrogen-bonding network in the active center. An inward proton pump has never been created naturally or artificially. This sounds reasonable, because inward proton pump competes the ATP synthase function, being dangerous for survival. Proton pumps must have a specific mechanism to exclude transport in the reverse direction to maintain a proton gradient, and in the case of BR, a highly hydrophobic cytoplasmic domain may constitute such machinery. Recently, we succeeded in creating an inward-directed proton transport from a bacterial rhodopsin by a single amino acid replacement [7]. Anabaena sensory rhodopsin (ASR) is a photochromic sensor in freshwater cyanobacteria, possessing little proton pump activity. When we replace Asp217 at the cytoplasmic domain (distance ~15 Å from the retinal chromophore) to Glu, ASR exhibited an inward proton transport activity. FTIR spectra clearly show an increased proton affinity for Glu217, which presumably controls the unusual directionality opposite to normal proton pumps. The newly designed inward proton transport may be useful as an application tool in neurobiology as well as channelrhodopsin, a light-activated cation channel, and HR. References [1] H. Kandori, Retinal binding proteins. in cis-trans Isomerization in Biochemistry; Wiley-VCH: Freiburg, pp 53-75 (2006). [2] M. Shibata, H. Yamashita, T. Uchihashi, H. Kandori and T. Ando, Nature Nanotechnology 5, 208 (2010). [3] H. Kandori, Biochim. Biophys. Acta 1460, 177 (2000). [4] K. Hashimoto, A. R. Choi, Y. Furutani, K.-H. Jung and H. Kandori, Biochemistry in press (2010). [5] Y. Furutani, M. Shibata and H. Kandori, Photochem. Photobiol. Sci. 4, 661 (2005). [6] J. Sasaki, L. S. Brown, Y.-S. Chon, H. Kandori, A. Maeda, R. Needleman and J. K. Lanyi, Science 269, 73 (1995). [7] A. Kawanabe, Y. Furutani, K.-H. Jung and H. Kandori, J. Am. Chem. Soc. 131, 16444 (2009). 113 www.msk.or.kr S13-1 Marine Brown Kelp Diversity Based on the Analyses of Multigenes from Plastid and Mitochondrion Ga Youn Cho Division of Non-Vascular Plants, National Institute of Biological Resources, Incheon 404-708 Brown algal kelp species are the largest seaweeds and provide a variety of microhabitats for marine organisms and are often used for food and medicine. A robust molecular systematic framework is yet to be established for these species largely due to insufficient taxon sampling. The present study aimed to examine molecular diversity, phylogenetic relationship, and evolution of the kep based on nucleotide sequence information. Here, we analyzed 42 species from 27 genera, which represent all genus-level taxa in the Laminariales described to date except Undariella and Feditia. Five protein-coding plastid genes (i.e., psaA, psbA, psbC, tufA, and rbcL) and a mitochondrial gene (i.e., cox3) were analyzed using phylogenetic methods. All trees from individual and combined data sets were congruent and supported the monophyly of all kelps. Within the Laminariales, 11 well supported lineages were identified by our study. These results do not support the long-held morphological classification of Laminariales into three families and 10 tribes. Instead, we propose a taxonomic revision of the Laminariales which is defined by a single family (i.e., the Laminariaceae) and 11 tribes. 114 Symposia S13-2 Functional Analysis in Escherichia coli of Biosynthesis Genes for Isoprenoids, Carotenoids, and Sesquiterpenes, and Their Production with Higher Plants Norihiko Misawa Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi-machi, Ishikawa 921-8836, Japan ([email protected]) Structural and functional diversity of isoprenoids Isoprenoids, also referred to as terpenes, are the most diverse class of natural products consisting of more than 23,000 structurally different compounds, which have been isolated from a variety of natural sources including higher plants and microorganisms. Since these compounds have a wide range of biological functions, they have extensively been applied to pharmaceuticals, nutraceuticals, fragrances, flavorings, cosmetics, colorants, or agrichemicals. For example, a monoterpene (C10) linalool, which is included in the essential oil of hop (Humulus lupulus), is an inevitable aroma ingredient for beer brewery. A sesquiterpene (C15) artemisinin, which is extracted from the shoots of annual wormwood Artemisia annua, has been used as an antimalarial drug. Taxol (paclitaxel), a diterpene (C20) including three benzene rings in the molecule, isolated from Pacific yew (Taxus brevifolia), has been used as an effective anti-cancer drug. Glycyrrhizin is a triterpene (C30) glycoside (triterpene saponin), extracted from licorice (the root and stolon of the Glycyrrhiza plants), is widely used as a natural sweetener as well as a pharmaceutical agent. Ginsenosides, a series of triterpene glycosides that are extracted from ginseng (the root of Panax ginseng), have been a traditional medicine due to their various beneficial effects on human health. Carotenoids (tetraterpenes; C40), which include astaxanthin, β-cryptoxanthin, lutein, zeaxanthin, lycopene, and β-carotene, have attracted great attention due to their beneficial effects on human health, e.g., their prevention of chronic diseases such as cancer, cardiovascular disease, and age-related macular degeneration, and used commercially as nutraceuticals for pharmaceutical and cosmetic purposes. Biosynthesis of isoprenoid basic structures Biosynthetic routes to isoprenoids are regularly and “simply” organized, despites their enormous structural diversity. They are all biosynthesized from the same basic isoprene units, isopentenyl diphosphate (pyrophosphate) (IPP) and its isomer dimethylallyl diphosphate (DMAPP) (Fig. 1). Most prokaryotes including Escherichia coli and plant plastids synthesize IPP and DMAPP through the non-mevalonate [2-C-methyl-Derythritol 4-phosphate (MEP)] pathway starting with the reaction between pyruvate and glyceraldehyde 115 www.msk.or.kr 3-phophate. DMAPP and IPP are condensed to generate geranyl diphosphate (GPP; C10), which is further converted with IPP into farnesyl diphosphate (FPP; C15), with FPP synthase (Fig. 1). FPP is similarly condensed with IPP to form geranylgeranyl diphosphate (GGPP; C20) with GGPP synthase that is CrtE in the case of carotenogenic (carotenoids-producing) bacteria. GPP and FPP are the precursors to volatile mono- and sesquiterpenes, respectively. GGPP is the precursor for diterpenes including gibberellins. Two molecules of FPP and GGPP are typically condensed to squalene and phytoene, which are the first substrates of triterpenes and carotenoids, respectively. Monoterpenes OPP IPP FPP synthase OPP IPP isomerase (type 1, 2) DMAPP IPP OPP GPP PPi IPP GGPP synthase PPi OPP OPP GGPP (2 X GGPP) FPP synthase PPi Diterpenes Carotenoids (Tetraterpenes) IPP FPP (2 X FPP) Triterpenes Sesquiterpenes Fig. 1. Biosynthesis of isoprenoid basic structures. Functional analysis of isoprenoid biosynthesis genes E. coli has been found to be the microorganism of choice for many cases of the functional analysis of a biosynthesis genes as well as biotechnological applications. This is because extensive molecular genetic resources are present and it is amenable to genetic manipulation, and considerably tolerant to solvents. Since 1990, the functional analyses of carotenoid biosynthesis genes have successfully been performed using E. coli.1,2 Recently, the functional analysis system for sesquiterpene synthase (cyclase) genes were developed using recombinant E. coli cells, in which the Streptomyces mevalonate pathway genes were co-expressed for the utilization of D-mevalonate (D-mevalonolactone) supplemented in the culture medium.2 Various sesquiterpene synthase genes from higher plants, which include the genes for α-humulene synthase, β-eudesmol synthase, and (S)-β-bisabolene synthase from the genus Zingiber, have been identified using this system. Biotechnological production of carotenoids with higher plants Many commercially relevant isoprenoids are present in minute quantities in nature or low yielding from their natural sources. Chemical synthesis of these isoprenoids has been an important target for chemists, although it is often difficult or costly because of their structural complexity. Biosynthesis of such isoprenoids with genetically modified heterologous hosts should be an alternative and promising approach. Biotechnology researches 116 Symposia towards efficient production of the isoprenoids have extensively been carried out using recombinant bacteria such as Escherichia coli and yeasts such as Saccharomyces cerevisiae, and using transgenic plants. We have performed genetic engineering of oil crops, rape (canola; Brassica napus), one of the major oil crops worldwide that includes large amounts of oleic acid (18:1), and linseed flax (Linum usitatissimum), an industrially important oil crop that includes plenty amounts of α-linolenic acid (18:3), to increase their carotenoid contents and to produce such industrially useful carotenoids in the seeds. The phytoene synthase (crtB) gene derived from soil bacterium Pantoea ananatis (formerly classified as Erwinia uredovora) was introduced into flax seeds under the control of the fatty acid elongation enzyme 1 gene (FAE1) or CaMV 35S promoter. Subsequent transgenic flax seeds synthesized 0.16 mg/g fresh weight of carotenoids as the total amounts (at the maximum), which corresponded to 19-fold increase compared with that of an untransformed control. The carotenoid composition was β-carotene, α-carotene, phytoene, and lutein. We expressed multiple (max seven) key genes for ketocarotenoid biosynthesis in rapeseed, which were the crtW and crtZ genes originated from marine bacterium Brevundimonas sp. strain SD212, idi from marine bacterium Paracoccus sp. N81106, and the P. ananatis crtE, crtB, and crtI genes. The transgenic rape plants were enriched with large amounts of carotenoids (1.37 mg/g fresh weight; corresponded to 39-fold increase at the maximum) in the seeds, which included ketocarotenoids such as astaxanthin, canthaxanthin and echinenone in addition to β-carotene, α-carotene, phytoene, β-cryptoxanthin, and lutein. References [1] Y. Nishida et al., Appl. Environ. Microbiol., 71: 4286-4296, 2005. [2] H. Harada and N. Misawa, Appl. Microbiol. Biotechnol., 84: 1021-1031, 2009. 117 www.msk.or.kr S13-3 5-Hydroxyindole-Type Alkaloids from Marine Organisms Hyi-Seung Lee*, Hee Jae Shin, and Yeon Ju Lee Marine Natural Products Laboratory, Korea Ocean Research and Development Institute, Ansan 425-600 Much attention has been focused on indole alkaloids owing to their potential utility and significant biological activity, including cytotoxic, antiviral, antimicrobial, antiparasitics, and anti-inflammatory. A variety of marine sources including sponges, tunicates, red alga, and symbiotic bacteria have been shown to generate indole alkaloids, which represent the largest number and most complicated of the marine alkaloids [1]. The alkaloids obtained from marine organisms frequently possess novel frameworks while in other cases terrestrially related compounds clearly exist. Their structure elucidation, chemical modification, and synthesis have received a great deal of interdisciplinary attention from areas of research other than chemistry and include pharmacology and medicine. In the course of our study on the chemical investigation of marine organisms [2], we found indol alkaloids from the Micronesian sponge Hyrtios sp. collected in Chuuk State, Federated States of Micronesia and Korean sponge Psammocinia sp. collected in Dokdo Island, Korea. Bioassay-guided separation of the crude extracts using various chromatographic techniques yielded a new β-carboline alkaloid together with known 5-hydroxyindol alkaloids. From the extracts of Hyrtios sp., 1-carboxy-6-hydroxy-3,4-dihydro-b-carboline (1), 6-hydroxy-3,4dihydro-1-oxo-β-carboline (2), 5-hydroxy-1H-indole-3-carboxylic acid methyl ester (3), serotonin (4), hyrtiosin A (5), 5-hydroxyindole-3-carbaldehyde (6), and hyrtiosin B (7) were isolated. And from the extracts of Psammocinia sp., bis-indole alkaloid, hyrtinadine A (8) together with the known metabolites were isolated. Their structures were elucidated on the basis of mass spectrometry and detailed 2D NMR spectroscopic data. The isolated compounds were evaluated for their inhibitory activities against ICL of C. albicans. The inhibitory potencies, expressed as IC50 values, of the tested compounds are compared to that of a known ICL inhibitor, 3-nitropropinate. Among the compounds tested, hyrtiosin B (7) was found to be a strong ICL inhibitor. 118 Symposia To investigate the structure-activity relationship on the inhibitory activities, the indol derivatives were synthesized by the Pitet-Spengler reaction. All of the synthesized compounds were tested for the inhibitory activities. The preliminary structure-activity relationship, to elucidate the essential structure requirements for the inhibitory activity, will be described. References [1] J. Kobayashi, T. Murayama, M. Ishibashi, S. Kosuge, M. Takamatsu, Y. Ohizumi, H. Kobayashi, T. Ohta, S. Nozoe, and T. Sasaki. Tetrahedron 46 (1990) 7699. [2] H.-S. Lee, K.-M. Yoon, Y.-R. Han, K. J. Lee, S.-C. Chung, T.-I. Kim, S-H. Lee, J. Shin, and K.-B. Oh. Bioorg. Medi. Chem. Lett. 19 (2009) 1051. 119 www.msk.or.kr S13-4 Marine Microbial Water Quality off Hawaii Hans Krock, Ph.D., P.E. Emeritus Professor, University of Hawaii Over the last four decades, studies in the deep waters off the Hawaiian Islands have defined several parameters relevant to understanding the dynamics of the microbial community in these waters. These studies were related to ocean thermal energy conversion (OTEC) research projects and to the disposal of municipal wastewater. The mid-ocean location of Hawaii, away from direct continental influences, allowed definition of the environmental conditions found in the open ocean – a large area of the world that has not been extensively studied with respect to microbiology. The OTEC related studies have helped define the essentially pristine deep ocean and mid-water environment while the studies related to municipal wastewater show the extent of the influence from wastewater discharges on the ocean surface layer environment near a tropical island. The deep ocean water, below the thermocline and above the influence of the bottom, was found to be very clear (turbidity ~ 0.03 NTU) with low levels of dissolved oxygen and essentially no influence from human activities. The ambient microbial population of these waters has not been studied in any significant detail. Modest concentrations of microbial colonies can be detected using heterotrophic plate counts. However, under in situ conditions, most of these bacterial populations are likely to not be metabolically active since there are almost no reduced organics in these waters. Detailed study and identification of the microbiological populations of these waters remains to be done. Such an effort will likely accompany the development of the thermal energy resource of the tropical ocean using OTEC. Much more is known about the microbial population of the nearshore tropical oceanic surface layer. In the mid-ocean, these waters are generally nutrient limited but, because of good mixing and transport, do not have a significant eutrophication response to municipal wastewater discharges located on open coastlines outside of embayments. Surface waters subject to solar UV radiation are relatively rapidly disinfected with respect to human pathogens and their indicator organisms. However, long term (days and weeks) wastewater related microbial contamination can be detected in these waters below the depth of UV penetration. UV disinfection of the wastewater before discharge effectively controls this contamination. 120 Symposia S13-5 Plant Growth-Promoting Fungi from the Sand-Dune Plants in East Coast of Korea Yung-Hyun You1, Sumera Afzahl Khan6, Jung-Sook Hwang2, Yeon-Sik Choo2, In-Jung Lee3, Sang-Dal Kim5, In-Koo Lee4 and Jong-Guk Kim1* 1 School of Life Science and Biotechnology, 2Department of Biology, 3Department of Agronomy, 4 Department of Agricultural Chemistry, Kyungpook National University, Taegu 702-010 5 Yeungnam University, Gyeongsan 6 Center of Biotechnology, University of Peshawar, Pakistan About half of sand dunes in coastal regions of South Korea are being destroyed due to anthropogenic disturbances such as military action as well as beach construction and recreational activities. The intensity of artificial activities had affected the speed and efficiency of their conservation and revegetation. We investigated root endophytic fungi of sand-dune flora for GAs production as such work had not been carried out in the past. 1,438 fungal strains were isolated from the root of sand-dune plant, and 3 strains were selected by plant gowth-promoting activity. One of these strain was identified as Penicillium citrinum KACC43900. Analysis of the culture filtrate of P. citrinum showed the presence of physiologically active gibberellins, GA1, GA3, GA4 and GA7 (1.95 ng/ml, 3.83 ng/ml, 6.03 ng/ml and 2.35 ng/ml, respectively) along with other physiologically inactive GA5, GA9, GA12, GA15, GA19, GA20 and, GA24. The plant growth promotion and gibberellin producing capacity of P. citrinum was much higher than the wild type Gibberella fujikuroi, which was taken as control during present study. When a sand dune plants were treated with the culture filtrate of P. citrinum KACC43900, their growth were promoted and the rate of photosynthesis was increased. And expression level of gibberellins 3-oxidase, cytochrome P450 family protein and ent-kaurene synthase was increased. Therefore P. citrinum KACC43900 can be used for growth promotion of sand-dune plants in destructed sea sand-dunes. References [1] S. A. Khan et al, Biotechnol Lett (2009) 31:283-287. [2] S. A. Khan et al, BMC Microbiology (2008) 8:231 doi:10.1186/1471-2180-8-231. 121 www.msk.or.kr 122 Symposia 2010 International Meeting of the Microbiological Society of Korea Colloquium 123 www.msk.or.kr 124 Colloquium C-1 Pyrene Metabolism and Enzymes Involved in Novosphingobium pentaromativorans US6-1 Yuanrong Luo1*, Sung Ho Yun2, Kae Kyoung Kwon1, Sang Jin Kim1, Seung Il Kim2, and Young Ho Chung2 1 Marine Biotechnology Research Centre, Korea Ocean Research & Development Institute, Ansan 426-744 2 Korea Basic Science Institute, Daejeon 305-333 Pyrene is a polycyclic aromatic hydrocarbon (PAH) commonly present in PAH-contaminated sites. Novosphingobium pentaromativorans US6-1 was isolated from sediment of Ulsan Bay and has broad substrates with two to five rings PAHs. Newly and increased expression of proteins were found with 1-D PAGE or 2-DE and LC-MS/MS. Presence of pyrene 1,2-monooxygenase, pyrene 4,5-monooxygenase and 4,5-dihydroxypyrene dioxygenase indicates that degradation of pyrene in N. pentaromativorans US6-1 proceeds via multiple metabolic routes initiated by mono-(C-1,2 and C-4,5) and dioxygenation (C-4,5) reactions. Further degradation entered phenanthrene metabolic pathway and via either o-phthalate pathway or salicylate pathway, both pathways were subsequently entered tricarboxylic acid (TCA) cycle and mineralized to CO2. A pyrene degradation pathway for N. pentaromativorans US6-1 was proposed by identifying the enzymes involved in the pyrene degradation [Supported by MEGRC]. 125 www.msk.or.kr C-2 Microarray Analysis of Knockout Mutant LuxS Relevant to the Quorum Sensing Mechanism in Escherichia coli K-12 Su-Wan Son1*, Jaisoo Su Kim1, Yong-Hyun Cho2, Ji-Youl Lee2, Seung-Ju Lee2, Kyong-Ran Peck3, and Sang-Seob Lee1 1 Department of Biological Engineering, Kyonggi University Department of Urology, School of Medicine, The Catholic University 3 Division of Infectious Diseases, Sungkyunkwan University School of Medicine 2 The LuxS synthesized an autoinducer to relate the quorum sensing (QS) mechanism. These autoinducer was a signal molecule and it preformed a biofilm from cell to cell community. In this study, we investigated that it was effect on the QS and virulence related gene expression level. First, we made the LuxS knockout mutant by pDM4 vector system. To investigate the role of luxS in Escherichia coli k-12, a luxS mutant strain was constructed by integration of the suicide plasmid (pDM4 vector) containing the kanamycin resistance gene into the chromosomal luxS gene. This mutant could be confirming to polymerase chain reaction (PCR) of genomic DNA and thin layer chromatography (TLC). N-acylhomoserine lactone (AHL) signal molecules were detected in E. coli K-12 strain with the Agrobacterium tumefaciens reporter strain NT1 (pDCI4IE33). On the basis of this mutant, we experimented on the Microarray for a study on the effect of luxS gene in the E. coli k-12. It was found from the results that luxS gene is an important factor in QS and virulence mechanism. Further research on the microarray results would clarify the correlation of QS and virulence mechanism. 126 Colloquium C-3 Osmotic Stress Inhibits the Cell Growth of Listeria monocytogenes by the Downregulation of PTS Genes Dongryeoul Bae* and Chinling Wang Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi 39762, USA The concern regarding Listeria monocytogenes is becoming increased due to their adaptable abilities and inherent resistances to environmental stresses. Osmotic stress can damage the bacterial cells via the disruption of the bacterial maintenance of osmotic pressure. The molecular mechanisms that L. monocytogenes adapt to high osmolarity are not fully understood. In order to examine the gene expression profiles of L. monocytogenes strain F2365 to osmotic stress, cDNA microarray was used. We have characterized genes mediated from the strain grown in BHI medium supplemented with 1.2% NaCl at the transcriptional level. Four and twenty four genes were upregulated and downregulated by the stress, respectively. The data from cDNA microarray analysis were confirmed by quantitative real time RT-PCR. Based on microarray data, the expression level of genes encoding ribosomal protein L2 and the ATP-binding cassette superfamily proteins involved in the uptake of glycine betaine/L-proline were upregulated, whereas the expression level of genes associated with PTS system, its metabolic enzymes, pathogenesis, and hypothetical protein were downregulated. The growth of L. monocytogenes in the medium with salt was significantly inhibited (P<0.01). The expression level of PTS transport genes involved in the uptake of glucose, fructose, mannose, and cellobiose were examined at various concentrations of NaCl. All genes were significantly downregulated, suggesting that osmotic stress may inhibit listerial cell growth through the downregulation of genes involved in sugar uptake. The current study reports global gene expression level in F2365 to osmotic stress. In advance, this study provides a baseline for the mechanism of the growth or osmotolerance of L. monocytogenes. 127 www.msk.or.kr C-4 Direct Activation of Transcription of Fur-Positive Regulon by Iron-Free Form of Fur Protein Hyun-Jung Lee Dept. Environ. Sci., Hankuk Univ. Foreign Studies and Dept. Environ. Med. Biol., Yonsei Univ. Coll. Med. Pathogenic bacterial ability to acquire iron from host environments is essential to maintain its growth and to elicit its virulence, which is mainly regulated by Fur. Recently, expression analysis of Vibrio vulnificus fur gene indicated that Fur acts as a transcription activator under the iron-deprived condition. This finding led us to question if this novel regulation is unique in autoregulation of fur expression, or it is widely distributed in V. vulnificus genome. Thus, the DNA microarray was utilized to screen the ORFs activated by Fur under iron-limited conditions. EMSA showed that the iron-free Fur directly and specifically bound the upstream regions of each candidate gene. DNase I footprint assays identified its binding sites, which located around -150 relative to the corresponding transcription start sites. Fusion assays showed that in vivo expression of each fusion in wildtype was increased in the presence of an iron-chelator, and this positive effect was dependent upon the presence of functional Fur protein. The requirements of the binding sites for activation by iron-free Fur were further examined via site-directed mutagenesis of the sites. Therefore, this study demonstrates that V. vulnificus Fur protein is dynamically involved in transcription of diverse genes via repression by iron-complex form and activation by iron-free form. 128 Colloquium 2010 International Meeting of the Microbiological Society of Korea Workshop 129 www.msk.or.kr 130 Workshop W1-1 Microbial Community Analysis Using Pyrosequencing (Pyrosequencing을 이용한 미생물 군집 분석) Jongsik Chun CEO, ChunLab, Inc. 시료 안에 존재하는 미생물의 종류와 양을 정확히 측정하는 것은 직접적인 연관이 있는 생태학자뿐만 아니라, 미생물학자에게 중요한 기술이다. 그 동안 denaturing gradient gel electrophoresis (DGGE)와 같은 간접적인 군집분석 기술이 많이 쓰여 왔으나, Next generation sequencing (NGS) 기술의 도래로 직접적인 DNA sequencing 에 의한 군집의 분석이 가능해 졌다. NGS 기술 중에서도 Pyrosequencing 은 400 bp 이상의 DNA 염기서열의 해독이 가능하고, 그 throughput 도 커서 Bacteria, Archaea, Fungi 등의 다양한 미생물의 군집 분석이 가능하다. 본 워크숍에서는 다음과 같은 내용으로 Pyrosequencing 을 이용한 미생물 군집 분석에 대해 소개하고자 한다. (1) Next generation sequencing 기술의 개요 (2) Pyrosequencing 원리 (3) 미생물 군집 분석 전략 (Phylogenetic marker, primer design 등) (4) 통계 처리의 문제점과 해결 전략 (5) 통합 분석 소프트웨어 소개 (6) Human microbiome, 물환경 시료, 식품 등의 분석 적용 예 (7) Genome 분석, Comparative genomics, Metagenomics 분야에서의 Pyrosequencing 적용 전략 131 www.msk.or.kr 124 Colloquium 2010 International Meeting of the Microbiological Society of Korea Poster Sessions A. Systematics B. Ecology and Environmental Microbiology C. Applied Microbiology D. Immunology and Microbial Pathogenesis E. Physiology and Biochemistry F. Genetics G. Biotechnology H. Others 133 www.msk.or.kr 134 Poster Sessions A001 A003 Brachybacterium squillarum sp. nov., Isolated from Salt-Fermented Seafood Pedobacter yonginensis sp. nov., Isolated from a Mesotrophic Artificial Lake in Korea Seong-Kyu Park*, Min-Soo Kim, Mi-Ja Jung, Eun-Jin Park, Young-Do Nam, Seong Woon Roh, and Jin-Woo Bae Department of Life and Nanopharmaceutical Sciences and Department of Biology, Kyung Hee University Yochan Joung*, Haneul Kim, and Kiseong Joh Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies A Gram-positive bacterium, strain M-6-3T, was isolated from salt-fermented seafood in Korea. The organism grows in conditions ranging from 0-10% (w/v) NaCl, and 25-37°C, with optimal growth occurring at 5% NaCl and 28-30°C. The polar lipid profile of M-6-3T consists of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), an unidentified phospholipid (PL), and an unknown glycolipid (GL). Strain M-6-3T contains MK-7 as the major component of the quinone system and anteiso-C15:0 (62.1%) as the predominant fatty acid. Based on 16S rRNA gene sequence similarity, strain M-6-3T is most closely related to Brachybacterium rhamnosum LGM 19848T (98.5%). The G+C content of the genomic DNA was measured at 71.5 mol% and DNA-DNA hybridization values were under 10% with reference strains. Based on phenotypic, genotypic and phylogenic analyses, the name Brahcybacterium squillarum sp. nov. is proposed for strain M-6-3T (= KACC 14221T = JCM 16464T). A002 A non-motile and red-pigmented bacterium, designated strain HMD1002T, was isolated from an artificial lake located within the campus of Hankuk University of Foreign Studies, Korea. The major fatty acids were iso-C15 : 0 (29.6%), Sum In Feature 3 (comprising C16 : 1 ω7c and/or iso-C15 : 0 2-OH;17.5%), isoC17 : 0 3-OH (12.5%), 15:1 w6c (8.3%), Sum In Feature 9 (comprising iso- C17:1 -w9c:6.9%), C15 : 0 (5.3%), iso-C15 : 0 3-OH (2.6%), anteiso-C15:0 (2.4%). The DNA G+C content was 41.0 mol%. A phylogenetic tree based on 16S rRNA gene sequences showed that strain HMD1002T formed a lineage in the genus Pedobacer and was closely related to Pedobacer terrae (96.3% sequence similarity) and Pedobacer suwonensis (95.8% ). On the basis of the evidence presented in this study, strains of HMD1002T represent a novel species of the genus Pedobacter, for which the name Pedobacter yonginensis sp. is proposed. The type strains are HMD1002T (=KCTC 22721T= CECT 7544T). A004 Leucobacter celer sp. nov., Isolated from Korean Fermented Seafood Gramella jeungdonese sp. nov., Isolated from Saltern in Korea Na-Ri Shin*, Min-Soo Kim, Mi-Ja Jung, Seong Woon Roh, Young-Do Nam, and Jin-Woo Bae Department of Life and Nanopharmaceutical Sciences and Biology, Kyung Hee University Haneul Kim*, Yochan Joung, and Kiseong Joh Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies A novel, Gram-positive, aerobic, rod-shaped, and non-motile bacterial strain NAL101T was isolated from gajami-sikhae, a traditional Korean fermented seafood made with flatfish. Growth occurs at 4-45°C, pH 5-10, 0-12% (w/v) NaCl. Optimum growth occurs at 30-37°C, pH 8, 0-1% NaCl. The cell-wall amino acids were 2,4-diaminobutyric acid, alanine, glycine, threonine and glutamic acid and the major fatty acid components were anteiso-C15:0, iso-C16:0 and anteiso-C17:0. The 16S rRNA gene sequence similarity value of strain NAL101T and Leucobacter chironomi DSM 19883, most closely related, is 97.7%. The DNA G+C content was 68.8 mol% and the highest DNA-DNA hybridization values were 22.2%. Phylogenetic analysis based on 16S rRNA gene sequence, physiological, and biochemical characteristics indicated that the strain NAL101T represents a new species of the Leucobacter (family Microbacteriaceae), for which we propose the name Leucobacter celer sp. nov.. The type strain is NAL101T (=KACC 14220T= JCM 16465T). A non-motile, Gram-staining-negative, yellow pigmented, rodshaped, strictly aerobic bacterium, strain HMD3159T, was isolated from a saltern in Korea. The major fatty acids were iso-C15:0(26.3%), iso-C17:0 3OH(12.1%), iso-C16:0(12.0%), Summed Feature 3(comprising C16:1 ω7c and/or C16:1 ω6c ; 11.0%) and Summed Feature 9(iso-C17:1 ω9c and/or 10-methyl C16:0; 10.0%). The major respiratory quinone was MK-6. The DNA G+C content was 40.9 mol%. Phylogenetic tree based on 16S rRNA gene sequence showed that strain HMD3159T formed a lineage within the genus Gramella and was closely related to Gramella portivictoriae(94.9% sequence similarity), Gramella echinicola(94.6%) and Gramella marina(93.6%). On the basis of the evidence presented in this study, strain HMD3159T represent a novel species of the genus Gramella, for which the name Gramella jeungdonese sp. nov., is proposed the type strain HMD3159T (=KCTC 23123T= CECT ingT). 135 www.msk.or.kr A005 Leucobacter salsicius sp. nov. from a Salt-Fermented Food Ji-Hyun Yun1*, Seong Woon Roh1,2, Min-Soo Kim1,2, Mi-Ja Jung1, Eun-Jin Park1, Kee-Sun Shin2, Young-Do Nam1, and Jin-Woo Bae1 1 Department of Life and Nanopharmaceutical Sciences and Department of Biology, Kyung Hee University, 2University of Science and Technology, Korea Research Institute of Bioscience and Biotechnology Strain M1-8T was isolated from a Korean fermented food. It exhibited optimal growth between 25-30°C at pH 7.0-8.0 and in 0-4% (w/v) NaCl. It can tolerate up to 10.0 mM Cr (VI). Phylogenetic analyses of 16S rRNA sequences indicated that strain M1-8T represents a new species in the genus Leucobacter. The 16S rRNA gene sequence of M1-8T exhibits 98.1% homology to that of Leucobacter chromiireducens subsp. chromiireducens L-1T. The chromosomal G+C content of strain M1-8T was 62.8 mol%. Its cell wall peptidoglycan contained DAB, glutamic acid, alanine, glycine and GABA. The major menaquinone was MK-11 and the predominant fatty acids were anteiso-C15:0 (63.6%), anteiso-C17:0 (16.7%) and iso-C16:0 (14.2%). The polar lipid profile of strain M1-8T contained diphosphatidylglycerol and one unknown glycolpid. Significant genotypic and phenotypic differences were found between strain M1-8T and other Leucobacter species. These differentiating characteristic indicate that strain M1-8T (= KACC 21127T= JCM 16362T) represents a novel species of the genus Leucobacter, for which the name Leucobacter salsicius is proposed. A007 Lentibacillus jeotgali sp. nov., a New Halophilic Bacterium Isolated from Traditional Korean Fermented Seafood Mi-Ja Jung*, Seong Woon Roh, Min-Soo Kim, and Jin-Woo Bae Department of Biology, Kyung Hee University A novel, Gram-positive, non-motile, endospore-forming and moderately halophilic bacterium, strain GrbiT, was isolated from a traditional Korean fermented seafood. The organism grew optimally in the presence of 10-15% NaCl, at 37°C and pH 8.0. The peptidoglycan of the cell wall consisted of mesodiaminopimelic acid, and the predominant menaquinone was MK-7. The major fatty acids of strain GrbiT were iso-C16:0 (36.4%), anteiso-C15:0 (30.3%) and iso-C14:0 (18.2%). The polar lipids were phosphatidylglycerol, diphosphatidylglycerol and an unidentified glycolipid. The genomic DNA G+C content was 42.5 mol%. Strain GrbiT was most closely related to the type strain Lentibacillus kapialis JCM 12580T, with which it shared 97.5% 16S rRNA gene sequence similarity. The DNADNA hybridization value between strains GrbiT and L. kapialis JCM 12580T was 8%. Based on phenotypic, genotypic and phylogenetic data, strain GrbiT should be classified as a novel species within the genus Lentibacillus, for which the name Lentibacillus jeotgali sp. nov. is proposed. The type strain is GrbiT (= KCTC 13300T = JCM 15795T). [Supported by TDPAF] [Supported by grants from the TDPAF] A006 A008 Microbacterium mitrae sp. nov., Isolated from Salted Turban Shell Oceanobacillus kimchii sp. nov. Isolated from a Traditional Korean Fermented Food Yun-Ji Kim*, Seong Woon Roh, Mi-Ja Jung, Min-Soo Kim, Eun-Jin Park, and Jin-Woo Bae Department of Life and Nanopharmaceutical Sciences and Department of Biology, Kyung Hee University Tae Woong Whon1*, Mi-Ja Jung1, Seong Woon Roh1,2, Young-Do Nam1, Eun-Jin Park1, Kee-Sun Shin2, and Jin-Woo Bae1 1 Department of Life and Nanopharmaceutical Sciences, and Department of Biology, Kyung Hee University, 2University of Science and Technology, Korea Research Institute of Bioscience and Biotechnology A novel bacterium (strain M4-8T) belonging to the genus Microbacterium was isolated from salted turban shell, which is a traditional fermented food in Korea. The cells of this strain were Gram-positive, non-motile, non-spore-forming rodshapes. The optimal growth condition was 1% (w/v) NaCl and 30°C. Phylogenetic analysis based on the 16S rRNA gene sequences. Within the phylogenetic tree, this novel strain shares a branching point with species Microbacterium hominis IFO 15708T (97.8%). G+C content was 71.3 mol%, and DNADNA hybridization experiments showed a low level (<29%) of DNA-DNA relatedness between M4-8T and its closest relatives. The major fatty acids were C15 : 0 iso and C15 : 0 anteiso and major cell-wall diamino acid is ornithine. Data obtained from DNA–DNA hybridization and chemotaxonomic phenotypic analysis supports the conclusion that strain M4-8T represents a novel species within the genus Microbacterium, and we would like to propose the name Microbacterium mitrae sp. The type strain is M4-8T (= KACC 21129T = JCM 16363T). [Supported by grants from the TDPAF (Technology Development Program for Agriculture and Forestry)] 136 Poster Sessions The Gram-positive strain X50T was isolated from mustard kimchi, a traditional Korean fermented food. The organism grew under conditions ranging from 0-15.0% (w/v) NaCl (optimal: 1-5%), 10-37°C (optimal: 25-30°C), and pH 5.3-9.3 (optimal: pH 7.5). Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain X50T belongs to the genus Oceanobacillus and is closely related phylogenetically to the type strain of O. iheyensis HTE831T (98.9%) and O. oncorhynchi subsp. Oncorhynchi 20AGT (97.0%). The cellular fatty acid profiles predominately included anteiso-C15 : 0, and iso-C15 : 0. The G+C content of the genomic DNA of the type strain was 37.9 mol%, and the major isoprenoid quinone was MK-7. Analysis of the 16S rRNA gene sequences, DNA-DNA relatedness, and physiological and biochemical tests indicated genotypic and phenotypic differences between strain X50T and reference species in the genus Oceanobacillus. Therefore, strain X50T was proposed as a novel species and named Oceanobacillus kimchii. The type strain of the new species is X50T. A009 Phycicoccus sp. Isolated from Soil of a Potato Cultivating Field Hyangmi Kim1,2*, Doo-Sang Park1, Hyun-Woo Oh1, Kang Hyun Lee1, Hee-Moon Park2, and Kyung Sook Bae1 1 Biological Resources Center, Korea Research Institute of Bioscience and Biotechnology, 2Department of Microbiology, Chungnam National University Two Gram-positive, motile, cocci bacterium, strains L1b-b9T and B5a-b5, were isolated from a potato cultivating field in Ochang. Strain L1b-b9T grew at 10-45°C, pH5.0-10.0 and in the presence of 8%(w/v) NaCl. Major menaquinone was MK8(H4) and the main cellular fatty acids were iso-C14:0, iso-C15:0 and anteiso-C15:0. Polar lipids in strain L1b-b9T consisted of diphosphatidylglycerol and phosphatidylglycerol. The G+C content of genomic DNA was 73.6 mol%(HPLC). Phylogenetic analysis based on 16S rRNA gene sequences showed that strains L1b-b9T and B5a-b5 shared 99.36% similarity and formed a robust clade with the type species of the genus Phycicoccus. Strains L1b-b9T and B5a-b5 were related most closely to Phycicoccus aerophilus 5516T-20 (97.13% 16S rRNA gene sequence similarity). The DNA-DNA relatedness values between the two novel isolates and Phycicoccus aerophilus (KACC20658) were below 70%. A010 Bifidobacterium stercoris sp. nov., Isolated from Human Faeces Min-Soo Kim1,2*, Seong Woon Roh1,2, and Jin-Woo Bae1,2 Department of Life and Nanopharmaceutical Sciences and Department of Biology, Kyung Hee University, 2University of Science & Technology, Biological Resources Center, Korea Research Institute of Bioscience and Biotechnology 1 Strain Eg1T, an anaerobic, Gram-positive, non-motile and nonspore-forming bacterium, was isolated from human faeces. F6PPK activity was positive. The end products of glucose fermentation were acetic acid and lactic acid, which were produced at a molar ratio of 1.76 : 1. The G+C content was 57.8 mol%. Comparative analyses based on the 16S rRNA and hsp60 gene sequences showed that the isolate was closely related to two species of the genus Bifidobacterium: B. adolescentis (98.36% and 99.35% sequence homology, respectively) and B. ruminantium (97.93% and 92.13% sequence homology, respectively). Despite this degree of sequence similarity being high enough for the isolate to be included at the same species level as B. adolescentis and B. ruminantium (96.5–100%), low level of DNA-DNA relatedness (41%) indicated that the isolate was distinguishable from these related strains, B. adolescentis and B. ruminantium. Based on phenotypic, genotypic and phylogenetic analyses, we propose that the isolate from human faeces be classified as Bifidobacterium stercoris sp. nov., and designated as strain Eg1T (= KCTC 5756T = JCM 15918T). [Supported by grants from KFDA] A011 Leifsonia moechotypicola sp. nov., a Xylanolytic Bacterium Isolated from the Gut of Hairy LongHorned Toad Beetle Moechotypa diphysis (Pascoe) Byung-Chun Kim*, Doo-Sang Park, Hyangmi Kim, Hyun-Woo Oh, Kang Hyun Lee, Kee-Sun Shin, and Kyung Sook Bae Microbial Resources Center, Korea Research Institute of Bioscience and Biotechnology A novel Gram-positive, non-motile, rod-shaped bacteria, designated strain RB-62T, was isolated during the study of culturable bacteria in the gut of Moechotypa diphysis (Pascoe). This isolates grew at 15-30°C and pH 5.0-7.5. The predominant isoprenoid quinone was MK-11, and the major components of its cellular fatty acids were anteiso-C15 : 0 (44.1%), anteiso-C17:0 (23.3%), and iso-C16:0 (20.3%). The DNA G+C content of the genomic DNA was 70.2 mol% (HPLC). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain RB-62T was affiliated with a cluster within the family Microbacteriaceae. The type strain that was most closely related to strain RB-62T was Leifsonia pindariensis PON10T (97.70%) and then Leifsonia ginsengi wged11T (97.63%). The DNA–DNA relatedness value among strain RB-62T, L. pindariensis PON10T and L. ginsengi wged11T was under 70%. The phenotypic and phyogenetic characteristics of the isolates allowed this isolate to be clearly distinguished from other Leifsonia species. Based on these polyphasic data, the strain RB-62T is considered to represent a novel taxon of the genus Leifsonia, for which the name Leifsonia moechotypicola sp. nov. is proposed. A012 Phylogenetic Diversity and Abundance of the Intestinal Bacterial Community in the Food Waste Reducing Larvae of Hermetia illucens Hyun Bum Jeon1*, So Young Park1, Jiyoung Choi2, Gil-Sang Jeong2, Jonggill Kim2, Youngcheol Choi2, and Sung-Jae Lee1 1 Department of Biology, College of Science, Kyung Hee University, 2 Laboratory of Environmental Entomology, National Academy of Agricultural Science, Rural Development Administration As it is discovered that food waste can be reduced by larval Hermetia illucens, the scientific and commercial value of larval BSF is increased recently. We extracted their intestinal metagenomic DNAs from each larva provided with rice, calf forage and food waste. We performed PCR by using bacterial 16S rRNA primers and pyrosequencing for the analysis of their intestinal microbial community. We got the useful data set of 9744, 9768, and 5986 sequences from the PCR products of food waste, rice, and calf forage fed samples, respectively. On the basis of the BLAST search of EzTaxon program, the bacterial community of the food waste fed gut was revealed that the phyrum Bacteroidetes had dominancy of 67.31%, Proteobacteria of 18.83%, and Firmicutes of 9.39%. The phylogenic groups from rice fed gut were two dominant phyrums of Firmicutes (42.09%) and Proteobacteria (53.77%). The phylogenic groups from calf forage fed gut were four similar portions of Firmicutes (23.39%), Actinobacteria (24.69%), Bacteroidetes (20.51%), and Proteobacteria (30.91%). We identified several organic compound degrading bacterial strains: Bacillus amyloliquefaciens, Paenibacillus xylanedens, Proteus mirabilis. 137 www.msk.or.kr A013 A015 Saccharospirillum aestuarii sp. nov., Isolated from Tidal Flat Sedimentin the Yellow Sea The Identification of Flavobacterium candidumensis KUDC1076 sp. nov., Isolated from Dokdo Island Ahyoung Choi*, Hyun-Myung Oh, and Jang-Cheon Cho Inha University Jong Myong Park1*, Seon-Ae Jeon1, Hye-Ri Sung1, Jung-Hoon Yoon2, and Sa-Youl Ghim1 1 School of Life Sciences, Kyungpook National University, 2 Korea Research Institute of Bioscience and Biotechnology A Gram-reaction-negative, non-motile, facultatively anaerobic and curved rod-shaped bacterial strain, IMCC4453T, was isolated from tidal flat sediment and subjected to a polyphasic taxonomic study. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain IMCC4453T belonged to the genus Saccharospirillum. DNA-DNA relatedness between strain IMCC4453T and S. salsuginis YIM-Y25T was 4.2%. The DNA G+C content of the strain was 56.5 mol%. The major polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and lyso-phosphatidylethanolamine and the major isoprenoid quinone was Q-8. Several physiological and biochemical characteristics between strain IMCC4453T and the two Saccharospirillum species, together with phylogenetic and genomic distinctiveness, differentiated the strain from members of the genus Saccharospirillum. On the basis of polyphasic data obtained in this study, it is concluded that strain IMCC4453T represents a novel species of the genus Saccharospirillum, for which the name Saccharospirillum aestuarii sp. nov. is proposed. The type strain is IMCC4453T (KCTC 22684T = NBRC 105825 T). A014 Optimization of Hexanoic Acid Production by Megasphaera sp. nov. BS-4 Byoung Seung Jeon*, Seil Kim, Youngsoon Um, and Byoung-in Sang Korea Institute of Science and Technology A new hexanoic acid-producing bacterium was isolated from cow rumen using RCM(reinforced clostridia medium) with hexanoic acid for isolation. The strain was designated Megasphaera BS-4 based on 16s rDNA sequence and phylogenetic tree. Physiological characteristics of the strain were investigated using API 50 CH and batch fermentation. Modified PYF medium was selected for cultivation of the strain. Megasphaera BS-4 used fructose as the carbon source and produced acetic acid, butyric acid, and hexanoic acid in liquid phase and hydrogen in gas phase. The medium composition for hexanoic acid production was optimized by RSM (response surface methodology). When acetic acid and butyric acid were added into the medium, concentration of hexanoic acid increased up to 10 g/L in batch culture without pH adjustment. The productivity of hexanoic acid was about 0.41 g/L/hr, which is higher than the result reported so far. [Supported by grants from KETEP] 138 Poster Sessions Strain KUDC1076 was isolated from the rhizosphere of a native plant Solanum nigrum L. in Dokdo island. Accorging to transmission electron microscopic observation, KUDC1076 has peritrichous flagella, fimbriae, pili. It showed rod shape, Gram negative and non spore forming. Phylogenetic analysis based on 16s rRNA gene sequences showed that KUDC1076 belongs to genus Flavobacterium, being closest related with F. johnsoniae (97.5%), F. anhuiense (96.9%), F. defluvii (96.4%) and F. denitrificans (96.2%). KUDC1076 produces the flexirubin type pigment which is a unique bacterial pigment of Cytophaga and Flexibacter group and its related genera. On the basis of the phenotypic and molecular data, strain KUDC1076 represents a novel species within the genus Flavobacterium, for which the name Flavobacterium candidumensis sp. nov. is proposed. A016 The Identification of Paenibacillus taehanensis U5-6 sp. nov., Isolated from The Rhizosphere of Acer okamotoanum, a Native Plant in Ulleungdo Island Ye-Ji Hwang1*, Seon-Ae Jeon1, Hye-Ri Sung1, Jung-Hoon Yoon2, and Sa-Youl Ghim1 1 School of Life Sciences, Kyungpook National University, 2 Korea Research Institute of Bioscience and Biotechnolgy Strain U5-6 was isolated from the rhizosphere of Acer okamotoanum, a native plant in Ulleungdo island. This strain comprised Gram positive, endospore-forming, flagellar rods and is capable of growth at 18~37°C with optimal growth temperature of 30°C. This strain is oxidase- and catalasepositive, and does not hydrolyze starch and casein. The length of this strain is from 2.0 to 2.5 μm. Phylogenetic analysis based on 16s rRNA gene sequences showed that strain U5-6 belongs to the genus Paenibacillus, being related to P. chondroitinus (97.8%), P. alginolyticus (97.6%), P. pocheonesis (97.5%), P. pectinilyticus (97.1%), and P. aestuarii (97.1%). On the basis of the analysis of cellular fatty acid composition, G+C contents and phenotypic data, strain U5-6 represents a novel species of the genus Paenibacillus, for which the name Paenibacillus taehanensis is proposed. A017 A019 Inferring Bacterial Species Tree Using Multilocus Sequence Data Genotyping of Bacillus anthracis Korean Isolates Using Molecular Typing Methods Mincheol Kim* and Jongsik Chun School of Biological Sciences and Institute of Microbiology, Seoul National University Sang Hoon Kim*, Sudipto Shahid, Kyoung Hwa Jung, Dong In Jeon, and Young Gyu Chai Division of Molecular and Life Sciences, Hanyang University Inferring species tree in bacteria is of intense interest because there is no clear species definition in bacteria and bacterial molecular phylogeny has relied mainly on 16S rRNA gene which is known for the lack of consistency over the whole bacterial taxa. Recently, the combined information from multiple genes has been used as information for inferring bacterial species trees. The method concatenating sequences from multiple genes has been widely used, but it frequently leads to poor estimation of the species trees. Several alternative approaches, such as Bayesian approach and consensus and so on, have recently been tried to infer species trees accurately without biases. Here we tested the applicability of several promising tools, which are based on multispecies coalescent, into multilocus sequence data of the gut symbiont Lactobacillus group. The result showed that bacterial species tree was estimated more precisely and accurately by the multispecies coalescent based methods compared to the concatenation method. The development of a new approach regarding horizontal gene transfer and recombination with coalescence is warranted for following study. Bacillus anthracis is the bacterium that causes fetal anthrax to a wide range of herbivores and even humans is closely related and often critical to differentiate from other members of the Bacillus cereus group that can causes diverse diseases. Identification and detection of these threatening bugs through molecular approaches is enhancing the insights in to the process of microbial population genetics and pathogenesis of infectious diseases. In our study we have employed several molecular approaches like single-nucleotide polymorphisms analysis, multilocus variable number tandem repeat analysis and single–nucleotide repeats for the molecular typing of B. anthracis Korean isolates. Using those efficient genotyping methods we screened the genetic diversity and have found highly similar with two major lineages (A and B) for the first time of those isolates. Furthermore, results suggest a greater understanding of the genetic diversity of B. anthracis in natural populations may be required to definitively distinguish a natural outbreak from an intentional use situation. A018 A020 Isolation of a New Alcohol-Producing Clostridium Species from Tidal Flat Chitiniphilus sp. nov., a Novel Chitin-Degrading Bacterium Isolated from Farming Field Kieun Choi1,2*, Byoung Seung Jeon1, Seil Kim1, Min-Kyu OH2, Youngsoon Um1, and Byoung-In Sang1 1 Korea Institute of Science and Technology, 2Department of Chemical and Biological Engineering, Korea University Jae-Chan Lee*, Dong-Jin Park, and Chang-Jin Kim Korea Research Institute of Bioscience and Biotechnology Butanol and ethanol are alternative biofuels for transportation. The bioproduction of butanol and ethanol is generally found in cultures of solventogenic Clostridium species through acetonebutanol-ethanol (ABE) fermentation. For the isolation of new alcohol-producing Clostridium species, tidal flat samples were collected from seashore area of Tae-An, Korea. The isolated bacterium was designated Clostridium sp. nov. BS-5 and its 16S rDNA revealed 96% similarity to Clostirdium algidixylanolyticum SPL73T. Products of Clostridium sp. nov. BS-5 were acetone, ethanol, butanol, acetic acid, and butyric acid from glucose as a carbon source. To improve alcohol production, P2 medium supplemented with 3% sea salt and 5 g/L yeast extract was used and sodium butyrate was initially added 5g/L to investigate butanol production. Optimized temperature for alcohol production was 30°C. At the optimized conditions, Clostridium sp. nov. BS-5 produced 26.1 g/L alcohol with the yield of 0.43 g/L. This result shows that Clostridium sp. nov. BS-5 is a promising new microorganism for alcohol production. Chitiniphilus sp. nov. is a bacterial strain capable of degrading chitin isolated from farming field in Daejeon, Korea. The strain was Gram-negative rod-shaped facultatively anaerobic and motile with a single flagellum. It grew well with chitin as a single carbon source. It grows best at 0-7% w/v NaCl. The predominant respiratory lipoquinone found in the strain is ubiquinone with eight isoprene units. Phylogenetic analyses strongly indicate that this strain forms a distinct line within a clade containing the genus Chitiniphilus and the closest similarity value to Chitiniphilus shinanonensis SAY3T with 95.9%. On the basis of the polyphasic evidence gathered in this study it is proposed that the isolate is a new species, Chitiniphilus nov. sp. [Supported by the Microbial Genomes & Application Center of 21st Century R&D Program, MEST] [Supported by grants from KEMCO] 139 www.msk.or.kr A021 A023 Identification and Characterization of Paenibacillus sp. nov., Isolated from Farming Field in Korea * Jae-Chan Lee , Dong-Jin Park, and Chang-Jin Kim Korea Research Institute of Bioscience and Biotechnology A Gram positive, non-motile, rod-shaped bacterium, strain BH038, was isolated from a farming field in Korea. Strain BH038 grew optimally at 30-33°C, pH 7·2-7·5 and 0% (w/v) of NaCl. The isolate showed oxidase- and catalase-negaitive reactions and did not reduced nitrate to nitrite. Anaerobic growth did not occur on TSA agar (Difco). The major cellular fatty acids were anteiso-C15:0, iso-C16:0 and iso-C15:0. The levels of 16S rRNA sequence similarity of strain BH038T to Paenibacillus chibensis JCM 9905T, Paenibacillus barengoltzii DSAFN-016T, Paenibacillus timonensis 2301032T, Paenibacillus chibensis NRRL B-142T, Paenibacillus motobuensis MC10T, Paenibacillus konsidensis LBYT were 96.3%, 96.0%, 95.9%, 95.5%, 95.5% and 95.3%, respectively. On the basis of combined phenotypic properties and phylogenetic and genetic data, strain BH038 should be placed in the genus Paenibacillus as the type strain of a novel species. Additions to Leprarioid Lichens in South Korea Yogesh Joshi*, Young Jin Koh, and Jae-Seoun Hur Korean Lichen Research Institute, Sunchon National University The presentation contributes to the study of new records of leprarioid lichens reported for the first time from this country as well as the species earlier reported from this part of the continent and expands the knowledge of lichen diversity in East Asia including China and Japan. Twenty taxa belonging to four different genera Chrysothrix, Lepraria, Lepraucaulon and Normandina respectively have been reported out. Except Lepraria coriensis and Normandina pulchella, all other species described here are new to South Korea. Among them, Lepraria caesiella, L. eburnea, L. leprolomopsis, L. lobata, L. pallida, L. texta and L. xerophila are reported for the first time in East Asia including China and Japan. Brief taxonomic description and comments of each species are provided. [Supported by the Microbial Genomes & Application Center of 21st Century R&D Program, MEST] A022 A024 A Novel Ornithinicoccus sp. nov., Isolated from Bigeum Island in Korea * Jae-Chan Lee , Dong-Jin Park, and Chang-Jin Kim Korea Research Institute of Bioscience and Biotechnology A Gram-positive coccoid, non-motile bacteria with L-ornithine as diagnostic diamino acid of the peptidoglycan were isolated from a sample of farming field of Bigeum island in Korea. This strain grew optimally at 28-33°C, pH 6.8-7.5 and 3% (w/v) of NaCl. This strain forms a distinct line within a clade the genus Ornithinicoccus and the cloestest type strain was Ornithinicoccus hortensis KHI 0125T with the similarity of 96.1%. The other close type strains to the strain were Oryzihumus leptocrescens KV-628T, Lapillicoccus jejuensis RAc013T, Konellia aerolata 5317S-21T and Tetrasphaera duodecadis ATCC 5116T with the sequence simililarity of 95.6%, 95.5%, 95.0%, and 94.9% respectively. On the basis of combined phenotypic properties and phylogenetic and genetic data, this strain should be placed in the genus Ornithinicoccus as the type strain of a novel species. [Supported by the Microbial Genomes & Application Center of 21st Century R&D Program, MEST] 140 Poster Sessions Additions to Foliicolous Lichen Flora of Vietnam Thi Thuy Nguyen1*, Yogesh Joshi1, Anh Dung Nguyen2, Young Jin Koh1, and Jae-Seoun Hur1 1 Korean Lichen Research Institute, Sunchon National University, 2 Plant Biological Department, Faculty of Agriculture, Tay Nguyen University, Vietnam Foliicolous lichens (i.e. lichens growing over leaves) have been well worked out in Neotropics, while fewer data are available from tropical Africa and SE Asia. Fewer publications have come out from Vietnam mentioning the occurrence of 70 foliicolous taxa from this place. The authors describes six additional new records of foliicolous lichens from Vietnam belonging to three different families-Gomphillaceae (Aderkomyces albostrigosus fo. albostrigosus, A. albsostrigosus fo. aggregatus and Asterotherium rotuliforme), Pilocarpaceae (Byssoloma vanderystii and Fellhanera emarginata) and Porinaceae (Trichothelium minutum), thus adding to the knowledge of the Vietnam lichen biota and increasing the tally of foliicolous lichens to 76. The specimens were collected at Thác Dray Sap (commonly called Dray Sac Waterfall) region situated in the Central Highland part of Vietnam. Most of the specimens were collected from understory leaves where high humidity and low light intensity prevail. This number represents only a small fraction of the actual foliicolous lichen diversity in the country, and further studies in this area will definitely increase the tally. A025 A027 Tenacibaculum luteolum sp. nov., Isolated from a Marine Sponge Sphingopyxis soli sp. nov., Isolated from Landfill Soil Sung-Hyun Yang1*, Olga I. Nedashkovskaya2, Hyun-Seok Seo1, Sung Hyuk Lee1, Jung-Hyun Lee1, Sang-Jin Kim1, and Kae Kyoung Kwon1 1 Marine Biotechnology Research Center, Korea Ocean Research & Development Institute, 2Pacific Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy of Sciences, Russia Jung-Hye Choi1*, Min-Soo Kim1,2, Mi-Ja Jung1, Seong Woon Roh1,2, Kee-Sun Shin2, and Jin-Woo Bae1 1 Department of Life and Nanopharmaceutical Sciences and Department of Biology, Kyung Hee University, 2University of Science & Technology, BRC, Korea Research Institute of Bioscience and Biotechnology A novel psychrotrophic bacterium, strain HJ103T, was isolated from a marine sponge collected in the East Sea, Korea (also known as Sea of Japan). Cells were Gram-negative, motile by gilding, strictly aerobic, catalase-, and oxidase-positive, rodshaped (0.3-0.5 μm×1.6-6.2 μm) and halophilic. Growth was observed between 5 and 33°C (optimum 26°C), at pH 6.5-9.0 (optimum 8.0) and in the presence of 2.0-3.5% (optimum 2.5%) NaCl. The strain requires Ca2+, Mg2+ and K+ ions for growth. The 16S rRNA gene sequence analysis revealed that strain HJ103T showed high similarity with members of the genus Tenacibaculum (94.4-96.4%), and phylogenetic analysis revealed that strain HJ103T shared a phyletic line with Tenacibaculum maritimum. The major respiratory quinone is MK-6. The DNA G+C content was 31.1 mol%. The major fatty acids were iso-C15:0, iso-C15:0 3-OH, C16:0 3-OH and summed feature 3 comprising C16:1 ω7c and/or iso-C15:0 2-OH. On the basis of this polyphasic taxonomic evidence, strain HJ103T should be classified as a species in the genus Tenacibaculum and the name as Tenacibaculum luteolum sp. nov. is proposed. The type strain is HJ103T (= KCCM 42328T = JCM 14618T). Yellow pigmented bacterium (on LB agar) plate was isolated from landfill soil in Pohang, Republic of Korea and designated as BL03T. Strain BL03T is Gram-negative, aerobic, rod-shaped, motile, oxidase- positive and catalase- negative. Temperature range of the strain is 15 to 42°C, and pH range is 5.0 to 9.5. Growth occurs at 0 to 3% (w/v) NaCl. Predominant ubiquinone of the strain characterized chemotaxonomically as containing Q-10 and the major cellular fatty acids were C17:1ω6c, C15:0 2OH and C18:1ω7c. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain BL03T belongs to the genus Sphingopyxis, showing high sequence similarity to the type strain Sphingopyxis taejonensis JSS54T (97.8%), Sphingopyxis alaskensis RB2256T (97.4%) and Sphingopyxis chilensis S37T (96.9%). The GC content of the DNA strain was 65.9 mol%. Several characteristics served to differentiate this isolate from recognized members of the genus Sphingopyxis. Strain BL03T (KCTC 22405T = JCM 15910T) should be classified as novel species in the genus Sphingopyxis, which the name Sphingopyxis soli sp. nov., is proposed. [Supported by MEGRC] A026 Agromyces atrinae Fermented Seafood [Supported by grant from the Eco-technopia 21] A028 sp. nov., Isolated from Screening and Identification of Antibacterial Lactic Acid Bacteria Isolated from Kimchi Eun-Jin Park*, Min-Soo Kim, Mi-Ja Jung, Seong Woon Roh, and Jin-Woo Bae Department of Life and Nanopharmaceutical Sciences and Department of Biology, Kyung Hee University Ji Eun An*, Hyoung Ro Lee, Ji Sun Kim, and Nam Soo Han Department of Food Science and Technology, Chungbuk National University A Gram-positive, aerobic, non-motile and rod-shaped bacterium, designated P27T, was isolated from a traditional fermented seafood. The isolate grew optimally in 0–2.0% (w/v) NaCl, pH 6–7, at 30°C. The predominant menaquinones were MK-12 and MK-11. The major cellular fatty acids were anteiso-C17:0, anteiso-C15:0 and iso-C16:0. The major cell wall sugars were galactose, mannose and rhamnose. The peptideglycan amino acids of strain 27T were 2,4-diaminobutyric acid, alanine, glutamic acid and glycine. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and an unidentified glycolipid. The genomic DNA G+C content of strain P27T was 69.0 mol%. Based on its 16S rRNA gene sequence, strain P27T showed highest pairwise similarity with Agromyces cerinus subsp. cerinus JCM 9083T with 97.0% similarity value. Based on phenotypic, genotypic and phylogenetic studies, strain P27T represents a novel species in the the genus Agromyces, for which the name Agromyces atrinae sp. nov. is proposed. The type strain is P27T (=KCTC 19593T= JCM 15913T). The purpose of this study was to screen and identify the antibacterial lactic acid bacteria from kimchi by using spot-onthe-lawn method. Listeria monocytogenes ATCC 19115, Staphylococcus aureus KCTC 1916, Escherichia coli KCTC 1467 and Salmonella typhimurium KCTC 2515 were used as indicators. Approximately 100 strains showed antimicrobial activity inhibiting growth of indicators on MRS and PEA media, and 21 strains were isolated by their cell morphology, gram stain and high antimicrobial activity. The 21 isolates were identified as Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Weissella cibaria, Lactococcus lactis and Enterococcus faecalis by 16S rRNA gene sequencing. [Supported by grant from TDPAF] 141 www.msk.or.kr A029 A031 Community Structure of Culturable Bacteria Associated with Sponge, Penares incrustans Mucilaginibacter dorajii sp. nov., a Novel Bacterium Isolated from the Platycodon grandiflorum Il-Gyo Jang* and Jin-Sook Park Department of Biotechnology, Hannam University Kee-Sun Shin*, Byung-Chun Kim, Kang Hyun Lee, and Mi Na Kim Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology A cultivation-based approach was employed to determine the culturable bacterial diversity associated with marine sponges, Penares incrustans. The diversity of culturable bacteria collected from seawater which the marine sponge is inhabiting, was also analyzed as control. The bacteria associated with sponge were cultivated using Zobell medium for 3 days in 25°C incubation. Community structures of the culturable bacteria were analyzed with PCR-RFLP based on 16S rDNA sequences. The sequence analysis of each RFLP type showed >97% similarity to known bacterial species in public databases. Overall, the microbial populations of Penares incrustans sponges investigated were found to be the members of the classes; Alphaproteobacteria (29.0%), Gammaproteobacteria (64.5%), and Actinobacteria (6.5%). The dominant bacterial group of seawater was also found to be the members of the Gammaproteobacteria. But, the bacterial community of seawater showed to be more simple than that of the sponge. In bacterial species composition of Gammaproteobacteria, Vibrio (43%) and Pseudoalteromonas (60.6%) were dominant in Penares incrustans and seawater respectively. A030 Community Analysis of Culturable Associated with Sponge, Asteripus sp. A032 Bacteria Choon-Soo Im* and Jin-Sook Park Department of Biotechnology, Hannam University A cultivation-based approach was employed to determine the culturable bacterial diversity associated with marine sponges, Asteripus sp. The diversity of culturable bacteria was also analyzed as control. The bacteria associated with sponge were cultivated using Zobell medium for 3 days in 25°C incubation. Community structures of the culturable bacteria were analyzed with PCR-RFLP based on 16S rDNA sequences. The RFLP fingerprinting of 16S rDNA digested with HaeIII and MspI, revealed 12 and 8 independent RFLP types from the sponge species and seawater respectively. The sequence analysis of each RFLP type showed >96% similarity to known bacterial species in public databases. Four other strains were potential candidates for new species. The dominant microbial populations of Asteripus sp. were found to be the members of the Alphaproteobacteria (78.5%). But, predominant microbial group of seawater was Gammaproteobacteria (81.8%). The bacterial community of the sponge and seawater indicated similar composition at phyla level, revealing difference in dominant bacterial species. MspI was more useful than HaeIII in PCR-RFLP analyzing for bacterial diversity of marine sponge, Asteripus sp. 142 Poster Sessions A Gram-negative, yellow-pigmented, rod-shaped bacterial strain, DR-f4, was isolated from the rhizosphere of Platycodon grandiflorum in a study of bacterial diversity in the rhizosphere, and its taxonomic position was investigated by a genotypic and phenotypic analysis. It grew 10-30°C, optimally at 20-25°C, and in the presence of 0–1% (w/v) NaCl. It contained MK-7 as the predominant menaquinone. It represented positive activity for catalase, oxidase, β-galactosidase and hydrolysis of esculin. The major cellular fatty acids were iso-C15:0 2OH/C16:1ω7c and iso-C15:0. The DNA G+C content was 42.6 mol%. This isolate belonged to the genus Mucilaginibacter based on phylogenetic analysis using 16S rRNA gene sequences. The nearest phylogenetic neighbours of strain DR-f4T were T Mucilaginibacter lappiensis ANJL12 and Mucilaginibacter rigui WPCB113T, with 16S rRNA gene sequence similarity levels of 96.9% and 96.4%, respectively. The genetic and phenotypic evidences suggest that strain DR-f4T should be classified as a novel species, for which the name Mucilaginibacter dorajii sp. nov. is proposed. The type strain for the novel species is DR-f4T (=KACC 14556T = CECT 7660T). Dietzia alimentaria sp. nov., Isolated from a Traditional Korean Food Jandi Kim*, Seong Woon Roh, Mi-Ja Jung, Min-Soo Kim, Eun-Jin Park, and Jin-Woo Bae Department of Biology, Kyung Hee University An actinobacterial strain 72T was isolated from a traditional salt-fermented seafood in Korea. Colonies were coral-red, and cells were Gram-positive, non-motile and rod shaped. Strain 72T grew in 0–10% (w/v) NaCl, at pH 7–10 and a temperature of 15-37˚C. Optimum growth conditions were 2% NaCl, pH 7.0 and 30˚C. Phylogenetic analysis indicated that strain 72T belonged to the genus Dietzia. The major cellular fatty acids were C16:0 (33.2%) and C18:1 ω9c (17.1%), both of which are characteristic of members of the genus Dietzia. Analysis of the 16S rRNA gene sequences and DNA-DNA hybridization, coupled with physiological and biochemical tests, determined genotypic and phenotypic differences between strain 72T and other members of the genus. Based on these data, strain 72T is a novel species of the genus Dietzia, and we propose the name Dietzia alimentaria sp. nov. The type strain is 72T (= JCM 16360T = KACC 21126T). A033 Algoriphagus jejuensis, sp. nov., Isolated from Seawater Ki Young Lee1*, Dong-Heon Lee1, Hyung-Yeel Kahng2, and Sun Bok Lee1,3 1 Department of Chemical Engineering, Pohang University of Science and Technology, 2Department of Environmental Education, Sunchon National University, 3Gyungbuk Sea Grant Institute, Pohang University of Science and Technology A Gram-negative, pink-pigmented, non-motile, strictly aerobic, rod-shaped bacterium, designated CNU040T, was isolated from seawater on the coast of Jeju Island in Korea, and subjected to a polyphasic taxonomic study. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CNU040T belongs to a distinct lineage in the Algoriphagus, with high sequence similarity to Algoriphagus terrigena DS-44T (98.3%). DNA-DNA relatedness values between the strain CNU040T and Algoriphagus terrigena KCTC 12454T were 44.5%. The DNA G+C content of the strain was 48.5 mol% and the major respiratory quinone was menaquinone-7. The major cellular fatty acids were iso-C15 : 0 (28.6%), summed feature 3 (iso-C15:0 2-OH/C16:1ω7c, 24.0%) and iso-C17:0 3-OH (4.7%). On the basis of phenotypic, phylogenetic and genotypic data, strain CNU040T represents a novel species within the genus Algoriphagus, for which the name Algoriphagus jejuensis sp. nov. is proposed. The type strain is CNU040T (=KCTC 22647T = JCM 16112T ). [This work was supported by the 21C Frontier Microbial Genomics and Applications Centre Program, Ministry of Education, Science & Technology, Korea]. A034 A035 Kopria litus gen. nov., sp. nov., of the Family Oxalobacteraceae, Isolated from Antarctic Coastal Seawater Eun Hye Kim1,2*, Hyun-Jeong Jeong1, Yoo Kyoung Lee1, Eun Young Moon3, Jang-Cheon Cho2, Soon Gyu Hong1, and Hong Kum Lee1 1 Polar BioCenter, Korea Polar Research Institute, Korea Ocean Research & Development Institute, 2Division of Biology and Ocean Sciences, Inha University, 3Institute of Microbiology, Seoul National University A Gram-negative, non-motile, catalase- and oxidase- positive, strictly aerobic and short-rod-shaped bacterium that was designated strain KOPRI 25157T was isolated from coastal seawater sample in around King Sejong Station, in King George Island, Antarctica. The temperature and pH ranges for growth on R2A agar were 10-20°C, and 5.0-10.0, respectively. Phylogenetic analyses of the 16S rRNA gene sequence of strain KOPRI 25157 T have shown that it belongs to the family Oxalobacteraceae of the class Betaproteobacteria, but it formed a distinct clade from other recognised members of the family. Major ubiquinone was Q-8. Predominant cellular fatty acids were C10:0 (0.2%), C10:0 3OH (3.2%), C12:0 (2.9%), C16:1 ω7c/15 iso 2OH (56.4%), C16:0 (30.5%), C18:1 ω7c (3.7%), C18:0 (0.7%), and 11 methyl C18:1 ω7c (2.4%). On the basis of these data, it is proposed that strain KOPRI 25157T is the representative of a novel genus, named Kopria gen. nov. is proposed in the family Oxalobacteriaceae. The type strain for Kopria litus sp. nov. is KOPRI 25157T (=JCM 16673T =KCTC 23040 T). A036 Notes on the Existence of Leucodecton desquamescens (Thelotremoid Graphidaceae) in South Korea Caenimonas terrae sp. nov., Isolated from a Soil Sample in Korea Yogesh Joshi*, Young Jin Koh, and Jae-Seoun Hur Korean Lichen Research Institute, Sunchon National University Soo-Jin Kim1*, Hang-Yeon Weon2, Yi-Seul Kim2, Jung-Im Bok2, and Soon-Wo Kwon2 1 Korean Agricultural Culture Collection, Agro-biodiversity Center, National Academy of Agricultural Science, Rural Development Administration, 2Korean Agricultural Culture Collection, Agro-biodiversity Center, National Academy of Agricultural Science, Rural Development Administration The presentation describes one new record of thelotremoid lichen (Leucodecton desquamescens) from South Korea, which is characterized by thick, bulging thallus with many calcium oxalate crystal inclusions, the immersed, round to irregular ascomata with free exciple, ellipsoid to ±roundish submuriform, brown ascospores and lack of secondary metabolites. A detailed taxonomic description and comments are presented for the taxa studied. Lichen genus Leucodecton is reported for the first time for this country. A white-coloured bacterium, SGM1-15T, was isolated from a paddy soil sample from Suwon, Republic of Korea. The cells were strictly aerobic, Gram-negative and curved rod-shaped (0.6-0.7×1.4-2.3 μm). A phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SGM1-15T was closely related to Curvibacter delicatus LMG 4328T (97.6% similarity) and Caenimonas koreensis EMB320T (97.5% similarity). The major respiratory quinone system was Q-8 and the predominant cellular fatty acids were C16:0 (39.9%), summed feature 3 (C16:1 ω7c and/or iso-C15:0 2-OH; 24.3%) and C17:0 cyclo (22.7%). The DNA G+C content was 68.7 mol%. On the basis of the phylogenetic, physiological and chemotaxonomic data, stain SGM1-15T represents a novel species of the genus Caenimonas, for which the name Caenimonas terrae sp. nov. is proposed. The type strain of Caenimonas terrae is SGM1-15 (=KACC 13365T=NBRC 106341T). [Supported by grants from RDA] 143 www.msk.or.kr A037 A039 Cohnella soil sp. nov. and Cohnella terrae sp. nov. Isolated from Two Different Soil Samples in Korea Psychroserpens gangjinensis sp. nov., Isolated from Seawater Soo-Jin Kim*, Hang-Yeon Weon, Yi-Seul Kim, In-Cheol Park, and Soon-Wo Kwon Korean Agricultural Culture Collection, Agro-biodiversity Center, National Academy of Agricultural Science, Rural Development Administration Young Sun Lee1*, San Ho Shon1, Jae Sung Oh1, Dong-Heon Lee2, Hyung-Yeel Kahng2, and Jae Sung Jung1 1 Department of Biology, Sunchon National University, 2 Department of Environmental Education, Sunchon National University Two bacterial were isolated from soil samples in Korea, strains YM2-7T and WD2-19T. The cells were strictly aerobic, Grampositive, motile with peritrichous flagella and rod-shaped. YM2-7T and WD2-19T showed 16S rRNA gene sequence similarities of 93.2-96.0% to type strains of recognized Cohnella species. The G+C contents of the DNA of strains YM2-7T and WD2-19T were 52.2 and 55.6 mol%, respectively. Major fatty acids of strain YM2-7T are anteiso-C15:0 (44.4%), C16:0 (19.2%) and iso-C16:0 (16.8%), and the major fatty acids of strain WD2-19T are anteiso-C15:0 (46.5%), iso-C16:0 (21.8%) and C16:0 (11.2%). Both strains contained MK-7 as the predominant quinone. Both strains had diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and lysylphosphatidylglycerol as the major polar lipids. Comparative analysis of phenotypic and phylogenetic traits indicated that strains YM2-7T and WD2-19T represented two novel species of the genus Cohnella. The names Cohnella soli sp. nov. (type strain YM2-7T = KACC 13346T = NBRC 106486T), and Cohnella terrae sp. nov. (type strain WD2-19T = KACC 13347T= NBRC 106485T) are proposed for these organisms. [Supported by grants from RDA] Gram-negative, catalase and oxidase-positive, aerobic, yellow pigmented strain, K7-7T, was isolated from sea water of the Gangjin bay (34°32'58.0"N, 126°46'15.7"E) in Korea. Based on 16S rRNA gene sequence comparisons, the novel strain was closely related to genera Bizionia (96.192-93.626%), Psychroserpens (95.551-95.326%), and Winogradskyella (95.05693.922%) of the family Flavobacteriaceae. According to Phylogenetic analyses, K7-7T 16S rRNA showed similarity with Bizionia, but it was more distantly related with genera Psychroserpens. It grew at 10-37°C (Optimum 30°C), at pH 78 and with 2-6% NaCl (Optimum 3%). Strain K7-7T was able to hydrolysis Casein, Tween20, tween60, Tween80, DNase. It is contained C15:0 ISO (31.2%), C15:1 ISO G (21.1%) and C17:0 ISO 3-OH (17.2%) as major fatty acid. The DNA G+C contents was 37.48 mol%. On the basis of polyphasic taxonomic data, strain K7-7T (=KCTC22734T = JCM16151T) should be classified as the type strain of a novel species within the genus Psychroserpens, for which the name Psychroserpens gangjinensis sp. nov. is proposed. A038 Thalassobacter haliotis sp. nov., Isolated from Abalone (Haliotis discus) Young Sun Lee1*, San Ho Shon1, Dong-Heon Lee2, Hyung-Yeel Kahng2, and Jae Sung Jung1 1 Department of Biology, Sunchon National University, 2 Department of Environmental Education, Sunchon National University A rod-shaped, yellow-pigment, aerobic, Gram-negative bacterium, strain JA14T, was isolated from the Haliotis discus collected from sea of Wando-island(34°25'30.88"N, 126°56' 23.37"E), Korea. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain JA14T shared 95.945%95.032% sequence similarity with species of the genus Thalassobacter, and also Roseovarius (95.752-92.939%), Pseudoruegerian (95.661-95.608%), Celeribacter neptunius (95.326%) and Sulfitobacter (95.090-93.160%). However, Phylogenetic analyses showed that it was most closely related to genena Thalassobacter. It grew at 10-37°C (Optimum 25°C), at pH 6-11 (Optimum 6) and with 1-4% NaCl (Optimum 1). Strain JA14T was able to hydrolysis tween60. Major fatty acids were C15:0 ISO (36.7%), C15:1 ISO G (12.4%), C17:0ISO 3-OH (8.4%), C16:0 (6.2%), C15:0 ISO 3-OH (5.7%) and C15:0 ISO 2OH and/or C16:1 (5.7%). The DNA G+C contents was 28.74 mol%. These data, as well as phylogenetic analyses, suggest that strain JA14T (=KCTC22732T =JCM16145T) should be classified as the type strain of a novel species within the genus Thalassobacter, for which the name Thalassobacter haliotis sp. nov. is proposed. 144 Poster Sessions A040 Genetic Diversity of Pseudomonas syringae pv. morsprunorum Strains Isolated from Prunus mume and Relatedness with Other Pathovars Based on the Comparative Sequence Analysis of the 16S rRNA Gene Young Sun Lee1*, Young Jin Koh2, and Jae Sung Jung1 1 Department of Biology, Sunchon National University, 2 Department of Plant Medicine, Sunchon National University Genetic diversity among Pseudomonas syringae pv. morsprunorum strains isolated from Prunus mume in Korea and Japan was investigated by comparative sequence analysis of the 16S rRNA gene. The strains included 24 field isolates recovered in Korea and 7 Japanese isolates. Two strains isolated from P. salicina in Japan, one strain from P. avium in United Kingdom and the pathovar reference strain isolated from Prunus domestica were also used for comparison of 16S rRNA gene sequence. A nearly complete sequences of the 16S rRNA genes were sequenced in 35 strains, and three types of sequence, designated Group I, II and III, were identified. The pathovar reference strain and 19 Korean strains belonged to Group I, while 5 Korean, 5 Japanese strains and one strain from United Kingdom belonged to Group II. Another 4 Japanese strains belonged to Group III. Five Korean strains belonging to Group II were isolated from only five among 24 orchards investigated. When the nucleotide sequences of the 16S rRNA genes of P. syringae pathovars were aligned, the sequence polymorphism was found to be focused on the variable regions, which were shown in pv. morsprunorum population. A041 Reclassification of Paenibacillus ginsengisoli as a Later Heterotypic Synonym of Paenibacillus anaericanus Kwang Kyu Kim*, Keun Chul Lee, and Jung-Sook Lee Korean Collection for Type Cultures, Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology The type strains of the species Paenibacillus ginsengisoli (KCTC 13931T) and Paenibacillus anaericanus (DSM 15890T) were compared in order to clarify the taxonomic relationship of the two species. On the basis of 16S rRNA, gyrB and rpoB gene sequence comparisons, the two strains shared 99.9, 100 and 99.4% similarity, respectively. The mean DNA-DNA relatedness value was 71% and the genomic DNA G+C contents were 42 and 41 mol%, respectively. Phenotypic data, including fatty acid patterns and acid production, enzyme activity and substrate utilization profiles, showed no pronounced differences between the type strains of the two species. These polyphasic data suggest that the two taxa constitute a single species. According to Rules 38 and 42 of the Bacteriological Code, they should be united under the name Paenibacillus anaericanus, with the name Paenibacillus ginsengisoli as a later heterotypic synonym. [Supported by a grant from the KRIBB Research Initiative Program] A042 A043 Microvirga aerophila sp. nov. and Microvirga aerilata sp. nov. Isolated from Air and Reclassification of Balneimonas flocculans Takeda et al. 2004 as Microvirga flocculans comb. nov., and Emended Description of the Genus Microvirga Hang-Yeon Weon1*, Soon-Wo Kwon1, Jung-A Son1, Eun-Hye Jo1, Soo-Jin Kim1, Yi-Seul Kim1, Byung-Yong Kim1, and Jong-Ok Ka2 1 Korean Agricultural Culture Collection, National Agrobiodiversity Center, National Academy of Agricultural Science, Rural Development Administration, 2Department of Agricultural Biotechnology, Seoul National University Two bacterial strains 5420S-12T and 5420S-16T isolated from air samples, were characterized using a polyphasic approach. 16S rRNA gene sequence analysis showed that strain 5420S-12T was phylogenetically related to Microvirga subterranea Fail4T (97.4% sequence similarity) and Microvirga guangxiensis 25BT (97.1% similarity), and strain 5420S-16T was closely related to Balneimonas flocculans TFBT (98.0% sequence similarity) and Microvirga guangxiensis 25BT (97.2% similarity). The G+C content of the genomic DNA was 62.2 mol% for strain 5420S-12T and 61.5 mol% for strain 5420S-16T. The results of DNA-DNA hybridization and phenotypic data showed that strains 5420S-12T and 5420S-16T could be distinguished from their phylogenetically related species, and represent two novel species within the genus Microvirga, for which the names Microvirga aerophila sp. nov. (type strain 5420S-12T = KACC 11743T = NBRC 106136T) and Microvirga aerilata sp. nov. (type strain 5420S-16T = KACC 11744T = NBRC 106137T) are proposed. Furthermore, the reclassification of Balneimonas flocculans as Microvirga flocculans comb. nov. is proposed and also emended description of the genus Microvirga is provided. A044 Bacillus composti sp. nov. and Bacillus suwonensis sp. nov., Isolated from Cotton Composts The First Report of Two Species of Polyporus (Polyporaceae, Basidiomycota) from South Korea Yi-Seul Kim1*, Soo-Jin Kim1, Rangasamy Anandham2, Seung-Hee Yoo1, Hang-Yeon Weon1, and Soon-Wo Kwon1 1 Korean Agricultural Culture Collection, Agro-biodiversity Center, National Academy of Agricultural Science, Rural Development Administration, 2Department of Agricultural Microbiology, Agricultural College and Research Institute, India Eun Ju Woo1*, Jin Sung Lee1, Kyoung Hee Oh1, Jae-Jin Kim2, and Young Woon Lim1 1 National Institute of Biological Resources, Environmental Research Complex, 2Division of Environmental Science and Ecological Engineering, College of Life Science and Biotechnology, Korea University Two bacterial isolates from cotton composts in Korea, designated strains 5M44T and 5M55T, were characterized using a polyphasic approach. The cells were strictly aerobic, Grampositive, motile, spore-forming and rod-shaped. Phylogenetic analysis of their 16S rRNA gene sequences revealed a clear affiliation with the genus Bacillus. Strain 5M44T showed the highest sequence similarities with Bacillus massiliensis 4400831T (97.3%) and Lysinibacillus xylanilyticus XDB9T (97.2%), and the 16S rRNA gene sequence of 5M55T revealed more than 97% with Bacillus massiliensis 4400831T (97.6%). The G+C contents of the DNA of strains 5M44T and 5M55T were 35.3 and 35.9 mol%, respectively. Major fatty acids (>10%) of strain 5M44T were iso-C15:0 (38.0%), anteiso-C15:0 (28.4%) and iso-C16:0 (10.2%), and those of strain 5M55T were iso-C15:0 (30.8%) and iso-C16:0 (18.1%). The major isoprenoid quinone of both strains was menaquinone 7 (MK-7). Comparative analysis of phenotypic and phylogenetic traits indicated that strains 5M44T and 5M55T represented two novel species of the genus Bacillus. Based on morphological examination, two species of Polyporus, P. dictyopus and P. tuberaster, were identified, which constitutes the first record of these species in South Korea. To confirm their affinity within the genus Polyporus, the phylogenetic relationships of Polyporus and allied genera were established from nuclear large subunit ribosomal DNA (nLSU rDNA) sequences, and a morphological diagnostic key is presented to clarify the Korean species of Polyporus. [Supported by the Korean indigenous species research project from NIBR.] [Supported by grants from RDA] 145 www.msk.or.kr A045 Characterization of Highly Unstable Phage SPG24 of Bacillus subtilis G24 and Its Long-Term Survival Strategy in the Nature Yu Ji Jang1*, Yong Sin Park2, Tae-Sook Kang3, and Oh Hyoung Lee1 1 Department of Life Science, Mokpo National University, 2 Medical Laboratory, Mokpo City Medical Center, 3Department of Biomedical Science Laboratory, Mokpo Science College A phage SPG24 was isolated from deteriorating Chungkookjang using Bacillus subtilis G24 as host. B. subtilis G24 has been isolated from grape to be used as soybean-fermenting agent into Korean traditional food Chungkookjang. This phage contains double-stranded DNA and seems to be a noble subtilis phage because either a 400 base-pair SmaI fragment or a 850 base-pair EcoRV fragment of phage DNA was found to have no match in Blast nucleotide sequence comparison. This phage is belonging to group AI phages of B. subtilis according to its morphology. This phage is highly virulent because it can cause a rapid lysis of actively growing hosts after infection. Although SPG24 is very unstable when it is exposed to outside of its host, it can survive long in nature by hiding its chromosome in the host spore and taking advantage of spore’s persistency. In spite of its high virulency, this phage never causes a complete demolition of its hosts in any growth volume. This would be the reason why both virus and its host can survive together until now evolutionally. 146 Poster Sessions B001 B003 Physiological and Metabolic Responses for Alkane Degradation in Acinetobacter sp. Strain DR1 Genetic Diversity of Escherichia coli Obtained from Yeongsan River Jaejoon Jung* and Woojun Park Division of Environmental Science and Ecological Engineering, Korea University Jeonghwan Jang1*, Tatsuya Unno1, Yaesul Suh1, and Hor-Gil Hur1,2 1 Department of Environmental Science and Engineering, Gwangju Institute of Science and Technology, 2International Environmental Research Center, Gwangju Institute of Science and Technology The hexadecane degradation of Acinetobacter sp. strain DR1 was evaluated with changes in temperature and ionic salt contents. Hexadecane degradation of strain DR1 was reduced markedly by the presence of sodium chloride (but not potassium chloride) and high temperature (37°C) was also shown to inhibit the motility, biofilm formation, and hexadecane biodegradation. The biofilm formation of strain DR1 on the oil-water interface might prove to be a critical physicological feature for the degradation of hexadecane. The positive relationship between biofilm formation and hexadecane could be expected with changes in salts at 30°C, but not at low temperatures (25°C). Alterations in cell hydrophobicity and EPS production by temperature and salts were not correlated with biofilm formation and hexadecane degradation. The results of proteomic analyses have demonstrated that proteins involved in fatty acid oxidation, the glyoxylate pathway, and gluconeogenesis are highly upregulated and many oxidative stress proteins have been identified as important for the efficient biodegradation of hexadecane. B002 Surface water samples were collected every month from 13 sites of Yeongsan river watershed from April to December 2009, and 60 E. coli isolates were obtained from each water sample being doubted about fecal pollution. All E. coli isolates were genotyped by Horizontal Fluorophore-Enhanced RepPCR DNA fingerprinting technique (HFERP) and discriminated at the strain level, then we surveyed the strain composition of E. coli depending on each month and each site. Overall, there was less diversity in isolates collected during winter season compared to the other seasons. Average of percentage of unique strain among isolate data sets on October, November and December were below 30%, but over 50% from April to September. Moreover the survey of genetic difference between E. coli isolates obtained from surface waters and sediments sampled on August and November 2009 was conducted and interesting results were drawn, indicating that there was no exchange between E. coli communities of surface water and sediment on August, but E. coli of surface water affected on the opposite side on November. B004 Comparing Microarrays and Next Generation Sequencing for Microbial Ecology Research Food Waste Treatment by Using Thermophilic Bacteria Seong Woon Roh1*, Guy Abell2, Kyoung-Ho Kim1, Young-Do Nam1, and Jin-Woo Bae1 1 Kyung Hee University, 2CSIRO You-Jung Jung*, In-ho Chang, Ahnna Cho, Dawoon Jung, and Tae-Seok Ahn Department of Environmental Science, Kangwon National University Recent advances in molecular biology have resulted in the application of DNA microarrays and next generation sequencing (NGS) technology to the field of microbial ecology. This review aims to: examine the strengths and weaknesses of each of the methodologies, including depth of analysis, throughput, ease of data analysis and cost effectiveness; highlight the optimal application of each of the individual technologies to the study of a particular environment; and identify potential synergies between the two technologies, whereby both sample number and coverage can be maximized. We suggest that the efficient use of both microarray and NGS technologies will allow researchers to advance the field of microbial ecology and allow an improved understanding of the role of microorganisms in their environment. [Supported by grants from the Environmental Biotechnology National Core Research Center, TDPAF, NMC0300837, CAER, 21C Frontier Microbial Genomics and Application Center Program, Eco-Technopia 21 project, KFDA and the Office of the Chief Executive, CSIRO.] For food waste treatment, we used thermophilic bacteria isolated from humus leaf layer. Bacteria could be cultured at 45°C and their enzyme activities were characterized. And the bacteria were identified based on their 16S rRNA gene sequences. Then we applied those bacteria to food waste treatment in pilot scale on various conditions. After than we did it in real machine. Fourteen strains of isolated bacteria were selected, based on their high enzyme activity. Those bacteria were classified as family Bacillaceae, genus Bacillus and genus Brevibacillus. In pilot scale, removal rate of food waste was 46.1%/day. Optimal humidity was 75% for those bacteria. Total bacterial number in treatment process was about 108 cells/g and pH about 4.5. In real machine, average of removal rate was 96%/day in the best condition. [This research was supported by Brain Korea21] 147 www.msk.or.kr B005 B007 Korean Human Gut Microbiota Revealed by Barcoded Pyrosequencing Flagella Motility is Involved in the High Current Production of Geobacter sulfurreducens KN400 Young-Do Nam1,2*, Min-Soo Kim1,2, Kyoung-Ho Kim1, and Jin-Woo Bae1 1 Department of Biology, Kyung Hee University, 2These authors contributed equally to this work Hana Yi*, Hoa Tran, Peng Zhou, Toshiyuki Ueki, Kengo Inoue, Ashley E. Franks, and Derek R. Lovley University of Massachusetts Amherst, USA Human gut microbiota plays important roles in energy harvest from diet, stimulation of intestinal epithelium, development immune system, and regulation of fat storage of host. Establishment of gut flora, however, has been limited to western people and previously acquired information has not been extensive enough to fully describe human gut microbiota. In this study, we investigated gut microbiota of 21 Korean using 454-pyrosequencing and compared with that of three American, as well as temporal stability of intestinal microbiota. Total 390,291 read sequences covering V1 to V3 hypervariable regions of the 16S rDNA gene were analyzed. Overall shape of microbial communities is dominated with previously mentioned four phyla: Firmicutes, Bacteroidetes, Fusobacteria, and Proteobacteria. PcoA (principal coordinate analysis) showed that gut microbiota of Korean and American differed from each other suggesting that genetic variation might affect the composition of microbial community. Weighted and unweighted clustering analysis in Fast UniFrac revealed that the composition of gut microbiota is maintained, but relative abundance in microbial community is changed for 2 months test period. B006 In a recent study, a newly isolated Geobacter sulfurreducens strain, designated as KN400, that dramatically increases power output than earlier genome sequencing strain (DL1) was reported. It was suggested that the enhanced current producing capacity of strain KN400 could be originated from the changes in outer surface components like as pili, flagella and exopolysaccharides. To evaluate the effect of motility system of strain KN400 in its increased power production ability, knockout mutants of flagella regulator, flagella hook protein and flagella motor protein were constructed and characterized. The resulted aflagellate and flagellated but nonmnotile mutants showed remarkably slower and lower current production in microbial fuel cell compared to wild type KN400. The pili and outersurface c-type cytochromes analysis also supported the hypothesis that the defective electrode reducing ability of nonmotile mutants was not coming from pili or cytochrome difference but from the absence of flagella motility. B008 Change of Total Bacterial Number in Nutrient Concentrating System Launched in Lake Soyang Formation and Localization of Tellurium Nanocrystals in Shewanella spp. In-Ho Chang1*, Eun-Young Seo1, You-Jung Jung1, Seung-Ik Choi2, and Tae-Seok Ahn1 1 Department of Environmental Science, Kangwon National University, 2Institute of Environmental Research, Kangwon National University Dong-Hun Kim1* and Hor-Gil Hur1,2 1 Department of Environmental Science and Engineering, 2 International Environmental Research Center, Gwangju Institute of Science and Technology Nutrients (nitrogen and phosphate) are necessary for plant growth but are dissolved as low state in aquatic ecosystem. For extracting nutrients from oligo-mesotrophic lakes, we invented new devices with hydrophobic media, such as sponge and rubberized coconut fiber (RCF). These devices were launched at Lake Soyang on March, 2009, and then pore water of both media and lake water were retrieved daily for 3 days and total bacterial number and nutrient concentrations were measured. Total bacterial numbers, nitrogen and phosphate concentration in RCF were 4.3~8.4 × 106 cells/ml, 4.3 mg/l, 0.35 mg/l which were about 11~26, 2, 4 and 2~3, 1.5, 2 times higher than those of lake water (control) and sponge (comparative case), respectively. These results suggest that RCF media have excellent ability to accumulate the nutrient in the lake water and bacteria are playing an important role for accumulating nutrient in oligo-mesotrophic lake. [Supported by grants from ECO-STAR project] 148 Poster Sessions Shewanella species are gram-negative facultative anaerobes capable of utilizing a broad range of electron acceptors, including several solid substrates such as Fe(III) and Mn(IV) oxides. Shewanella species can reduce tellurite [TeO32-] to elemental tellurium [Te(0)] under anaerobic conditions. This reduction results in the formation of unique crystalline Te(0) nanoarchitechtures as end products. The needle-like shaped granules of element tellurium are localized in the cytoplasm or near the cytoplasmic membrane, and each species forms a similar structures. Several different S. oneidensis MR-1 deletion mutants which are related with electron transfer cytochrome c genes, produced tellurium nanostructures. However, they are show different reduction rates. These results indicate that deleted genes play a significant role and different mechanisms are related in tellurite reduction. [Support for this work from the 21C Frontier Microbial Genomics and Applications Center Program (M102KK01001108K1101-01110), Ministry of Education, Science and Technology, Republic of Korea.] B009 B011 Comparison of the Bacterial Community in Drinking Water Purification Systems Isolation of Plant Growth Promoting Bacteria from Rhizosphere of Wild Plant at Barren Soil Ok-Sun Kim*, Yunmin Kim, Kihyun Lee, Suk-Hwan Yoon, Mincheol Kim, and Jongsik Chun School of Biological Sciences, Seoul National University Kyung-Mi Kim* and Hong-Gyu Song Department of Biological Science, Kangwon National University Despite the importance of its implication for human health, the bacterial community composition in drinking water purification systems has not well described. In the present study, we compared bacterial communities in 20 different systems based on 16S rRNA gene by traditional cultivation and barcoded pyrosequencing. Sphingomonas spp., Staphylococcus spp., Mycobacterium spp., and Blastobacter spp. were isolated with the sequence similarity of 98.1 to 100.0%. Although the bacterial diversity in this habitat was relatively low compared to natural ones, 19 known divisions and 18 unknown divisions were detected, where Alphaproteobacteria with 54.8% were overwhelmingly dominant. We also reveal the presence of bacterial families with the members including environmental relatives retrieved from diverse environments such as different types of soil habitats, drinking water biofilm, freshwater habitats and the atmosphere. Finally, we expect that complex environmental factors could affect shaping the microbiological composition of this environment. In conclusion, bacterial community in the water purification system can be readily monitored by pyrosequencing of 16S rRNA gene. B010 This study was conducted with some rhizobacteria able to exert beneficial effects upon plant growth. Many bacteria were isolated from the rhizosphere of a wild plant Isachne globosa (Thunb.) O. Kuntze, and among them two strains (AG and ED) of plant growth promoting rhizobacteria (PGPR) were selected. They could solubilize 531.5 mg l-1 of 0.5% insoluble phosphate Ca3(PO4)2 in 3 days of incubation. Productions of indole-acetic acid (IAA) by strain AG and ED under the addition of tryptophan were 2.4 and 1.0 mg mg protein-1, respectively. Strain AG and ED also produced other phytohormones, such as gibberellin, abscisic acid (ABA) which were measured by HPLC. They produced 270.1 and 271.7 mg l-1 of gibberellin, respectively. Isolated bacteria were applied to the germination test of wild plant in soil. Strain ED could enhance the germination rate of seed of Abutilon avicennae by 26.6%. Their plant growth promoting capabilities were examined in the pot and microcosm composed of barren soil collected from lakeside of Lake Paro, Kangwon-do. These results suggest that the isolated strains of PGPR may be utilized as environmentally friendly biofertilizer for the revegetation of barren lands. B012 Isolation and Characterization of Glycerol-Utilizing and Solventogenic Anaerobic Bacteria Plant Growth Promotion by Isolated Strains of Enterobacteria and Streptomyces Jae-Hyung Ahn*, Chuloo Moon, Kyung-Duk Kim, Byoung-In Sang, and Youngsoon Um Clean Energy Center, Korea Institute of Science and Technology Young-Mi Kim* and Hong-Gyu Song Department of Biological Science, Kangwon National University Glycerol is a byproduct of bioethanol and biodiesel production processes and can be converted to more valuable products such as ethanol, butanol, 1,3-propanediol, and 2,3-butanediol by various anaerobic bacteria. In this study, we isolated glycerolutilizing anaerobic bacteria to obtain ones having a superior ability to produce butanol. By adding acetic and butyric acids into the mineral medium containing glycerol, we could enrich butanol-producing bacteria from soil samples. Isolates showed >99% 16S rRNA gene similarities with Clostridium diolis/ beijerinckii (group 1), C. butyricum (group 2), C. arbusti (group 3), and Klebsiella oxytoca (group 4). The isolates belonging to group 3 produced up to 9.3 g/L of butanol and the isolates belonging to group 2 produced up to 17.1 g/L of 1,3propanediol from 30 g/L of glycerol. When glucose was used instead of glycerol, the isolates belonging to group 1 produced 12.0 g/L of butanol and 6.9 g/L of ethanol and the isolate of group 4 produced 21.0 g/L of 2,3-butanediol and 8.0 g/L of ethanol from 60 g/L of glucose. This result shows the diversity of glycerol-utilizing anaerobic bacteria and their potential for industrial application. Plant growth promoting bacteria have a number of mechanism of growth enhancement such asvariousvarious phytohormone production, phosphate solubilization, inhibition of plant pathogen and others. We isolated enterobacteria Kluyvera intermedia strain GBI and Ewingella americana strain GBL and actinomycetes Streptomyces mirabilis strain GBQ from the soil collected from Mt. Gyebang. These bacteria could produce the plant growth hormones such as indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and gibberellin. They also inhibited growth of Fusarium oxysporum f. sp. lycopersici and Fusarium oxysporum f. sp. niveum which are important pathogenic fungi to many agricultural crops. E. americana strain GBL showed the highest production rates of phytohormones, including 1105.1 µg mg protein-1 of IAA, 1313.9 µg mg protein-1 of IBA and 799.3 mg l-1 of gibberellins (GA3). The lengths of shoot and root of germinated tomato seedling treated with strain GBL increased by 47.09 and 57.12%, respectively in 21 days of incubation compared to the unionculated control. Dry weight of tomato plants grown was also higher in the inoculated soil by 60.75% compared to that in the uninoculated soil. 149 www.msk.or.kr B013 Plant Growth Promoting Capability of Isolated Photosynthetic Bacteria Hyeok-Do Kwon* and Hong-Gyu Song Department of Biological Science, Kangwon National University Environmental concern over conventional agricultural fertilezation and disease control measures has led to increased interest in finding environmentally friendly alternatives. This study was conducted with some photosynthetic bacteria for the plant growth promotion. Some photosynthetic bacteria are able to exert beneficial effects upon plant growth by production of phytohormones. Phytohormones have essential roles in coordination of many growth and behavioral processes in the plant life cycle. Several photosynthetic bacteria were isolated from river sediments. Isolated photosynthetic bacteria, strain JFR cultured in the modified Biebl and Pfenning medium, and productions of indole acetic acid (IAA) and indole butyric acid (IBA) were determined by colorimetric assay with Salkowski’s reagent. Among the isolated bacteria, strain JFR showed the highest production rate of 1388.1 µg mg protein-1 of IAA and 1251.4 µg mg protein-1 of IBA in the modified Biebl and Pfenning medium. The effect of strain JFR on the germination and growth of tomato seedlings were examined in a pot test. This photosynthetic bacterium may be utilized as environmentfriendly biofertilizer in the agriculture. B014 Plant Growth Promoting Effect of Isolated Rhizobacteria and Endophytic Bacteria Sang-Yual Lee* and Hong-Gyu Song Department of Biological Science, Kangwon National University This study was conducted for the plant growth promotion by some isolated rhizobacteria inhabiting the rhizospere of various plants and endophytic bacteria residing within plant hosts without causing disease symptoms. Rhizobacteria were isolated from rhizosphere of mugwort, and endophytic bacteria were isolated from the interior tissue of corn root using nutrient broth medium. Indole-acetic acid (IAA) and indole-butyric acid (IBA) production was determined by Salkowski’s reagent test after cultivation of bacteria in nutrient broth containing tryptophan, precursor of IAA and IBA. Rhizobacterial strain LSY28 showed the highest production rates of 546.8 µg IAA mg protein-1 and 609.2 µg IBA mg protein-1. Isolated endophytic bacteria were tested the antagonistic effects against fungal pathogen. When the bacterial isolates were streaked onto petri plate containing potato dextrose agar medium, strain E23-2-2 showed the highest growth inhibition against Fusarium oxysporum. This study demonstrated that bacterial endophytes may also have beneficial effects on host plants through biological control of plant pathogens. 150 Poster Sessions B015 Findings of Microbial Community Structure and Dominant Species in Soils Polluted with Various Contaminants Jaisoo Kim1*, Sujeong Yang1, Hyunjung Kim2, Jee-Young Kim2, and Sang-Seob Lee2 1 Department of Life Science, Kyonggi University, 2 Department of Biotechnology, Kyonggi University This study is to examine what factors influence microbial community structure by DGGE analysis and comparison using soil samples collected from various contaminated sites. As a result, the similarities based on DGGE band profiles showed the closer relationship in regional properties than in pollution characteristics, probably due to the degree of weak contamination. The highly contaminated samples with oil revealed lower similarities with others in the same region or far relationships with all the other samples in the other regions. Thus the microbial community structure would more be affected by region-based environmental factors than by contamination factors in case of not serious pollution. All the dominant culturable bacterial species were involved in firmicutes or high GC Gram+ in a major portion of soil samples and the highly oil-contaminated samples contained Arthrobacter, Bacillus, Methylobacterium, Clavibacter, Streptomyces and Nocardia as reported genera, and Leifsonia as an unreported genus. B016 Biodegradation by Permeable Reactive Barrier Using The Nano-Bio Hybrid Technology to Remediate BTEX Contaminated Groundwater/Soil Hye-Jung Lee*, Jaisoo Kim, Yoo-Taek Kim, Seung-Gu Kang, and Sang-Seob Lee Kyonggi University Recently developed materials such as nano scale of zero valent iron (NZVI) and emulsified NZVI droplet (ENZVID) have been used in PRB process, but they can be inefficient process that is not practical in many situations. It is believed that the PRB using the nano-bio hybrid technique is the most economical and reliable method of response to this problem. The technique is the fusion technology using the multi-nanopore ceramic carriers and BTEX-degrading bacteria. To immobilize cells, three kinds of carriers (compression molding, foaming molding and circular carrier) were fabricated by pelletizing and press molding methods. The compression molding carrier showed the highest absorption rate among three carriers, 1.30×109, 1.15×108 and 5.05×108 cell/cm3 with P. putida BJ10, E41, and their mixture after 5 days incubation at 28°C, respectively. Immobilized BJ10 and E41 (mixture) could degrade 76.4% of 40 mg/L BTEo-X for 4 days in a batch system and 42.1% of 50 mg/L BTEo-X within 5 hours at 28°C with 1.00×108 cell/cm3 (initial) in a continuous reactor system. Furthermore we are going to experiment on reactor using the waste-made ceramic carrier absorbed with BTEX- degrading bacteria. B017 B019 Heat Shock-Induced Plasmid Conformational Changes in Rhodococcus sp. Strain DK17 Biogenic HgSe Nanoparticles Produced by Shewanella putrefaciens SP200 Kyungsun Kim1*, Sunme Shin1, Hae Youn Park1, Dockyu Kim2, and Eungbin Kim1 1 Department of Biology, Yonsei University, 2Polar BioCenter, Korea Polar Research Institute, Korea Ocean Research & Development Institute Cuong T. Ho1* and Hor-Gil Hur1,2 1 Department of Environmental Science and Engineering, 2 International Environmental Research Center, Gwangju Institute of Science and Technology Rhodococcus sp. DK17 possesses three linear megaplasmids (380-kb pDK1, 330-kb pDK2, and 750-kb pDK3). The alkylbenzene-degrading genes are present on pDK2 while the phthalate operons are duplicated and are present on both pDK2 and pDK3. DK 17 with a growth temperature optimum at 30°C showed no growth at 37°C. When transferred to 30°C, however, the 37°C culture began to grow immediately. This indicates that the temperature of 37°C is not lethal but stressful to DK17. Thus, to investigate the environmental factors affecting the structural stability of its megaplsmids, cells of DK17 were exposed to a heat shock of 37°C. Colonies were randomly picked for colony PCR using primers specific for pDK2 and pDK3, respectively. After screening 200 colonies a total of 30 strains were found to lose at least one of the three genes. PFGE analysis revealed six different cases of plasmid conformational changes: pDK2 loss (9), pDK3 loss (1), pDK2 loss with appearance of a new plasmid (~700 kb)(10), pDK3 loss with appearance of a new plasmid (~400 kb)(8), pDK3 loss with appearance of a new plasmid (~600 kb)(1), and both pDK2 and pDK3 loss with appearance of two new plasmids (~400 and ~700 kb)(1). B018 Gram-negative facultative Shewanella are able to use metals such as U(VI), Fe(III), As(V), Se(IV), Cr(III) as electron acceptors, which have a great potential to apply in the environment technology for heavy metal treatment. Shewanella putrefaciens SP200, the highest resistance strain to Hg (II), in this report was used to investigate the ability of co-removing Hg(II) and Se(IV) from aqueous environment, and the production of HgSe nanoparticles as recovered nanostructure products. This bacterium was incubated with 10 uM of Hg(II) and 100 uM of Se(IV) at 30°C under aerobic condition in defined medium amended with 10 mM of lactate. The SEM and TEM images of samples collected after 5 days confirmed spherical nanoparticles of Se(0) produced by Shewanella putrefaciens SP200, and the EDX data showed the peak of elemental Hg incorporated with Se nanospheres. Our results suggest the possibility of recovering the toxic metals, Hg(II) and Se(IV), as the advanced material by Shewanella putrefaciens SP200. [Support for this work from the 21C Frontier Microbial Genomics and Applications Center Program, Ministry of Education, Science and Technology, Republic of Korea.] B020 Metagenomic Analysis of Uncultured Marine RNA Viral Communities Detection Method and Occurrence of Helicobacter pylori in Water Bandamaravuri Kishore Babu, Gui Hwan Han, Jang Min Park, and Si Wouk Kim* Pioneer Research Center for Controlling of Harmful Algal Blooms (HAB) and Department of Environmental Engineering, Chosun University Eun-Sook Lee*, Gun-Seung Lee, Mok-Young Lee, and Sun-Hee Han Waterworks Research Institute, Seoul Metropolitan Government Algal virus contributes a major role in controlling of algal blooms in coastal environments. In the present study, metagenomic library has been constructed for an estimated average RNA viral genome size of 10 kb at 5X coverage. Total genetic material was extracted from the concentrated viral community and was treated with DNase1 to remove DNA contamination. The pure viral RNA was reverse transcribed using random primers. The resulting cDNA was ligated with EcoR1 adapters and amplified with adapter specific primers. The PCR product of 600 bp-1200 bp size was cloned into TA vector and transformed into E. coli. To check the insert, nearly about 20 primary clones were picked and sequenced. BLAST analysis of the sequences obtained so far showed wide genetic diversity and had no significant homology with the sequences in any database. Further, we are continuing the effort to develop molecular markers to study the distribution and abundance of algal RNA viruses in costal aquaculture environments. We studied the occurrence and method to detect H. pylori in water using real-time PCR and selective cultivation. We collected 103 samples containing surface water at the Han River and wastewater from September 2009 to March 2010. H. pylori were detected in two of 103 environmental water samples using real-time PCR assay and their concentration was 5¯100 and 5.2¯101 cells. We used selective culture technique to investigate the occurrence of live H. pylori for finished water at the six water treatment plants and tap water at the terminal sites of the distribution systems in Seoul. The concentrated samples were spreading on columbia agar containing horse blood and H. pylori-selective supplement and were incubated at 37°C for 7 days under microaerophilic atmosphere condition. Any H.pylori was not detected from 120 finished water and tap water samples. [This study was supported by grants from the Waterworks Research Institute, Seoul Metropolitan Government.] [Supported by the Pioneer Research Program for Converging Technology of the Ministry of Education, Science and Technology, Republic of Korea] 151 www.msk.or.kr B021 B023 Isolation and Identification of Streptomyces sp. AN090571 Producing Quorum Sensing Inhibitor Distribution Patterns of the Members of Phylum Acidobacteria Jong-Ok Jang1,2*, Jin Hee Choi1, Jae-Chan Lee1, Dong-Jin Park1, Chang-Keun Sung2, and Chang-Jin Kim1 1 Korea Research Institute of Bioscience and Biotechnology, 2 Department of Food Science and Technology Chungnam National University Sang-Hoon Lee* and Jae-Chang Cho Department of Environmental Sciences, Hankuk University of Foreign Studies Quorum Sensing (QS) is community genetic regulation mechanism that controls microbiological functions of medical, agricultural and industrial importance. To screen novel quorum sensing inhibitor (QSI) from actinomycestes, well diffusion assay was used with Chromobacterium violaceum (CV026) as a test organism and an isolate designated AN090571 was selected. A comparative 16S rRNA gene sequence analysis revealed that the strain AN090571 was belonged to the genus Streptomyces and closest to Streptomyces misionensis NBRC 13063T with 99.01% of sequence similarity. AN090571 was characterized morphologically and physiologically by the given methods in the international Streptomyces project. It has gray or yellow aerial hypha and showed high growth rate with glucose and rhamnose. To investigate optimal carbon and nitrogen sources for the high production of QSI, 20 carbohydrates and 14 nitrogen sources were tested separately and carbon/nitrogen ratio was selected. [Supported by technology Development Program for Agriculture and Forestry, Ministry of Agriculture and Forestry, Republic of Korea] B022 Unexpectedly High Diversity of Bacterial Community in Continental Shelf Sediment Jin-Kyung Hong* and Jae-Chang Cho Department of Environmental Sciences, Hankuk University of Foreign Studies We investigated the phylogenetic diversity of bacterial community in deep marine sediment. Bacterial 16S rDNA was amplified from community DNA extracted from continental shelf sediment, and a 16S rDNA clone library was constructed. The majority of the cloned sequences belonged to novel taxa, and high diversity at phylum-level was observed. The most dominant members of the clone library were phylogenetically affiliated with Proteobacteria (35%) and minor groups included Deferribacteres (3.8%), Gemmatimonadetes (1.9%), Nitrospira (1.9%), and Planctomycetes (5.7%). These findings demonstrate high diversity of bacterial community in the continental shelf sediment, and expand our knowledge of microbial diversity in deep marine sediment. 152 Poster Sessions The distribution pattern of the phylum Acidobacteria, a previously uncultured bacterial group, was investigated by molecular ecological analysis. Acidobacterial 16S rDNAs were observed in almost all soil samples, and members of acidobacterial primer group A were detected in all samples that harbored the phylum Acidobacteria. Other primer groups, Y, G, and O, showed limited distribution patterns. We further divided the primer groups into acidobacterial subdivisions (class-level). Subdivisional distribution patterns were determined by comparing the observed T-RFs with theoretical TRFs predicted by in silico digestion of acidobacterial 16S rDNAs. Consistent with the PCR results obtained with subgroup-specific primers, T-RFLP analyses showed that acidobacterial subdivision 1 belonging to primer group A was present in the majority of the soil samples. At the subdivisional level, acidobacterial subdivision 1 might be the most widely distributed group in this phylum, indicating that members of subdivision 1 might be adapted to various soil environments, and members belonging to other subdivisions might be restricted to certain geographic regions or habitats. B024 Simple and Rapid Detection of Listeria monocytogenes in Fruit Juice by Real-Time PCR without Enrichment Culture Hye-Jin Kim* and Jae-Chang Cho Institute of Environmental Sciences and Department of Environmental Sciences, Hankuk University of Foreign Studies We developed a simple, rapid procedure for the detection of Listeria monocytogenes in fruit juice using real-time PCR without enrichment culture. PCR inhibitors were removed from fruit juices using Chelex resin followed by gel filtration with a Sephadex column. The purification step can be completed in 20 minutes, and purified juice samples can be directly used as PCR templates without further dilution. PCR conditions were optimized and maximum sensitivity (ca. 1 cell/reaction) was achieved. This convenient method should prove useful for high-throughput surveillance for L. monocytogenes as well as other food-borne pathogens that may contaminate fruit juices. B025 B027 Changes of Cultivable Bacterial Community of Marine Sponges Depend on Media Composition and Storing Period Chatacteristics of Effective Dye Substance Degrading Bacteria Obtained from the Complex Dyeing Wastewater Treatment Plant Hyun-Hee Cho* and Jin-Sook Park Department of Biotechnology, Hannam University Seon-Yeong Jin1*, Eun-Jung Heo1, Hong-Jae Choi1, Eun-Joo Lee2, Young-Ok Lee1, and Gwang-Hui Lim2 1 Department Biological Science, College of Natural Science, Daegu University, 2Department Environmental Engineering, College of Engineering, Daegu University The cultivable bacterial communities associated with three Jeju Island Oedolgae marine sponges -Spirastrella abata, Spirastrella panis, and an association of the sponges Poecillastra wondoensis and Jaspis coreana in mixed cultures using F media with inorganic composition and C media with organic compositionwere investigated by microbial community DGGE and 16S rDNA phylogenetic analysis. Diverse bacterial group such as α-, γ-, δ-proteobacteria, Bacteriodetes, Firmicutes were cultured. δ-proteobacteria and Bacteriodetes were specific bacteria cultivated in C media, but they were not cultivated in F media which is inorganic media. The common bacterial group was found to be α-proteobacteria, and the association of two sponges showed the highest diversity among 3 sponge samples. One month after marine sponges were collected and freezed, the diversity of cultivable bacterial community decreased. The common community was also changed to Firmicutes. Therefore, the much difference of bacterial diversity depending on media composition was not found. Also we were able to confirm that the diversity of cultivable bacterial community decreased depending on storing time of sponge samples. B026 Cultured Bacterial Diversity and the Impact of Human Activity in Cryoconites Samples from Alps Yung Mi Lee1*, So-Yeon Kim2, Jia Jung2, Eun Hye Kim1, Kyeung Hee Cho1, Rosa Margesin3, Franz Schinner3, Soon Gyu Hong1, and Hong Kum Lee1 1 Polar BioCenter, Korea Polar Research Institute, Korea Ocean Research & Development Institute, 2Korean Minjok Leadership Academy, 3Institute of Microbiology, University of Innsbruck, Austria The relationship between human impact and the diversity of culturable bacteria in cryoconite samples from Alps was investigated. Growth temperature, activity of protease and lipase, and diversity were studied for 260 isolates. The ratio of isolates which grew below 20°C was higher in samples with no or weak human impact compared to that in samples with strong human impact. In contrast, the ratio of isolates which grew as high as 37°C were higher in strongly affected samples. In most cases, protease activity at 10°C and 20°C seemed similar, but the ratio of isolates with higher protease activity at 10°C than 20°C was higher when human impact were strong. In contrast, correlation of lipase activity with human impact was not evident. Isolates were included in the Actinobacteria, Bacteroidetes, Firmicutes, α-proteobacteria, ß-proteobacteria, and γproteobacteria and diversity was affected by human impact. Isolates included in the Arthrobacter, Bacillus, and Pseudorhodobacter were detected only from samples with no human impact while isolates included in the Enterobacteraceae, Oxalobacteraceae, Pedobacter, Rhodococcus, and Sphingomonas were detected from samples with human impact. In order to screen the complex dye degrading bacteria in the wastewater of Daegu Dyeing Industrial Complex, bacteria were isolated using dyeing wastewater itself as nutrient-medium supplemented with mineral salts medium(MSM) and agar (15g/L). The strong high pH of dyeing wastewater (pH 13) was adjusted to pH7 for preparation of culture medium. After 3days cultivation at 30°C, 2 strains grown on the dyeing wastewater agar were pure cultured. For their identification 16S rRNA of each strain was extracted and PCR was performed using both 341F and 518R primer. Those PCR products were sequenced (~177 bp) and then compared with 16S rRNA sequences by use of the EzTaxon. As a result, those bacteria was identified as Pseudomonas monteilii (98.6%), P. taiwanensis (98.2%), P. plecoglossicida (98.2%), P. mohhnii (98.1%), P. brenneri (97.9%). Furthermore, optimal growth factors of those bacteria in the complex dyeing wasterwater treatment plant such as temp, pH and c-source will be discussed. [This work (Grants No. 000367620109) was supported by Business for Cooperative R&D between Industry, Academy, and Research Institute funded Korea Small and Medium Business Administration in 2009.] B028 Phenotypic and Physiological Changes as a Result of Natural Transformation in Acinetobacter oleivorans DR1 Jungsoon Park* and Woojun Park Division of Environmental Science and Ecological Engineering, Korea University The genus Acinetobacter is well-known to uptake exogenous DNA from the environment. In this study, we provided frequency of natural transformation of thirteen Acinetobacter species along with novel oil- degrading Acinetobacter oleivorans DR1 using a broad host range plasmids, pRK415 (tetA as selective marker). Many factors including cell-density, DNA amounts, aeration are proven to be important for efficient natural transformation. Interestingly, A. oleivorans DR1 (pRK415) displayed phenotypic and physiological differences such as motility, ability for biofilm formation, response to oxidative stress compared to wild type strain: transformed cells became more sensitive to hydrogen peroxide and cumene hydroperoxide and their motilities and biofilm formation decreased. Oil biodegradation ability and growth fitness on various substrates of transformed cells will be discussed. Our data suggested that exogenous plasmid could influence on either genes expression of host chromosome or membrane integrity due to membrane-bound tetracycline resistant gene. [This work was supported by a grant from NCRC (grant #: 20090091491).] 153 www.msk.or.kr B029 B031 Characterization of a Positive Transcriptional Regulator of Isoeugenol Monooxygenase Gene from Pseudomonas nitroreducens Jin1 1* 2 3 Ji-Young Ryu , Joong-Hoon Ahn , Michael J. Sadowsky , and Hor-Gil Hur1,4 1 Department of Environmental Science and Engineering, Gwangju Institute of Science and Technology, 2Department of Bioscience and Biotechnology, Konkuk University, 3Department of Soil, Water, and Climate; and BioTechnology Institute, University of Minnesota, USA, 4International Environmental Research Center, Gwangju Institute of Science and Technology We currently isolated about 140-kb DNA fragment containing isoeugenol monooxygenase (iem) gene and its transcriptional regulator gene, iemR gene from Pseudomonas nitroreducens Jin1, which produced vanillin from isoeugenol. IemR played a role as a positive transcriptional regulator for iem gene, which encodes Iem involved in the biotransformation of isoeugenol to vanillin in the presence of an inducer such as isoeugenol. In this study, comparison of β-galactosidase activities of two E. coli strains harboring pMC-IemR, which contains iemR and iem-lacZ fusion gene, induced by isoeugenol showed that strain BL21 (DE3) exhibited about 2 times higher activity than strain DH5α. In addition, β-galactosidase activity in BL21(DE3) was significantly induced even 0.1 µM of isoeugenol. Results from βgalactosidase assay of BL21(DE3)(pMC-IemR) incubated with various aromatic compounds indicated that isoeugenol was the best inducer for the expression of iem gene. β-Galactosidase assay was also performed with sigma factor mutated E. coli strains carrying pMC-IemR during culture. The sigma factor RpoH was likely to be involved in the transcription of iemR or iem genes. Effects of Ionizing Radiation and UV Irradiation on Mutation Rate in Microorganisms Ji-Hye Shin1*, Ji-hyun Nam1, Kyong-Hee Oh2, Seungho Yu3, Myunju Lee3, and Dong-hun Lee1 1 Department of Microbiology, Chungbuk National University, 2 Department of Environmental Engineering, Chungbuk National University, 3Korea Atomic Energy Research Institute Advanced oxidation process (AOP) has shown substantial potential as a substitute for the process treating wastewater effluents containing toxic chemicals, xenobiotics, and etc. AOPs well-known for their effectiveness include ozonation, UV, γ-ray or electron beam (E-beam) irradiation. However, these treatments can lead to serious secondary problems by causing genetic changes in microorganisms. The goal of this study was to evaluate the change in mutation rate of bacteria exposed to ionizing radiation and UV. Mutation rates were measured by Ames test and E. coli mutation test. DNA damage was also estimated by umu-test. Death rate of bacteria exposed to one of the ionizing radiations or UV was proportional to irradiation dose. When bacteria were exposed with 5 mJ/cm2 UV, 0.5 kGy γ-ray and 0.2 kGy E-beam, the number of mutants were 300, 150 and 60 times higher than that of spontaneous mutation, respectively. The effects of E-beam on both mutation rate and DNA damage were less than those of γray and UV irradiations. Therefore, application of ionizing radiation, especially E-beam, appears to be more efficient for wastewater treatment system. [Supported by grants from MEST/ KOSEF] B032 B030 Bacterial Communities in Oak-Sawdust Composting Process for Lentinus edodes Cultivation Seo-Yeon Yoon1*, Ji-Hyun Nam1, Chang-Duck Koo2, and Dong-Hun Lee1 1 Department of Microbiology, Chungbuk National University, 2 Department of Forest Science, Chungbuk National University Oak-sawdust composting process has been used as an advanced method for Lentinus edodes cultivation. In spite of its successful application to culture medium for L. edodes cultivation, the bacterial population of leading the composting process remains unknown. In this study, changes in composition of bacterial communities in the sawdust composting were analyzed by PCR-T-RFLP and nucleotide sequences of 16S rRNA gene. Temperature of sawdust piles has risen up to 60°C during the composting process, and the numbers of total bacteria and thermophilic bacteria in interior parts of sawdust piles were higher than those in exterior parts. Structures of bacterial communities were classified into three stages; the sawdust composting stage, the pasteurization step, and the mycelium growth phase. Predominant populations in the composting process were members of Clostridium and Amycolatopsis. Therefore, anaerobic fermentation appears to be mediating oak-sawdust composting process. [Supported by grants from KFS] Fungal Community Analysis of the Samples Collected from the Stone Chamber and Its Neighboring Environment of the Takamatsuzuka Tumulus in Nara, Japan Kwang-Deuk An1*, Junko Tomita2, Tomohiko Kiyuna2, Rika Kigawa3, Chie Sano3, Moriya Ohkuma1, and Junta Sugiyama4 1 Japan Collection of Microorganisms, RIKEN BioResource Center, Japan, 2TechnoSuruga Laboratory Co., Ltd., NCIMB group, Japan, Independent Administrative Institution, National Research Institute for Cultural Properties, Japan, 4TechnoSuruga Laboratory Co., Ltd., Chiba Branch Office & Lab, Japan 3 The fungal community analysis using the denaturing gradient gel electrophoresis (DGGE) and clone library was applied to 50 and 22 samples were collected from the stone chamber interior, spaces between the stone walls, and stone chamber exterior of the Takamatsuzuka Tumulus (TT). The phylotaxa of Ascomycota, Basidiomycota, Zygomycota, Chytridiomycota were detected from these samples. Moreover, Eurotiales, Hypocreales, Chaetothyriales, Helotiales, Saccharomycetales of the Ascomycota were detected in a high ratio same as culture-dependent methods. Eurotiales and Hypocreales were detected in a high ratio in each sample, but Chaetothyriales was detected only interior a stone chamber and its environment. The different phylotypes in Helotiales were detected in neighboring environment of the stone chamber and burial mound’s samples. Molecular phylogenetically, there were the various ecological groups in samples derived from the TT stone chamber and its neighboring environment. [Supported in part by a Research Grant from the Institute for Fermentation, Osaka (IFO), and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.] 154 Poster Sessions B033 Genomic Analysis of a Shigella sonnei Bacteriophage Kyoung-Ho Kim1*, Ho-Won Chang2, Young-Do Nam2, Seong Woon Roh2, and Jin-Woo Bae2 1 Department of Microbiology, Pukyong National University, 2 Department of Life and Nanopharmaceutical Sciences and Department of Biology, Kyung Hee University Genomic structure of a bacteriophage that infects Shigella sonnei was analyzed. The bacteriophage showed the morphology similar to the family Myoviridae, and the phylogenetic analysis of sequences of g23 gene, major capsid gene showed that it belonged to T4-like phage. The bacteriophage genome was fully sequenced using 454 pyrosequencing and was ca. 170kb in length. Genomic comparison indicated that JS98, an enterophage isolated from human stool was the closest relatives. We compared two bacteriophage genomes and observed several differences such as deletion, insertion and duplication. Genomic sequencing helped us to understand the evolution and relationship of bacteriophages. [This work was supported by the 21C Frontier Microbial Genomics and Application Center Program and the EcoTechnopia 21 project] B034 Roles of Effective Microorganisms in Bioremediation of the Contaminated Harbor Sediments K. I. Ekpeghere1*, B.-H. Kim2, and S.-C. Koh3 Department of Civil and Environmental Engineering, Korea Maritime University, 2Environmental Biotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, 3Department of Environmental Engineering, Korea Maritime University 1 B035 Enrichment and Competition of Methanogens and Sulfate-Reducing Bacteria in Sterilized Tidal Flat Under Diverse Incubation Conditions Kihyun Lee*, Ok-Sun Kim, and Jongsik Chun School of biological sciences and Institute of Microbiology, Seoul National University Tidal flat is known to contain dissovlved methane upto hundreds nM concentration. Microorganisms mediating methane fluxes have been intensively studied in deep-sea sediments, but rarely in tidal flats. We aimed to cultivate naturally abundant methanogens in tidal flats, and observed competition between methanogens and sulfate reducers under diverse incubation conditions. The hypothesis was that physical structure of sterilized bulk sediment could generate various realizable niche in the bottle and prevent radical simplification of community structure, so that we can enrich naturally abundant microorganism by means of dilution. Bacterial and archaeal 16S rRNA, mcrA and dsrA genes were quantified in each natural and incubated sample. In natural samples, bacterial 16S rRNA gene was 7-20 times more abundant than archaeal 16S rRNA gene and correlated with dsrA abundance, while archaeal 16S rRNA gene did not with mcrA. After incubation for over a month, archaeal dominance was observed in hydrogen-enriched incubations, not exactly correlated by mcrA abundance. In various conditions, dsrA and mcrA abundance showed a roughly complementary pattern except in a methane-enriched incubation. B036 Idiomarina taeanensis sp. nov., a Novel Marine Bacterium Isolated from Crude Oil-Contaminated Seawater Young-Hyun Park1*, Dong-Heon Lee1,2, Young Sun Lee1, Jae Sung Jung1, and Hyung-Yeel Kahng1 1 Sunchon National Univeristy, 2Pohang University of Science and Technology We applied Effective Microorganisms (EM) carried on loess to the treatment of sediments contaminated with organics. We measured the physiochemical, biochemical and microbial parameters during the remediation process at a lab scale. Treatment with higher concentrations of EM loess balls (4%) and with a lower concentration of EM (0.1%) containing molasses (0.05%) contributed to a more rapid removal of malodor. Acetic acid, propionic acid, valeric acid, caponic acid, and lactic acid were rapidly removed when molasses (0.05%, w/w) were used as a supplemental nutrient source, indicating the EM activity was enhanced by molasses. Fermentation of molasses by EM showed that acetic acid was produced much higher than the other organic acids and most of the organic acids appeared to be eventually converted through intermediate metabolites to acetate. This shows that the rapid reduction in malodor could be due to the reduction of these organic acids by the molasses-assisted EM. It was concluded that EM might be directly involed in biodegradation of orgninc materials in the sediments. A novel, strictly aerobic, motile, Gram-staining-negative bacterium, designated strain MWD-44T, was isolated from the coastal seawater of Taean in Chungnam, Korea. It grew at 1042°C, at pH 5.0-10.0 and in the presence of 1-12% NaCl. A comparative 16S rRNA gene sequences study revealed that strain MWD-44T belonged to the genus idiomarina and shared significant similarity with Idiomarina aestuarii (99.5%) and Idiomarina salinarum (97.1%), respectively. DNA-DNA relatedness values between strain MWD-44T and the type strains of Idiomarina aestuarii KYW314T and Idiomarina salinarum CCUG 54359T were 44.5 and 31.8%, respectively. The predominant cellular fatty acids were iso-C15:0 (22.6%), Summed feature 3 (iso-C15:0 2-OH and/or C16:1ω7c, 13.7%), and C16:0 (10.4%). The G+C content of the genomic DNA was 47.9 mol% and the major respiratory quinone was ubiquinone 8 (Q-8). Based on phenotypic and genotypic data, strain MWD-44T represents a novel species within the genus Idiomarina, for which the name Idiomarina taeanensis sp. nov. is proposed. The type strain is MWD-44T (= KCTC 23070T = JCM 16452T) [Supported by grant from Center for Yeongdo Coastal Research, Korea Maritime University] [Supported by the Ministry of Environment (Grant No. 200805001-0033-0)]. 155 www.msk.or.kr B037 B039 Biodegradation of Oils by Pseudomonas taeanensis MS-3 Isolation and Characterization of Lactococcus lactis HK-9 Derived from Feces of a New Born Infant Dong-Heon Lee1,2*, Kye-Heon Oh3, and Hyung-Yeel Kahng1 1 Sunchon National University, 2Pohang University of Science and Technology, 3Soonchunhyang University Hyun Baek*, Hye-Ran Ahn, Yun-Jin Song, Yun-Seok Cho, and Kye-Heon Oh Department of Biotechnology, Soonchunhyang University Pseudomonas taeanensis MS-3T isolated from oil-contaminated seashore site has been reported as a novel species to degrade oils, which shared sequence similarity to Pseudomonas marincola KMM 3042T (97.9%), Pseudomonas cuatrocienegasensis 1NT (97.8%), Pseudomonas borbori LMG 23199T (97.3%), and Pseudomonas lundensis KACC 10832T (97.1%) as the closest neighbors (in press, Int. J. Sys. Env. Microbiol., 2010). GC-MS analysis revealed that approximately 80% of 0.5% gasoline was removed by strain MS-3T with 7-days incubation at 20°C. Strain MS-3T was also able to completely degrade 0.5% diesel and 0.5% kerosene at the same incubation time point. Analysis of substrate-dependent growth pattern revealed that strain MS-3T grew to an optical density of 0.25 at 600nm without lag phase with kerosene as a sole carbon source, while in BM medium containing 0.5% gasoline or 0.5% diesel as a sole carbon source it grew after approximately 2-days lag phase. PAH ring-hydroxylating dioxygenase and alkane hydroxylase responsible for the biodegradation of oils in strain MS-3T were also cloned and characterized The purpose of this work was to characterize the bacterium, Lactococcus lactis HK-9 isolated from the feces of a 2-day newborn infant. Initially, the physiological and biochemical characteristics of the strain HK-9 was examined. Both BIOLOG system and phylogenetic analysis using 16S rRNA sequencing were performed to identify and designated as L. lactis HK-9, and registered in GenBank as [GU936712]. Phylogenetic analysis of L. lactis HK-9 was plotted based on 16S rRNA sequence comparisons. The changes of bacterial growth, production of lactic acid and acetic acid as metabolites, and pH were monitored. Maximum concentrations of lactic acid and acetic acid reached 495.6 mM and 104.3 mM, respectively, and the initial pH 7.0 of the cultures decreased to 4.1 after incubation of 60 hrs. Significant antimicrobial activity of the concentrated supernatants against 4 Gram-positive and 5 Gram-negative bacteria was demonstrated by the plate diffusion method. The results of this work are applicable to the elucidation of defense mechanism of various pathogens in newborn infant. [Supported by Ministry of Environment, Grant No. 200805001-0033-0]. B038 B040 Antibacterial Effects and Cellular Responses of Imipenem-Resistant Pseudomonas aeruginosa Exposed to Green Tea Polyphenols * Yu-Jin Song , Su-Hee Cho, Yun-Seok Cho, and Kye-Heon Oh Department of Biotechnology, Soonchunhyang University The aim of this work was to investigate the bactericidal effects and cellular responses of green tea polyphenol (gTPP) and imipenem on Pseudomonas aeruginosa. Imipenem-resistant Ps. aeruginosa was isolated from patient in hospital. The bactericidal effects of gTPP and imipenem were evaluated on the basis of its minimum inhibitory concentrations (MIC). The combined use of gTPP and imipenem resulted in ~16-fold reductions in the MICs of imipenem for the imipenemsusceptible/resistant Ps. aeruginosa, respectively. The bactericidal effects of the imipenem and gTPP was evaluated using the time-kill assay. Synergetic effects of the combinations of gTPP and imipenem against Ps. aeruginosa were confirmed. Western blot was performed to investigate the expression of stress shock proteins in the strains exposed to gTPP. SDSPAGE revealed that the amount of lipopolysaccharides increased or decreased in the strain treated to different concentrations and exposing periods of gTPP. SEM demontrated the presence of umbilicated and wrinkled surfaces for cells treated with gTPP or imipenem. 156 Poster Sessions The Effect of Methane on The Microbial Community in The Sediment of Ulleung-Basin Jin-Woo Lee1*, Kae Kyoung Kwon1, Jang-Jun Bahk2, and Jung-Hyun Lee1 1 Marine Biotechnology Research Centre, Korea Ocean Research & Development Institute, 2Korea Institute of Geoscience and Mineral Resources Geophysical survey has shown that gas hydrate exists in deepsea sediments in the East Sea, Korea. Bacterial community analysis in methane hydrate-bearing deep-sea sediments (ca. 8m below the seafloor) obtained from the Ulleung-Basin was made by T-RFLP and 16S rDNA pyrosequencing. In the TRFLP analysis, it was shown that the community structure in the 'possible' sulfate methane transition zone (SMTZ) that was characterized as depleted sulfate and increased methane, was different from that of the upper and deeper region of the SMTZ. And then, pyrosequencing analsys has been conducted to show the bacterial community structures in high-resolution. Among the total sequences, about 62% reads were sorted and analysed. The homology searches revealed that the most of those sequences, more than 50% affiliated into the Chloroflexi and candidate division JS1, which were characteristic in the deepsea marine sediments containing methane hydrate and that the proportion of sequences within δ-proteobacteria was relatively higher in the SMTZ (100∼150cm) and surface region (0∼20cm). This result implied that microbial community structure in the SMTZ characteristic in microbial community structure. B041 B043 Biodiversity and Phylogenetic Analysis of Antibiotic Streptomyces Isolated from Various Environments Hyo-Jin Lee1* and Kyung-Sook Whang1,2 Department of Microbial & Nano Materials, Mokwon University, 2 Institute of Microbial Ecology & Resource, Mokwon University 1 We have hitherto isolated large numbers of antifungal and antibacterial actinobacteria from various environments, including different layers of bamboo forest soils, livestock composts and heavy metal polluted soils. We enumerated the actinobacterial densities, the number of actinobacteria in litter layers of bamboo and some of composts were two to three orders of magnitude greater than those enumerated in other environmental samples. In the course of screening of antibiotic activity against seven plant pathogenic fungi and two plant pathogenic bacteria of 530 isolates, 110 strains showed antibiotic activity. Ninety-five percent of the total antibiotic isolates were Streptomyces by 16S rRNA gene sequence analysis. The 105 Streptomyces, categorized into four clusters based on the Benjamin’s classifying (2005). The Streptomyces from bamboo forest soils were belonging to the cluster II (72%), I (6%), III (8%), IV (14%). On the other hand, 86% of total Streptomyces from livestock composts was cluster I. Phylogenetic analysis indicated that bamboo forest soil valuable in detecting diverse antibiotic Streptomyces in the various environments. Comparative Analysis of Microbial Community Depending on the Cell Size from the Bathypelagic Layer of Ulleung-Basin, Korea Mi-Ree Kim*, Kae Kyoung Kwon, and Sang-Jin Kim Marine Biotechnology Research Center, Korea Ocean Research & Development Institute The size of microorganisms varies considerably. At the smallest end of this size spectrum are the ultramicrobacteria(UMB) which has a cell volume of less than 0.1μm3, irrespective of growth conditions. UMB are highly abundant in marine environments and they play important roles in the global nutrient cycle and in the marine food chains. In the present study the microbial diversity in the bathypelagic layer of Ulleung-basin was compared by the size fraction which is lager than 0.2μm(non-UMB) or smaller than 0.2μm(UMB). A total 420 clones of archaeal group and 303 clones of bacterial group were analyzed by 16S rRNA gene target sequence. All archaeal clones belong to Crenarchaeota and Euryarchaeota and a majority of both fractions was affiliated with Crenarchaeota marine group I by 82-85%. Bacterial clones were derived from 5 major lineages, α-,β-,γ- and δ-Proteobacteria and Chloroflexales. γ-Proteobacteria represents a dominant bacterial group in the fraction of non-UMB and UMB by 85% and 78%, respectively. Most clone of UMB is grouped into the uncultured group and these results support UMB possess high frequency of unique microbial community compared to non-UMB. [Supported by MEGRC] B042 B044 Phylogenetic Characteristics of Exopolysaccharide Producing Bacteria from Root System of Angelica sinensis 1* 1 1,2 Hae Ran Lee , Song Ih Han , and Kyung Sook Whang 1 Department of Microbial & Nano Materials, Mokwon University, 2 Institute of Microbial Ecology & Resources, Mokwon University In the previous study, we have been detected exopolysaccharide(EPS) producing bacteria in rhizosphere soils of domestic medicinal herbs. Half of the total isolates from rhizosphere soil of Angelica sinensis was EPS producing bacteria, suggesting the dominance of EPS producing bacteria in rhizosphere soil of Angelica sinensis. In the present work, 35 EPS strains were isolated from root system (rhizosphere, rhizoplane, endophyte) of Angelica sinensis. The 16 EPS strains from rhizospere soil belonging to the genus Burkholderia, Paenibacillus, Mucilaginibacter, Rhodococcus, Variovorax, Achromobacter, Shinella, Rhizobium, and Mesorhizobium. Forty-two percent of the total EPS strains from rhizoplane was dominantly distributed Rhizobium-related genera. Three EPS strains were genus Chitinophaga from inside of root. In particular, DR14 and DRP35 produced high levels of exopolysaccharide in 4% of glucose (6,555mpa.s) and 2% of sucrose (3,275mpa.s), respectively. When determine by homology relationship of the 16S rRNA gene sequence with the relative taxa, the DR14 strain was closely Burkholdria caribensis (99%), and DRP35 strain was closely Terriglobus sp. (96.8%). Proteome Analysis of Halophilic Bacterium, US6-1 Isolated from PHAs Contaminated Environment Sung Ho Yun1*, Chi Won Choi1, Sang Oh Kwon1,2, Dong Gyu Lee1, Kae Kyoung Kwon3, Young Ho Chung1, and Seung Il Kim1 1 Division of Life Science, Korea Basic Science Institute, 2 Graduate School of Analytical Science and Technology, Chungnam National University, 3Microbiology Laboratory, Korea Ocean Research & Development Institute Contamination of marine sediments with PHAs (polycyclic aromatic hydrocarbons) is critical environmental problem because PHAs have toxic, carcinogenic, and genotoxic properties. Therefore, bioremediation of PAHs from contaminated environments is very important. Halophilic bacterial strain US6-1 is a gram-negative bacterium and has high sequence homology with Novosphingobium. This strain US6-1 can utilize several polycyclic aromatic hydrocarbons(PAHs) such as pyrene, benzo[a]pyrene, phenanthrene, et al. Recently, genome sequencing of strain US6-1 was finished and the draft data is available. Purpose of this project is to search of enzymes of PHAs biodegradation pathways in stain US6-1. In this study, proteomes of stain US6-1 cultured under several PAHs as carbon sources were analyzed by 2DE and MS/MS analysis. We detected about 500 protein spots on the 2-D gel in control medium(MM2) and found several differentially induced protein spots in different PAHs conditions. Identification of these proteins and their biological significance were presented. We suggest that these proteins are related to the PHAs biodegradation pathway and physiological characterization of stain US6-1. 157 www.msk.or.kr B045 B047 Metagenomic Analysis of Microbiomes of a Korean Traditional Fermented Kimchi Bacterial Community Analysis of Asian Dust: Cultivation-Dependent Approach Ji Young Jung1*, Hyo Jung Lee1, Jin-Woo Bae2, Yoonsoo Hahn1, and Che Ok Jeon1 1 Department of Life Science, Chung-Ang University, 2 Department of Biology, Kyung Hee University Hang-Yeon Weon1*, Jung-A Son1, Wan-Gyu Kim2, Soon-Wo Kwon1, Soo-Jin Kim1, Yi-Seul Kim1, and Jong-Ok Ka3 1 Korean Agricultural Culture Collection, National Agrobiodiversity Center, National Academy of Agricultural Science, Rural Development Administration, 2Agricultural Microbiology Division, National Academy of Agricultural Science, Rural Development Administration, 3Department of Agricultural Biotechnology, Seoul National University Kimchi is well known as a traditional and popular Korean fermented food. In this study, we monitored biological states during a kimchi fermentation process using a metagenomic analysis and we mainly focused on microbial community change and their metabolism. For metagnomic analysis, the kimchi soups containing microorganisms were periodically collected for genomic DNA extraction. The extracted DNA samples were analyzed using the 454-FLX technology, which generated total 701,556 sequences with an average read length of 433.4 bp. Community analysis based on reads containing 16S rRNA genes showed that the Kimchi microbiome was predominated by the genera Leuconostoc, Lactobacillus, and Weissella and the microbial community was changed dramatically during the fermentation periods. We analyzed COGs of the metagenomes, which showed that their metabolisms corresponding to COG categories were also changed during the fermentation. And comparison of random metageome reads with previously sequenced several LAB genomes showed high mapping coverage. [This work was supported by Technology Development Program for Agriculture and Forestry and EB-NCRC program.] B046 Much attention has been given to the nature of Asian dust and its effect on the Korean Peninsula in the recent years due to an increase in the intensity and frequency of Asian dust events. However, there has been little research on the distribution characteristics of airborne bacteria in Asian dust. To address this issue, the quantities and compositions of airborne bacteria were monitored by using a culture-dependent technique. The bacterial concentrations of the normal atmosphere (NA) varied with the sampling dates and locations, and were in the range 41,148 CFU/m3 during the study period. However, when an Asian dust event occurred, the bacterial concentrations increased sharply at a rate proportional to the intensity of the Asian dust event. A rarefaction curve and diversity index analysis showed that the bacterial diversity of the normal atmosphere was higher than that of the dust samples from Korea and China. In a community tree, the Asian dust samples were clearly separated from NA and source-region soil samples. Consequently, the airborne microorganisms detected in Asian dust events were quantitatively and qualitatively different from those in the normal atmosphere. B048 Microbial Diversity of Sediments at Gas Seep Area of Kagoshima Bay as Determined by Pyrosequencing Bacterial Community Analysis Using Pyrosequencing in Chonggak Kimchi during Fermentation Hyun Hee Cho1*, Kae Kyoung Kwon1, Chiaki Kato2, Takako Sato2, and Sang-Jin Kim1 1 Korea Ocean Research & Development Institute, 2Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology Hyo Jung Lee1*, Ji Young Jung1, Eun Jin Park2, Jin-Woo Bae2, and Che Ok Jeon1 1 Department of Life Science, Chung-Ang University, 2 Department of Biology, Kyung Hee University Bacterial community structure was compared between two different sediments based on the 16S rRNA gene sequence analysis by pyrosequencing method. The sediment samples were collected from tube worm inhabited area (MEGC0044) and caldera region (MEGC0046), which are located at shallow water of marine hydrothermal vent area in Kagoshima Bay, Japan. The phylotypes, the class δ-, ε- and γ-Proteobacteria, and Flavobacteria are predominant in both sediments but the proportion was different. Other phylotypes, Acidobacteria, Chloroflexi, Planctomycetes, and the candidate division WS3, also could be detected by more than 1%. The predominance of δ- and ε-proteobacteria represents distinct characteristics compared with the bacterial community structure previously reported from other cold seeps or hydrothermal vent areas where the γ-Proteobacteria is dominant. The proportion of εproteobacteria in MEGC0044 (28.6%) was much higher than that of δ-proteobacteria (14.8%) but vice versa in MEGC0046 by 13.5 and 20.6%, respectively. The results imply that the sulfur oxidizing is major biogeochemical process in MEGC0044, however, MEGC0046 is dominant by the sulfur reducing process. [Supported by MEGRC] 158 Poster Sessions Chonggak kimchi is a Korean traditional fermented food using radish. In order to analyze bacterial communities of Chonggak Kimchi,kimchi soup samples containing microorganisms were collected every three days for one month via through of sterilized gauzes. The pH decreased from 5.8 to 4.5 during 21 day-fermentation, and was then maintained constantly around pH 4.5 ± 0.05. The multiplex barcoded pyrosequencing was performed in a single run, with multiple samples tagged uniquely by multiplex identifiers, using universal primers 27F and 517R. Phylotypes were identified by using naïve Bayesian rRNA classifier at the Ribosomal Database Project website (http://rdp.cme.msu.edu/classifier/classifier.jsp). The results showed that various bacterial groups such as Lactobacillus, Streptophyta, Leuconostoc, Bacillus, Sphingomonas, Pseudomonas, and Serratia were existed in Chonggak Kimchi at 0 day. On the process of fermentation, bacterial community became simple; the most predominant bacterial group was the genus Leuconostoc, and the second was Lactobacillus. [This work was supported by Technology Development Program for Agriculture and Forestry and EB-NCRC.] B049 Bacterial Community Analysis of Asian Dust: Cultivation-Independent Approach Hang-Yeon Weon1*, Jung-A Son1, Chan-Won Park2, Jae-Ho Joa3, Hyung-Jun Noh4, Chang-Muk Lee5, Soon-Wo Kwon1, Jong-Ok Ka6, Soo-Jin Kim1, and Yi-Seul Kim1 1 Korean Agricultural Culture Collection, National Agrobiodiversity Center, National Academy of Agricultural Science, Rural Development Administration, 2Soil & Fertilizer Management Division, National Academy of Agricultural Science, Rural Development Administration, 3 Agricultural Research Center for Climate Change, National Institute of Horticultural & Herbal Science, Rural Development Administration, 4 Mushroom Research Division, National Institute of Horticultural & Herbal Science, Rural Development Administration, 5Functional BioMaterial Division, National Academy of Agricultural Science, Rural Development Administration, 6Department of Agricultural Biotechnology, Seoul National University To elucidate the bacterial diversity and their relative abundance in Asian dust samples collected in Korea and China, we used massively parallel pyrosequencing The diversity and composition of the dust-borne bacterial communities were relatively static at the phylum level. These dominant phyla were represented in the source region soils, reflecting that dust bacteria might have mainly originated from the source regions soils, but the composition was significantly different. OTU-based analyses such as rarefaction, diversity indices, and Venn diagrams showed that many differences were observed across sampling locations and year of Asian dust. In addition, community trees showed that differences between the sampling locations were more pronounced than differences between years. Hence, our results indicated that Asian dust might harbor a unique community that possibly originates from the source region soils and is shaped by selective forces during long-range transport and may vary depending on sampling sites and dates. B050 B051 Efficient Bioremediation of High Concentrated Tetrachloroethylene by Mixed Cultures of Bacteria in Anaerobic Condition Jin Ha Lee*, Jaisoo Kim, and Sang-Seob Lee Department of Biological Engineering, Kyonggi University in Korea Tetrachloroethylene (PCE) is widely used in many industrial fields, usually as a mixture with other chlorocarbons, which can cause central nervous system depression. Bioremediation is one of the efficient process to overcome this pollutant evidently. We isolated a few bacterial mixtures which showed degradation of PCE in anaerobic condition. Among those, we selected high efficient mixtures named as Pe1, Pm1, Pm2, Pm3 and Po3. Ethanol and methanol were used as a carbon source for the growth of the bacteria. The efficiency to degrade high concentrated (50 mg/L of initial concentration in minimal medium with yeast extract(1g/L), cell concentration 1.0 g/L, pH 7 and temperature 30°C) PCE under strict anaerobic condition was 75.8~ 80.1% in 30 days. We also found some fermentable bacterial mixtures which could produce hydrogen gas. The Po3 and fermentable mixtures showed more efficient removability of PCE. Furthermore we also screened the capability with mixed bacterial culture in aerobic condition, some mixtures turned PCE to trichloroethylene (TCE) with methanol, phenol and toluene as a carbon source, which has not been reported earlier. B052 High Throughput Cultivation of Major Marine Bacterial Lineages Using Various Culture Conditions from the East Sea A Metagenomic Screen Identifies Genetic Elements that Encode Novel Determinants for Bacterial Biofilm Formation Seung-Jo Yang*, Kwang-Hyun Rhee, Hyun-Myung Oh, and Jang-cheon Cho Division of Biology and Ocean Sciences, Inha University Mi Young Yoon* and Sang Sun Yoon Class of Microbiology, College of Medicine, Yonsei University High-throughput cultivation (HTC) based on dilution-toextinction has been successfully used to cultivate marine uncultured bacterial lineages from Oregon coast and the Sargasso Sea. In this study, we applied a modified version of HTC to cultivate major marine bacterial lineages in the East Sea for one year. Microbial growth was detected in 1446 wells among 7872 inoculated wells and 325 colony-forming isolates were obtained on agar plates by transferring extinction cultures. Based on phylogenetic analyses of 16S rRNA gene sequences from pure isolates, cultured strains were generally assigned to the SAR11 groups 1 and 3, Roseobacter clade and SAR92 group. The isolates belonging to the OM42, OM60, OM182, K189A clades and the phylum Lentisphaerea were also recovered. Interestingly many strains were affiliated to genera of Fucophilus, Coraliomargarita and Pelagicoccus in the phylum Verrucomicrobia. This study suggests that efficient cultivation of novel members of major marine bacterial groups could be facilitated by a long-term approach using various culture conditions mimicking natural environments. [Supported by the 21C Frontier Program of Microbial Genomics and Applications] Metagenomics is a new research field, in which a collection of genetic materials recovered from a certain microbial ecosystem is studied.Bacteria propagate as complex, highly organized communities known as biofilm. We sought to identify novel genetic elements, presence of which resulted in the enhanced biofilm formation. To this end, we screened a library that consists of 5,760 E. coli DH10B clones, each of which harbors a bacterial artificial chromosome prepared from DNA extracted from the large-bowel microbiota of BALB/c mice. We successfully identified 7 clones. Interestingly, the insert DNA from three clones possibly originated from uncultivated bacteria, but the gene from the remaining clone resembled sequences reported in Bacteroides sp. and Filobasidiella sp., with low similarity. Our results represent a successful application of metagenomic approach to reveal a novel mechanism for bacterial biofilm formation. [This study was supported by Yonsei University College of Medicine, Intramural Grant Support for Starting Faculty, 62009-0055 to SSY and National Reserach Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology, 7-2009-0524 to SSY.] 159 www.msk.or.kr B053 B055 Diversity of Fungal Endophytes in Leaves and Stems of Red Pepper (Capsicum annuum L.) Cultivated in Greenhouses and Open Fields Fusarium spp. Isolated from Wilted Gypsophila (Gypsophila elegans B.) in the Greenhouses Located in Yeosu, Jeonnam Province, Korea Je Yong Jeong, Chang Jeon Kim, Hye Yeon Mun, and Hyang Burm Lee Chang Jeon Kim1, Hye Yeon Mun1, Hyo-Suk Choi2, Young-Hyuk Lee2, Hye-Ok Yun2, Yu-Kon Kim2, and Hyang Burm Lee1 Division of Applied Bioscience & Biotechnology, College of Agriculture & Life Sciences, Chonnam National University In total, 106 isolates of endophytic fungi were isolated from leaves and stems of red pepper (Capsicum annuum L.) cultivated in greenhouse and open field from 2008 to 2009. Isolation rate of the endophytes in the open fields (77%) were higher than that in greenhouses (39%). The isolation rates in greenhouses were 42% and 35% in environment-friendly and conventional cultivation sites, respectively. All the isolates were identified on the basis of their morphologies as well as 18S rDNA sequence analysis. Alternaria (62%) and Cladosporium (32%) were dorminant species in the open fields and greenhouses, respectively. Chaetomium (15%) and Alternaria (13%) were prevalent in the greenhouses while Cladosporium (5%) and Chaetomium (3%) in the open fields. Thirty nine isolates comprising 10 genera both from greenhouses and open fields included Athrinium phaeospermum, Cercospora zebrinae. Fusarium spp., Gibberella moniliformis, Hypoxylon howeanum, Microascus triqonosporus, Nigrospora sp., Phaeosphaeriopsis musae, Sordariomycetes spp., and Xylaria spp. et al. B054 First Report of Powdery Mildew Caused by an Erysiphe Species on Quercus phillyraeoides Chang Jeon Kim*, Hye Yeon Mun, and Hyang Burm Lee Division of Applied Bioscience and Biotechnology, College of Agriculture & Life Sciences, Chonnam National University In May 2009, a powdery mildew on Quercus phillyraeoides A.Gray has been first observed at the campus of Chonnam National University, Gwangju, Korea. White powdery growth was abundant on the upper leaf surfaces. Leaf symptoms appeared commonly white in May to October. Along with the typical white powdery mildews, spot and/or necrotic symptoms with irregular violet to wine red-colored surfaces were also frequently observed on overwintered leaves. A voucher specimen has been deposited in EML herbarium collection, Chonnam National University (EML-QUP1). Conidia were formed singly and the size was 36.6-42.4 (av. 36.0) µm in length x 11.9-17.1 (av. 14.7) µm in width. Sequence analysis by BLAST indicated that EML-QUP1 was closest to an Erysiphe species, E. quercicola (accession no. AB292693) with 96% sequence similarity. So far, it has been known that Cystotheca lanestris, Erysiphe heraclei, Microsphaera alphatoides and Phyllactinia quercus cause powdery mildews on Quercus plants. To our knowledge, this is the first report of leaf powdery mildew caused by an Erysiphe sp. different with E. heraclei on Q. phillyraeoides in Korea. 160 Poster Sessions 1 Division of Applied Bioscience and Biotechnology, College of Agriculture & Life Sciences, Chonnam National University, 2 Agricultural Technology Center Gypsophilas are grown both as garden plants and also valuable as a cut flower in floristry to add as a filler to flower bouquets. Two forms of gypsophilas are commonly cultivated: the annual Gypsophila elegans, and the perennial G. paniculata. In September of 2009, a severe wilt disease was observed on G. elegans in the greenhouses located in Yeosu, Jeonnam province, Korea. Ten isolates were isolated from the stems and roots of wilted gypsophila. Three Fusarium isolates (EML-GYP1, 2 and 3) showing different colony types were selected and studied. Sequence analyses by BLAST indicated that two isolates of EML-GYP1 and 2 were closest to F. proliferatum with 99% sequence similarity and another isolate of EML-GYP3 was related to F. oxysporum with 97% sequence similarity. Pathogenicity tests with the three isolates were performed on G. elegans. Seven days after inoculation, similar symptoms to those observed in the greenhouses were seen on the inoculated plants. The causal fungus was re-isolated from the artificial lesions fulfilling Koch's postulates. Control plants showed no wilt symptoms. EML-GYP2 isolate was more pathogenic to the gypsophila plants than EML-GYP1 and 3. So far, only F. oxysporum has been known to cause wilt disease on gypsophilas in Korea. However, this study showed that another Fusarium species along with F. oxysporum could cause the wilt disease on gypsophila. B056 Pantoea spp. Related to Leaf Blights on Rice in Paddy Fields Located in Gwangyang and Naju, Jeonnam Province, Korea Jin Pyo Hong1, Seung Bum Kim2, and Hyang Burm Lee1 1 Division of Applied Bioscience & Biotechnology, College of Agriculture & Life Sciences, Chonnam National University, 2 Department of Microbiology, Chungnam National University In September 2009, leaf blights were observed on rice (Oryza sativa L.) in paddy fields located in Gwangyang and Naju, Jeonnam province, Korea. Early lesions began as water soaked stripes or light brown to slightly reddish spots on upper blade of leaves, ultimately causing leaf blights. Four strains (EMLORY1 to 4) were isolated from the leaf lesions and all the isolates formed yellow colonies on LB medium. The strains were identified based on the 16S rDNA sequence analyses. Three strains of EML-ORY1, 2 and 3 belonged to genus Pantoea and were closest to Pantoea ananatis (ATCC19321) sharing >99% sequence similarity while EML-ORY4 strain was mostly related to P. agglomerans (AJ233423), sharing 98.8% sequence similarity. Pathogenicity tests were performed on 2-weeks-old rice seedlings with bacterial suspensions containing 108 cfu/ml with 0.001% tween-20. Out of them, EML-ORY3 showed the strongest pathogenicity to rice. B057 Effect of Water Activity (aw) and Temperature on Mycelial Growth of Endophytic Pestalotiopsis spp. Hyoung Ryoul Jang, Hye Yeon Mun, Chang Jeon Kim, and Hyang Burm Lee Division of Applied Bioscience & Biotechnology, College of Agriculture & Life Sciences, Chonnam National University The effect of water activities (0.995, 0.98, 0.95 aw and control) and temperatures (18, 23, 27 and 32°C) on mycelial growth of 6 endophytic isolates of Pestalotiopsis were investigated on V-8 juice and malt extract agar media. The six isolates (JUN-1 and 2, QUE-1 and 2, PIN-1 and 2) were isolated from Chinese juniper (Juniperus chinensis Linn.), oriental chestnut oak (Quercus aliena Blume) and Japanese red pine (Pinus densiflora Sieb. et Zucc.), respectively.The mycelial growth of different isolates of Pestalotiopsis varied with water activity and temperature. Out of six isolates tested, QUE-1 isolated from Q. aliena grew fastest while PIN-2 from P. densiflora slowest. Decrease (0.95 aw) in water activity caused a significant reduction in mycelial growth of all the six isolates. Mycelial growths of Pestalotiopsis spp. were highest on MEA and at 27°C. At 32°C, the growth rates of QUE-1 and 2 from Q. aliena in comparison with other isolates were higher at 0.98 aw than other water activity levels. 161 www.msk.or.kr C001 Isolation and Characterization of HistamineProducing Bacteria in Fermented Fish Products in Korea Jin Seok Moon1*, Kyung-Ju Cho2, Seung-Joon Yang2, Gun-Mook Yoon2, So-Young Kim3, Seung Kee Cho1, Yu Jin Kim1, and Nam Soo Han 1 1 Department of Food Science and Technology, Research Center for Bio Resource and Health, Chungbuk National University, 2 Chungbuk Institute of Health & Environment Research, 3 Functional Food & Nutrition Division, Department of Korean Food Research for Globalization, National Academy of Agricultural Science The objective of this study was to isolate and characterize histamine-producing microorganism in fermented fish products in Korea, including anchovy sauce, sand lance sauce, squid paste, clam paste, and shrimp paste. For screening of histamine-producing bacteria, histamine detection agar (HDA) medium was used and the multiplex PCR was done for genomic analysis. As a result, two histamine-producing bacteria were isolated from anchovy sauce and sand lance sauce, and they were identified as Bacillus licheniformis and Bacillus coagulans. The multiplex PCR analysis showed existence of histidine decarboxylase (HDC) the chromosome of those bacteria. At the same time, their abilities to produce biogenic amines in histidine-containing media were confirmed by HPLC analysis. Further application of these culture dependant/ independant methods will permit wider monitering of histamine-producing bacteria in various fermented foods. [Supported by the MKE and KIAT] C002 C003 Antimicrobial Treatment of Grapes Using Sodium Hypochlorite (NaOCl) in Winemaking and Its Effects on Chemical and Sensory Characteristics of Wine Ki-Seon Yoo*, Ji Eun Kim, Hwa Young Choi, Eun-Young Seo, Yu Jin Kim, and Nam Soo Han Department of Food Science and Technology, Research Center for Bioresource and Health, Chungbuk National University The aim of this study was to evaluate the use of NaOCl as an alternative antimicrobial compound in winemaking. Analyses of microorganisms, chemical changes and sensory characteristics were carried out during the fermentation periods of three wines; non-treated, SO2-added or NaOCl-treated wines. Treatment of grapes with NaOCl reduced the initial contaminating microbial population in red wine and this resulted in higher growth of yeast and LAB in alcoholic and MLF periods. After 200 days incubation, the chemical analysis of NaOCl-treated wine showed that ethanol content, redness (a*) in color, concentrations of fruity-ester compounds were higher and the total acidity was lower than the non-treated. In the sensory analyses, the NaOCl-treated wine obtained significantly (p<0.05) higher overall acceptability score (5.70) than the SO2-added wine (4.26). These results revealed that NaOCl acted as an alternative of SO2 in winemaking giving an inhibitory effect against the contaminating microorganisms and it might cause an environmental change toward a better wine fermentation condition for yeast and LAB. [This study was supported by grants from TDPAF and NRF] C004 Microbial Community Associated with Internal Organs of Snow-Crabs Identification of Antibiotics against Pathogenic Fungi from Bacillus subtilis Jin-Uk Lee1*, Na-Yeon Kim2, Gyeong-Jin Jo2, Jeoung-Hyun Hong1, Young-Eun Lee1, Nyun-Ho Park1, Choong-Gon Kim 1, and Jong-Shik Kim1 1 Gyeongbuk Institute for Marine Bioindustry, 2Yeungnam University Haemin Kim*, Yong Hoon Lee, Bong-Sik Yun, Byung-Taek Oh, Kui-Jae Lee, and Jong-Chan Chae Division of Biotechnology, College of Environmental & Bioresource Sciences, Chonbuk National University The purpose of this study was to analyze microbial community associated with the organs of Uljin snow-crab(Chionoecetes opilio), Red tanner crab(Chionoecetes japonicus) and NeodoDaege(Chionoecetes sp.). To our knowledge, there have yet studied what kinds of microbes are associated with their organs. Here, we have been analyzing the microorganisms of their organs using 6 different media for isolation. Microbes from organs of three crabs have been isolated (369 isolates) and identified by sequence analysis of 16S rRNA and ITS gene. From Uljin snow-crab, Acinetobacter, Stenotrophomonas and Pseudomonas were isolated mainly, and from a red tanner crab, Psychrobacter and Acinetobacter and from a Neodo-Daege snow-crab, Rhodococcus, Leifsonia and Bacillus. In addition, antibiotic susceptibility tests showed that Lactobacillus, Pseudomonas and Enterobacter from Uljin snow-crab and Claribacter, Psychrobacter, Lactobacillus from red tanner crab were resistant to tested all nine antibiotics, mainly isolates from Uljin snow-crab were high resistant to penicillin, erythromycin, vancomycin and ampicillin. 162 Poster Sessions A halotolerent bacterial strain was isolated from Suncheon Bay exhibiting strong antagonistic activity against various plant pathogenic fungi such as Alternaria alternate, Aspergillus niger, Botrytis cinerea, Collectotrichum acutatum, Collectotrichum coccodes, Diaphorina citri, Penicillium digitatum, and Penicillium italicum. Additionally, in vivo antagonistic assay on postharvest peppers showed inhibition activity against C. acutatum. The strain was identified as a Bacillus subtilis based on 16S rDNA sequences and produced antifungal compounds on Luria-Bertani media at 30°C. The crude compounds were precipitated by adjusting pH to 2.0 with 6 mol of HCl in the cell free culture broth, which were extracted with methanol. They were classified as lipopeptides according to the molecular masses by MALDI-TOF mass spectrometry. The estimated molecular weight peaks 1,080, 1,058 and 1,485 (m/z) indicate that the compounds belong to iturin, surfactin and fengycin families. C005 C007 Screening for Cold-Active Protease-Producing Bacteria from the Culture Collection of Polar Microorganisms and Protease Characterization * Ha Ju Park , Dockyu Kim, Yung Mi Lee, Soon Gyu Hong, Hong Kum Lee, and Joung Han Yim Polar BioCenter, Korea Polar Research Institute KOPRI has assembled a culture collection of cold-adapted bacteria from the Arctic and Antarctic. To identify excellent protease-producers among the proteolytic bacterial collection (874 strains), 78 strains were selected according to their relative activities and were subsequently re-examined for their extracellular protease activity on ZoBell/skim milk plates. This screening method permitted the selection of a small group of 15 cold-adapted bacteria, belonging to the genus Pseudoalteromonas or Flavobacterium, that showed proteolytic activities at 5-15°C. The cold-active proteases from these strains were classified into four categories according to the extent of enzymatic inhibition by a class-specific protease inhibitor. Since highly active and cold-adapted proteases have the potential for commercial enzyme development, the bacteria selected in this work are studied as a valuable natural source of new proteases. At present, a protease from one strain (KOPRI 21717), which is highly active at low temperatures and resistant to protein-denaturing SDS, is being studied for future application in detergent. Development of Functional Probiotics for Prevention of Dental Caries Donghwan Kim*, Yesl Sin, Kijong Yu, and Hongik Kim R&D Division, VITABIO, Inc. Dental caries is one of the most common oral diseases in human and Streptococcus mutans is its major causative microorganism. Glucosyltransferase (GTase) secreted by S. mutans produces insoluble glucan that plays an important role to form the biofilm. S. mutans under the biofilm demineralizes of tooth enamel by acids, finally induces dental caries. Therefore, inhibition of S. mutans growth and GTase activity will be effective in prevention of dental caries at the early stage of biofilm formation. In this study, 6 probiotic bacteria were isolated from kimchi and human feces. These strains had more potent antimicrobial activity against S. mutans compared to other reference strains. They effectively inhibited the formation of biofilm, in vitro. These probiotics were identified as Lactobacillus plantarum, Weissella confusa and W. cibaria by phylogenetic analysis using 16S rDNA. In addition, these isolates were resistant to gastric acidity and bile, and possessed antimicrobial activity against E. coli and Salmonella typhimurium. Therefore, it is expected that these probiotics may prevent dental caries and also show good probiotic effects through oral administration. [This work was supported by a grant to KOPRI (PE09050).] C006 C008 Silver and Zinc Coated Silica as a Preservative for Personal Care Product Study on Regarding a Hypha Culture Characteristic of a Naematoloma sublateritium Hyun Sang Lee1*, Yun Jeong Kim1, Ji Hyun Son1, Neri Geum2, and Jong Woo Chun1,2 1 R&D Center, ACT Co., Ltd., 2Department of Chemistry, Dankook University Jongwoon Choi1*, Jihong Kim2, Dusik Jeon1, Gilhwa Jeong1, Jiyoung Choi1, Sanghoon Chung2, Sunga Choi3, and Sangjun Sim1 1 Forest Research Institute of Gangwon-do, 2Department of Forest Management, Kangwon National University, 3 Department of Life Science, Hallym University Silver and zinc ion coated silica (ACTOSIL) was synthesized for the purpose of using a preservative for personal care product. To evaluate the antimicrobial activity of ACTOSIL, MIC tests were performed against various microorganisms. The MIC values of ACTOSIL against various bacteria and fungi were 12.5 to 100 ppm and 100 to 500 ppm respectively. Challenge test were conducted to test the antimicrobial effect of ACTOSIL in various personal care products. At the concentration of 15 ppm, ACTOSIL meets the recommendation of CTFA microbiological guideline. Furthermore, the result of MTT assay with ACTOSIL showed low cell cytotoxicity. As shown these results, ACTOSIL has a potential to use a safe preservative for personal care products. This study executed it in order to analyze a characteristic in a hypha culture of a Naematoloma sublateritium. It was excellent, and a GP medium, a YMA medium was investigated, but existing other study to a PDA medium it was investigated. Suitable temperature and provisional results regarding ph of a Naematoloma sublateritium, proper temperature appeared with 25 degrees, and proper ph was investigated to 6.5~7.0. This study showed to excellent hypha growing at Glucose in circular the carbon which was suitable for the third, hypha growing of a Naematoloma sublateritium provisional results carbon circle regarding a nitrogen circle, and cultures of a Naematoloma sublateritium looked at malt extract in case of organic nitrogen circles. This study looked in a phosphoric acid circle to affect the fourth, a mycelium of a Naematoloma sublateritium to growth and development. Also, If this study added it was investigated p-aminobenzoic acid in a vitamin circle so that hypha growing was comparatively good. [This paper consisted by assistances of a technology development assignment scientific forest. 163 www.msk.or.kr C009 Co-Culturing Bacillus lichinoformis with Clostridium tyrobutyricum for Production of Butyric Acid from Sucrose Mohammed Dwidar1,2*, Se-il Kim1, Jae-Yeon Park1, Byung-Seung Jeon1, Youngsoon Um1, and Byoung-In Sang1,2 1 Korea Institute of Science and Technology, 2University of Science and Technology An isolated strain of Bacillus lichinoformis was used in a coculture with Clostridium tyrobutyricum ATCC 25755 in serum bottles containing RCM supplemented with sucrose as the sole carbon source for the production of butyric acid. Although C.tyrobutyricum alone is unable to use sucrose as a carbon source, this co-culture was able to use sucrose and to produce butyric acid up to 10 g/L which is even higher than that produced from C.tyrobutyricum alone in serum bottles when glucose is used as a carbon source under the same conditions. B.lichinoformis alone did not show any butyric acid production, implying that B.lichinoformis degrades sucrose into monosaccharides by specific enzymes and then C.tyrobutyricum uses them for butyric acid production. Butyric acid was the main product in this co-culture. Also, the final acetic acid concentration in the medium was significantly lower than that produced from C.tyrobutyricum alone when glucose is used as a carbon source. In addition, this co-culture could grow in the RCM broth in serum bottles without initial purging with inert gas for providing anaerobic conditions. C011 Use of Bacteria Isolated from Dok-do Island for Improving Compressive Strength in Cement-Sand mortar Sung-Jin Park1*, Sung-Tae Kim2, Wha-Jung Kim2, and Sa-Youl Ghim1 1 School of Life Sciences, Kyungpook National University, 2 School of Architecture & Architectural Engineering, Kyungpook National University This study shows an application of microbiologically induced carbonate precipitate for strength improvement of cement-sand mortar. Nine calcium carbonate forming bacteria (CFB) were isolated from Dok-do and partially identified by performing sequence analysis of the 16s rRNA gene. Crystal aggregates were apparent in the bacterial colonies grown on an agar medium. The crystals on urea-CaCl2 agar medium were captured by a Zentech digital camera equipped with a stereomicroscope (Sw 804425; Samwon, Korea). These strains showed strain specific CaCO3 precipitation on urea-CaCl2 medium. To verify an effect of improved compressive strength in cement-sand mortar, the CFB were inoculated in cementsand mortar. [Supported by grants from KOSEF and KRF] [Supported by grants from SK energy] C010 C012 Synthesis of Chalcogenide Ternary and Quaternary Nanotubes through Directed Compositional Alterations of Bacterial As-S Nanotubes Purification of Determinants of Ochrobactrum lupini KUDC1013 Inducing Systemic Resistance against Soft-Rot Pathogen in Tobacco Shenghua Jiang1*, Fang Liu2, Min-Gyu Kim3, Jae-Hong Lim2, Kun-Jae Lee4, Yong-Ho Choa4, Kyung Song5, Michael J. Sadowsky6, Wilfred Chen2, Nosang V. Myung2, and Hor-Gil Hur1,7 Marilyn Sumayo1*, Mi-Seon Hahm1, Ye-ji Hwang1, Choong-Min Ryu2, and Sa-Youl Ghim1 1 School of Life Sciences, Kyungpook National University, 2 Laboratory of Microbial Genomics, Systems Microbiology Research Center, Korea Research Institute of Bioscience and Biotechnology 1 Department of Environmental Science and Engineering, Gwangju Institute of Science and Technology, 2Department of Chemical and Environmental Engineering, University of California at Riverside, 3 Pohang Accelerator Laboratory, 4Department of Fine Chemical Engineering, Hanyang University, 5Division of Electron Microscopic Research, Korea Basic Science Institute, 6Department of Soil, Water, and Climate; and BioTechnology Institute, University of Minnesota, 7 International Environmental Research Center, Gwangju Institute of Science and Technology Eco-friendly biological syntheses of nanomaterials have been recently introduced as alternative protocols for the conventional chemical routes. Previously we reported that Shewanella sp. HN41 can produce photoactive arsenic sulfide (As-S) nanotubes by biological reduction of As(V) and thiosulfate. In this study, we demonstrate the synthesis of chalcogenide ternary (i.e. As-S-Se and As-Cd-S) and quaternary (i.e. As-Cd-S-Se) composited nanotubes through bacterial Se(IV) reduction by strain HN-41 and/or abiotic ion exchange of As-S with Cd under ambient conditions. Microscopic observations and electrical measurements revealed that morphology, crystal structures, and electrical properties can be significantly altered by controlling the composition. These strategies may provide environmental friendly routes to manufacture other nanoengineered materials and devices in costeffective matter. [This work was supported by the 21C Frontier Microbial Genomics and Applications Center Program (M102KK010011-08K1101-01110), Ministry of Education, Science & Technology, Republic of Korea.] 164 Poster Sessions Ochrobactrum lupini KUDC1013 is a plant growth promoting rhizobacterium (PGPR) isolated from the rhizosphere of Solanum nigrum L. plants in Dokdo island and has the capability to induce systemic resistance (ISR) of tobacco against the leaf soft-rot pathogen Erwinia carotovora subsp. carotovora strain SCC1. In order to distinguish the bacterial determinants that elicit systemic resistance, extracellular components produced by KUDC1013 were extracted with series of solvents, fractionated, purified and were tested for their protective effects. ISR bioassay revealed that resistance elicitors were retained in the cell free supernatant and was partitioned into n-butanol. Further purification and identification analyses of the ISR elicitor from KUDC1013 will increase the range of factors from PGPR recognized to induce plant resistance. C013 Base Ranking Analysis on Regarding a rDNA ITS Part of a Naematoloma sublateritium Jongwoon Choi1*, Dusik Jeon1, Gilhwa Jeong1, Sanghoon Chung2, Jihong Kim2, Sunga Choi3, Sangjun Sim1, and Jiyoung Choi1 1 Forest Research Institute of Gangwon-do, 2Department of Forest Management, Kangwon National University, 3 Department of Life Science, Hallym University Condition of PCR cycling repeated 30cycle to extension for annealing/72 degrees, 90 seconds for denaturation/50 degrees, 30 seconds for 95 degrees, 30 seconds, and I performed it, and in 95 degrees, three minutes and last extension used it the first denaturation 72 degrees, 10 minutes. Primer ITS1 and ITS4 (White et al.)I used 1990), and analysis of PCR amplication product is Mupid 21(COSMO Bio Co.) use, 1.I executed electrophoresis at 508b3536p-1022garose gel (1X TAE buffer) for 100V 30 minutes. Marker 1kb DNA ladder (Promega Co.)I used), and I confirmed amplication product to Gel Documentation System 2000 (Bio-Rad) after I dyed it to ethidium bromide dynamic an electric area for 20 minutes, and washing it to a distillation number for five minutes. The Naematoloma sublateritium isolate sympathy and phylogenetic tree analysis results are EF530927. It is Hypholoma capnoides internal transcribed spa. one 1277,0.I appeared to 0.0. [This paper consisted by assistances of a technology development assignment scientific forest. C014 Chemical Structures and Apoptosis Inducing Effect of Diketopiperazine Disulfides Produced by Aspergillus sp. KMD 901 Isolated from Marine Sediment on Colon Cancer Cell Lines HCT116 Eun Ju Choi*, Jin-Soo Park, Young-Joo Kim, Ji-Hee Jung, Hak Cheol Kwon, and Hyun Ok Yang Korea Institute of Science and Technology, Gangneung Institute The objective of this research was to determine chemical structures and to estimate apoptosis inducing effect of diketopiperazine disulfides produced by Aspergillus sp. KMD 901 isolated from marine sediment, which collected from the East Sea of Korea. The ethyl acetate extract of strain KMD 901 exhibited the strongest cytotoxic activity on cancer cell lines. The cytotoxic compounds were purified from the ethyl acetate extract of Aspergillus sp. KMD 901 using reverse-phase high performance liquid chromatography. Structures of isolated cytotoxic compounds were elucidated diketopiperazine disulfides by means of spectroscopic analyses, including extensive 2D NMR and mass spectrometry. The diketopiperazine disulfides induced apoptosis of HCT116 cells, characterized by cleavage of apoptosis-related proteins using Western blotting analysis. In vivo xenograft test, acetylapoaranotin (2) showed antitumor effect. These results suggested that these diketopiperazine disulfides from Aspergillus sp., KMD 901, could be a candidate for the development of antitumor agents. [Supported by Korea Institute of Science and Technology institutional program, Grant number, 2Z03270, Republic of Korea.] C015 Biotransformation of Heterocyclic Aromatic Compounds by Naphthalene Dioxygenase from Pseudomonas sp. Strain NCIB 9816-4 in Escherichia coli Cung Nawl Thawng1* and Hor-Gil Hur1,2 1 Department of Environmental Science and Engineering, 2 International Environmental Research Center, Gwangju Institute of Science and Technology The naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 is a multicomponent enzyme system that carries out the initial step in the biodegradation of naphthalene. This enzyme has a broad substrate range and catalyzes several types of reactions including cis-hydroxylation, monooxygenation, and desaturation. In this study, E.coli JM109 (pDTG141) harboring NDO gene of Pseudomonas sp. strain NCIB 9816-4 was used for whole cell reaction. Totally eight chemicals which are heterocyclic aromatic compounds were used for this biotransformation experiments. NDO was found to be produced metabolites from all tested chemicals. The possible products of each metabolite were identified by HPLC and LC/MS analysis. Future study by LC-NMR will confirm the structures of metabolites of each compound. [This work was supported by a grant from the MEST/NRF to the Environment Biotechnology National Core Research Center (grant #: 20090091491), Korea] C016 Characterization of a High Salt Resistant pHydroxybenzoate Hydroxylase from Chromohalobacter sp. HS-2 in E. coli Hee Gon Kim1*, Yu Ri Park1, Wonduck Kim2, and Si Wouk Kim1 1 Department of Environmental Engineering, BK21 Team for Biohydrogen Production, 2Pioneer Research Center for Controlling of Harmful Algal Bloom, Chosun University The gene encoding p-hydroxybenzoate hydroxylase (pobA) from moderately halophilic bacteria, Chromohalobacter sp. HS-2, was cloned into the expression vector pCRT7-CTTOPO. The pobA gene was expressed in E. coli BL21 (DE3) by 0.75 mM IPTG. Expression in E. coli BL21 resulted mainly in the formation of inclusion bodies. To enhance the production of soluble PobA protein, chaperone plasmid (dnaKdnaJ-grpE) was co-expressed with E. coli harboring pobA gene. SDS-PAGE and Western blot analysis showed an estimated 44 kDa protein band corresponding to the recombinant p-hydroxybenzoate hydroxylase. The HPLC and LC/MS analysis revealed that resting cells of E. coli BL21 harboring pobA and chaperone plasmid oxidized phydroxybenzoate to protocatechuate. The expressed PobA protein was purified by Ni-NTA column and characterized. The optimal pH and temperature for enzyme activity was determined to be 9.0 and 35°C, respectively. The Vmax and Km values were 53.8 μmol/min/mg protein and 78.9 μM. The relative activity of PobA protein was remained 50 and 20% in the presence of 0.5 and 1M NaCl, respectively. 165 www.msk.or.kr C017 C019 The Isolation and Characterization of Antifungal Compounds from Soil Bacteria Ke Dong1*, Hyun-Ju Lee2, Sung-Kee Kim2, Jaisoo Kim1, and Sang-Seob Lee1 1 Kyonggi University, 2GyeonggiDo Agricultural Research & Extension Services A plant fungal pathogen, Fusarium oxysporum (Fox) is the causal agent of root rot or wilt diseases in several plant species, including some cash crops. We want to extract some antifungal compounds against Fox from soil bacteria. At the present, we screened 242 strains from 42 species, and the strain 2252-010 showed the highest antifungal activity. The strain 2252-010 was identified as Psedomonas chlororaphis based on 16S rDNA sequences. The strain 2252-010 showed efficient antifungal activities on all of the five different pathogenic strains of Fox. The growth conditions of the strain 2252-010 for antifungal compound production on potato dextrose agar (PDA) plate are pH 7.0, 28°C and 3 days incubation. We are going to extract, purify and characterize the antifungal compound. If the compound has an efficient antifungal and economical performance, we may use it as a novel, environmental-friendly antifungal agent. Lactobacillus reuteri Probio-16 Isolated from Pig Feces Inhibits Porcine Rotavirus and Enteric Bacterial Pathogens 1* 1 1 Byeong Joo Seo , Mi Ran Mun , V. J Rejish Kumar , Chul-Joong Kim2, Insun Lee1, Young-Hyo Chang3, Jeongheui Lim1, and Yong-Ha Park1 1 Department of Applied Microbiology and Biotechnology, Yeungnam University, 2Collage of Veterinary Medicine, Chungnam National University, 3Korean Research Institute of Bioscience and Biotechnology Bacterial strain Probio-16 was isolated from the swine excrements and characterized by morphology and biochemical characteristics. The strain was further identified by 16S rRNA gene sequencing and phylogeneitc analysis. The strain was tested for antiviral activity by porcine rotavirus inhibition in vitro in African green monkey epithelial cell line TF-104. Phenotypically and through 16S rRNA gene sequences, Probio-16 was identified and named as Lactobacillus reuteri Probio-16. This strain was resistant to pH 2.0, 5% bile acids and exhibited antimicrobial activity against all the thirteen enteric bacterial pathogens tested. Probio-16 supernatant inhibited porcine rotavirus in vitro in TF-104 cell lines. Except for erythromycin and penicillin G at a concentration of 4 µg/ml, Probio-16 showed resistance to all other thirteen antibiotics tested. This study indicates L. reuteri Probio-16 as a novel strain with its tolerance to low pH and bile, antimicrobial activity, antibiotic resistance and antiviral activity against rotavirus, and an ideal probiotic candidate for animal and human application after the proper in vivo experiments. [Supported by the grants from Yeungnam University] Poster Sessions Eunyoung Seo* and Nam Soo Han Chungbuk National University Leuconostoc sp. is a dominant lactic acid bacterium in kimchi and it catabolizes glucose, sucrose, and fructose producing lactate, dextran, and mannitol, respectively. The aim of this study was to investigate the protein expression patterns in L. mesenteroides ATCC8293 in various carbon sources (glucose, sucrose, and fructose). For this, L. mesenteroides was cultured in MRS medium containing 2% of reach sugars and proteomic analysis was performed using LC-MS. As results, about 600 proteins were isolated in the glucose-medium and the distinctive proteins expressed more than two folds in the sucrose-medium were identified as glycosidase, ABC-type transport system (2 types), sugar kinase, ribosomal proteins (two types), and heat shock proteins. The protein expression pattern in the fructose-medium was generally same with that of sucrose. The most highly expressed proteins in glucosemedium were enzymes or regulatory proteins involved in the central metabolic pathway, DNA replication and protein expression, and they were mainly house-keeping proteins, as expected. This information will be used to construct the sugarinducible expression vector system or the in silico metabolic pathway. C020 C018 166 Proteomic Expression Analysis of Leuconostoc mesenteroides Subsp. Mesenteroides ATCC 8293 in Glucose and Sucrose and Fructose Medium Isolation of Compounds Inhibiting Growth of Pathogenic Fungi Ah-Ra Goh*, Yi-Na Kim, Hyun-Gook Hwang, and Sang Ki Choi Department of Biological Sciences, Sunchon National University Many pathogenic fungi tend to acquire resistance to the antifungals although a number of antifungal drug has been developed. Thus it is necessary to develop novel drug based on new target proteins in fungi. To identify substances that inhibit growth of pathogenic fungi. we attempted to screen appr. 6,800 core chemicals which were provided from Korea Chemical bank. six hundreds eighty compounds inhibited by 80% at 4.5 μg/ml the growth of Candida grabrata, Cryptococcus neoformans and Aspergillus niger. Finally we selected five compounds which inhibited the growth of the pathogenic fungi at about 0.45 μg/ml significantly and tested further. The chemicals inhibited the growth of C. glabrata proportionally as concentration increased from 2 μg/ml to 32 μglml. MIC of a compound for C. albicans, C. glabrata, C. lusitaniae and C. tropicalis was each 16, 0.5, 0.06, and 0.25 respectively, which is similar with that of itracoazole and ketoconazole. We are under investigation how these compounds influence fungal growth. C021 Optimization of Antibacterial Activity of Perilla frutescens var. acuta Leaf against Pseudomonas aeruginosa Using Evolutionary Operation-Factorial Design Technique Ung Kyu Choi*, HanBoreum Lee, Hee Sun Bae, and Dae Hyun Kim Pohang Center for Evaluation of Biomaterials This study was under taken to optimize the extraction condition using evolutionary operation-factorial (EVOP) design technique to elicit the antibacterial activity of Perilla frutescens var. acuta leaf against Pseudomonas aeruginosa. Higher antibacterial activity was achieved at higher extraction temperature and in a longer extraction time. Antibacterial activity was not affected by differentiation of the ethanol concentration in the extraction solvent. The maximum antibacterial activity of ethanolic extract of P. frutescens leaf against P. aeruginosa determined by the EVOP factorial technique was obtained at 80°C (R = -0.8000**) extraction temperature, 26 h (R = -0.73**) extraction time, and 50% (R= -0.075) ethanol concentration. The population of P. aeruginosa also decreased from 6.660 log CFU/ml in the initial set to 4.060 log CFU/ml in the third set. Also the scanning electronic microscopic study of the ethanolic extract of P. frutescens revealed potential detrimental effect on the morphology of P. aeruginosa. C022 C023 The Melanogenesis Activity on Fermented Extract with Lactobacilli Min Sik Kwon*, Dae-Myoung Yun, Tae-Young Yoon, and Gyoseon Yeom Skin Science Laboratory, R&D Center, iPEERES Cosmetics LTD. Previous work, we identified tyrosinase activity of Ephedra sinica and so we should investigate melanogenesis inhibition and cytoxicity of fermented Ephedra extract on melanoma B16F10 cell. And we expect that fermented extract has lower cytoxicity than non-fermented extract and it has little whitening effect in skin. Ephedra was extracted with 70 w/v% ethanol and extract was fermented using lactobacilli such as Lactobacillus helveticus KCTC 3545. The cytoxicity was measured by MTT(tetrazolium salt 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) assay and skin whitening was evaluated by melanogenesis assay. Cell viability of fermented extract was higher than non-fermented extract. The melanogenesis inhibitory activity of fermented extract was the highest among those of various extracts. Therefore, this study suggests that fermented extract may be useful as whitening agent and irritation-reduction agent in the skin. C024 Comparison of Skin Penetration on Fermentation with Lactobacilli Inhibitory Effects of Broccoli Extract on Quorum Sensing of Escherichia coli O157:H7 Dae-Myoung Yun*, Tae-Young Yoon, and Gyoseon Yeom iPEERES, Skin Science Laboratory Kang-Mu Lee1*, Jeesun Lim2, Mi Young Yoon1, Yongjin Park1, Wasimul B. Chowdhury1, Sungsu Park2, and Sang Sun Yoon1 1 Department of Microbiology, College of Medicine, Yonsei University, 2Department of Chemistry and Nano Sciences, Ewha Womans University Effective material is to be ferment, nutritive component increase and particle of component become smaller. Therefore it is evaluated penetration in skin. We should identify penetration rate (PR) of Ephedra (Ma huang) extract by fermentation with lactobacilli. In the work, Ephedra was extracted with 70% w/v% ethanol (E) and extract was fermented using lactobacilli such as Lactobacillus helveticus KCTC 3545 (H) and Lactobacillus plantarum KCTC 3104 (P). Lactobacilli were incubated in appropriated condition and activities were measured at OD560 by ELISA reader.For confirm penetration into skin, we used Franz diffusion system. We prepared time by receptor-sample (10, 30, 60, 120, 240 min) and quantified amount by HPLC at each 10 v/v% extract solution. As a result, PR of H and P were higher than E. Also, we measured reactive activities of tyrosine-tyrosinase and LDOPA-tyrosinase in H and P. Although fermented extract is not superior to ethanol extract, we verified dose dependent inhibitory activities.We will make clear optimizing concentration of lactobacilli at once and we eagerly look forward to tyrosinase inhibition and distinguished penetration in skin. Quorum sensing is a key process for the production of virulence determinants in pathogenic bacteria. In this study, we investigated the effects of BE on quorum sensing (QS) and expression of QS-controlled virulence genes in Escherichia coli O157:H7. In assays using Chromobacterium violaceum CV026 and Vibro harveyi BB170, the BE suppressed the production of violacein and autoinducer-2, respectively in a does-dependent manner. Quantitative real-time PCR and GFPpromoter fusion analysis indicated the transcriptional level of many QS-regulated virulence genes in E. coli O157:H7 was decreased in the presence of BE. Furthermore, C. elegans fed on E. coli O157:H7 in the presence of BE survived longer than those fed solely on the pathogenic bacteria. These data suggest that the BE exerts inhibitory effects on QS-associated virulence in E. coli O157:H7 and thus has potential to be developed as an anti-infective agent. [This study was supported by Yonsei University College of Medicine, Intramural Grant Support for Starting Faculty, 62009-0055 to SSY and National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology, 7-2009-0524 to SSY.] 167 www.msk.or.kr C025 Identification of a Gene Encoding Endoglucanase That Belongs to Glycosyl Hydrolase Family 45 from the Brown-Rot Basidiomycete Fomitopsis palustris Ju-Hee Cha* and Chang-Jun Cha Microbial Biotechnology Lab, Department of Biotechnology (BK21-program), Chung-Ang University The brown-rot basidiomycete Fomitopsis palustris is known to degrade lignocellulosic materials and produce the three major cellulases such as endoglucanases, exoglucanases and βglucosidase. In this study, we identified a gene encoding endoglucanase (EG) that belongs to glycoside hydrolase (GH) family 45 from the fungus. mRNA was extracted from the fungus grown on a medium containing 2.0% Avicel and cDNA was synthesized from mRNA by reverse transcription. About a 650 bp fragment was obtained by 3’-RACE (Rapid Amplification of cDNA Ends) and sequenced. After the 5’-RACE, the full-length cDNA was obtained. The cDNA fragment consisted of 621bp nucleotides, including an open reading frame of 206 amino acid residues. Analysis of the deduced amino acid sequence of the fragment revealed that the protein has the highest sequence similarity to EG from Phanerochaete chrysosporium (71%) which belongs to the GH family 45. Functional studies are currently in progress to evaluate the enzyme for industrial applications. 168 Poster Sessions C026 Characterization of a Novel β-Glucosidase Transforming Ginsenoside Rb1 to rare Gypenoside XVII and Gypenoside LXXV from Terrabacter ginsenosidimutans sp. nov. Song-Gun Kim* and Dong-Shan An Korea Research Institute of Bioscience and Biotechnology A new β-glucosidase, which catalyzes the conversion of ginsenoside Rb1 to more pharmacologically active rare ginsenosides gypenoside XVII, gypenoside LXXV, was characterized and its gene bgpA (1947 bp) was cloned in Escherichia coli from a novel Terrabacter ginsenosidimutans Gsoil 3082T. BLAST search using the bgpA sequence revealed significant homology to family 3 glycoside hydrolases. The βglucosidase expressed in E. coli showed the apparent Km and Vmax values of 4.2mM and 100.6 μmol•min-1•mg protein-1 against p-nitrophenyl- β-D-glucopyranoside. Molecular weight was estimated as 70,000 by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The enzyme could hydrolyze the two glucose moieties from the C-3 position and the outer glucose from the C-20 site of ginsenoside Rb1. These cleavages occurred in a defined order, with the outer glucose of C-3 cleaved first, followed by the inner glucose of C-3, and finally the outer glucose was cleaved from the C-20 site. Therefore, BgpA converts selectively ginsenoside Rb1 to gypenoside XVII to gypenoside LXXV to C-K. D001 D003 The yiiR Gene in Escherichia coli O157:H7 Regulates O-Antigen Synthesis Role of yjhK Gene on Pathogenicity of Salmonella typhimurium χ3339 Jeesun Lim*, Min Yoon, Yidan Cui, Kang-Mu Lee, Seong-Won Nam, and Sungsu Park Ewha Womans University Junghwa Kang1*, Saehun Kim1, Jeesun Lim2, Kangmu Lee2, and Sungsu Park2 1 Division of Food Science, Korea University, 2Department of Chemistry and Nano Sciences (BK21), Ewha Womans University It is known that the O-antigen, which consists of repeating oligosaccharide subunits, plays crucial roles in the hostmicrobe interaction in Escherichia coli O157:H7. However, little information is available with regard to the regulation of O-antigen synthesis. In order to investigate the regulation mechanism, we screened out genes regulating the O-antigen synthesis using Caenorhabditis elegans, a host model for E. coli O157:H7 infection. Among about 900 random mutants, a yiiR mutant was shown to have reduced virulence against C. elegans. The microarray analysis showed that the expression of O-antigen synthesis operon in the mutant increased over twofold than the wild type. In agglutination assay, the yiiR mutant formed 40% less agglutination with O157 antiserum than its wild type. In addition, atomic force microscope (AFM) showed that yiiR mutant had shorter chain than the wild type. These results suggest that the yiiR gene play an important role in the regulation of O-antigen synthesis. It was shown that C. elegans was killed by Salmonella enterica serovars through the persistent infection in the intestine. In this study, about 1,000 S. typhimurium mutants were constructed through random mutagenesis and fed into the nematodes in order to screen out mutants showing reduced pathogenicity to C. elegans. The yhjK mutant killed less nematodes than its wild-type χ3339. It is known that the yhjK gene encodes a phosphodiesterase, but its role is unknown. 2D proteome analysis showed that the mutant produced 50% less thiolperoxidase (Tpx), compared to χ3339. The yhjK mutant also survived less in the macrophage than χ3339. The mutant was less tolerant to oxidative stress exerted by hydrogen peroxide, compared to the wild-type strain. Conclusively, these results suggest that the phosphodiesterase plays an important role in protecting the pathogen from its host innate immunity. [This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MEST) (Basic Science Research Program R012008-000-20593-0).] D002 D004 The Nematode Caenorhabditis elegans Can be Killed by Escherichia coli O157:H7 Through Infection in the Intestine Testing Synergistic Effect of Patulin with Antibiotics Against Burkholderia cepacia Using Whole-Body Fluorescent Imaging in C. elegans and Mouse Min Youn1*, Jeesun Lim1, Kang-Mu Lee1, Younghoon Kim2, Sae-Hun Kim2, Jang W. Yoon3, and Sungsu Park1 1 Department of Chemistry and Nano Sciences (BK21), Ewha Womans University, 2Division of Food Science, Korea University, 3Department of Microbiology and Research Institute for Translational System Biomics, Chung-Ang University College of Medicine Seong-Won Nam*, Jeesun Lim, Min Yoon, and Sungsu Park Department of Chemistry and Nano Sciences (BK21), Ewha Womans University Caenorhaditis elegans is a model host for identifying novel virulence factors in bacterial pathogens. A previous study1 showed that nematodes fed on enterohemorrhagic Escherichia coli (EHEC) O157:H7 were killed within 3 hours. This killing is mediated by a toxin secreted from the pathogen grown in the medium containing tryptophan. In this study, we report that the nematodes fed on E. coli O157:H7 were killed in the absence of tryptophan and the killing was related to infection in their intestine. Compared to the wild-type E. coli O157:H7, the strains (ΔperA, Δtir, Δeae, ΔpO157) which have a defect on the intimate adhesion to the intestinal cells were less pathogenic to C. elegans. The microscopic observation showed that the mutants were less persistent in the intestine than the wild-type. C. elegans mutants (sek-1 and ced-3 and ced-4) were hypersensitive to the pathogen, indicating that programmed cell death (PCD) and MARK pathway mediating innate immunity are required for C. elegans to protect it from bacterial infection. Conclusively, these results suggest that E. coli O157:H7 also kill C. elegans through the infection. In recent years, the mortality rate in the cystic fibrosis (CF) patients infected with Burkholderia cepacia complex (Bcc) species have been increasing because of their intrinsic resistance against many antibiotics. Previously, we visualized cyanide accumulated in the nematode C. elegans using a chemosensor which display a green fluorescence upon its binding to cyanide ion (CN-). In this study, combinations of patulin, a QS inhibitor, and several antibiotics were tested for antibiotic susceptibility. A combination of ceftazidime and patulin inhibited biofilm formation of Bcc species, while ceftazidime or patulin alone failed to inhibit biofilm formation. In vivo efficacy test using a whole animal imaging system indicated that the fluorescence in the mouse lung treated with the combination was significantly lower than that in the lung treated with only a single antibiotic. This synergistic effect was supported by the ex vivo bacterial counting. These results suggest that the whole-body fluorescent imaging of infected small animals is highly useful for testing in vivo antibiotic efficacy. [Supported by NRF grant funded by MEST (Basic Science Research Program R01-2008-000-20593-0).] 169 www.msk.or.kr D005 D007 Influence of Gene Expression by Escherichia coli LuxS Mutant Using Microarray Analysis Antibiotic Genotype and Resistance-Acquired Mechanism of Vancomycin-Resistant Enterococcus spp. (VRE) Isolated from Stool Hyun-Ah Choi1*, Jaisoo Kim1, Yong-Hyun Cho2, Ji-Youl Lee2, Kyong-Ran Peck3, and Sang-Seob Lee1 1 Department of Biological Engineering, Kyonggi University of Korea, 2Department of Urology, School of Medicine, The Catholic University of Korea, 3Division of Infectious Diseases, Sungkyunkwan University School of Medicine Hee-Jeong Kim*, Kang-Lim Kim, Hyo-Jin Lee, Soo-Myung Hwang, and Kyung-Soo Chang Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan Bacteria can detect their own cell density by quorum-sensing mechanism that coordinates the expression of genes. The QS signal autoinducer-2 has beenknown to promote interspecies signaling in a broad range of bacterial species at a critical threshold concentration of cells. LuxS is responsible for production of AI-2, which is involved in QS response of Escherichia coli. In this study, we examined how expression of virulence gene is affected by AI-2. E. coli were isolated from patient’s catheters and luxS mutants were constructed. To confirm mutations, we performed TLC using supernatants extracted from mutant cultures and found mutants did not produce detectable AI-2 through activity test. Also, to study the roles of LuxS/AI-2 on whether this system is functional in virulence, mutants were investigated using microarray. As a result, LuxS-dependent signal seems to be a variety of roles at transcriptomes level. We found that mutation of luxS altered the gene expression directly involved in other genes associated with virulence. Our results suggest AI-2 is an important signal in E. coli infection of the catheter-associated urinary tract infection. Vancomycin-resistant Enterococcus spp. (VRE) isolated from clinic stools were analyzed for antibiotic phenotype and genotype. Interactions between insertion of IS1216V and IS1542 genes and antibiotic susceptibility to teicoplanin were investigated. Consistence of van gene in VRE cultured in broth without vancomycin was determined. VRE strains isolated from stools were E. faecium and E. gallinarum, but not E. faecalis. There were 10 VanA and 1 VanB phenotypes among 11 VRE isolates which were classified as vanA genotype. Isolated VRE showed multi-drug resistant to antibiotics such as β-lactamase and imipenem, but sensitive to quinupristin/ dalfopristin. IS1216 gene in VRE showing sensitive intermediate to teicoplanin was not detected or weakly detected. Copy numbers of vanA were decreased in E. gallinarum but not in E. faecium cultured in broth without antibiotics. Copy numbers of vanA were immediately recovered by addition of vancomycin. Growth time of reference E. faecium is faster than that of reference E. faecalis in broth with vancomycin. Reference strains cultured in broth with vancomycin showed antibiotics resistant or intermediate without acquisition of van genes. [Supported by grants from MEST.] D006 D008 Isolation Frequency and Antimicrobial Resistance of Bacterial Pathogens Isolated from Physical Therapeutic Instruments in Geriatric Care Hospitals 1* 2 1 Min-Joo Kim , Laurentius Jongsoon Kim , Hee-Jeong Kim , Hyun-Jung Jo1, and Kyung-Soo Chang1 1 Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, 2Department of Physical Therapy, College of Health Sciences, Catholic University of Pusan Though geriatric care hospitals continuously are extending, studies on microorganisms on physical therapy equipment and their effects on hospital-acquired infections are currently lacking. The purpose of this study is to examine isolation frequency and antimicrobial resistance of bacterial pathogens isolated from physical therapeutic room in geriatric care hospitals. Specimens for this study were collected from physical therapy rooms in geriatric care hospitals and examined bacteria isolation and their antimicrobial resistance patterns. Most of the bacteria isolated from these specimens were Acinetobacter baumannii. which showed resistance to ceftazidime, imipenem, amikacin, ciprofloxacin, and tobramycin. They showed highest antibiotics resistance to cephalothin and ampicillin. The isolated Enterobacteriaceae showed high resistance to ampicillin, ampicillin/sulbactam, cefazolin, and cefoxitin. MR-CNS was frequently isolated than MRSA. By the results, it is possible to appearance of multi-resistance bacteria. To control nosocomial infections in physical therapy room in geriatric care hospitals, a continuous investigation and hygiene control have become more important. 170 Poster Sessions Immunomodulatory Activity from Pinus palustris Part I: Isolation and Biological Assessment In Vitro Gyeong-Im Choe*, Byung-Jae So, Jooyea Hwang, Hee Jung Hwang, Cheong-Up Choi, and Jae-Myung Kim National Veterinary Research & Quarantine Service MIFAFF Pinus palustris has been widely used as a drug and food, has been examined bioactivities, such as antioxidant, antimicrobial and anticancer. In the present study, the immunomodulatory activities as cell migration, cytotoxicity and IL-6 of the various fraction from P. palustris were investigated in vitro. The dired plant was extracted with 80% methanol to generate its methanol extract (PME). For some experiments, ethyl acetate (PEA), n-butanol (PB), and aqueous (PA) were prepared in succession from PME. Among the fractions, PEA fraction was further separated to 8 groups by silica gel 60 G (PEA-1 ~ PEA8), and PEA-7 was identified to immunomodulatory activities. Neutrophil migration activity of PEA-1(5 ug), PEA-2(5 ug) and PEA-7(5 ug) fractions compared with control was 149.2%, 142.2% and 135.2%, respectively. Furthermore, PEA-7 fraction was inhibited growth of tumor cell(B16F10) and stimulated production of IL-6 in RAW 264.7 cells. These results indicate that the PEA-7 fraction contains potential immunomodulatory substance and it need to research more various study. [This work was supported by the grants from NVRQS of MIFAFF.] D009 D011 Polymorphisms in VacA and CagA Affect the Severity of Gastric Diseases An Association Between Polymorphism in the CagA EPIYA Motif and Development of Gastric Cancer Sungil Jang1,2*, Kathleen R. Jones3, Cara H. Olsen4, Young Min Joo1, Yun-Jung Yoo1,2, In-Sik Chung5, Jeong-Heon Cha1,2, and D. Scott Merrell3 Sungil Jang , Kathleen R. Jones , Young Min Joo , 1 3 3 4 Yun-Jung Yoo , Hak-Sung Lee , In-Sik Chung , Cara H. Olsen , 2 2 1 Jeannette M. Whitmire , D. Scott Merrell , and Jeong-Heon Cha 1 1 Department of Oral Biology, Oral Science Research Center, BK21 project, Yonsei University College of Dentistry, 2Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, USA, 3Division of Gastroenterology, Department of Internal Medicine, College of Medicine, the Catholic University of Korea, 4Department of Preventive Medicine and Biometrics, Uniformed Services University of the Health Sciences, USA Department of Oral Biology, Oral Science Research Center, BK21 Project, Yonsei University College of Dentistry, 2Research Center for Orofacial Hard Tissue Regeneration, College of Dentistry, Yonsei University, 3Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, USA, 4Department of Preventive Medicine and Biometrics, Uniformed Services University of the Health Sciences, USA, 5Division of Gastroenterology, Department of Internal Medicine, College of Medicine, the Catholic University of Korea Helicobacter pylori infection is regarded as a cause of diverse gastric diseases. But why some individuals develop more severe forms of disease remains unclear. Herein, we genotyped and analyzed 225 Korean strains for vacA to determine if particular genotypes varied across disease state, sex, or cagA allele. Of these strains, 206 strains carried an s1/i1/m1 allele, 11 strains carried an s1/i1/m2 allele, and 8 strains carried an s1/i2/m2 allele. By using Fisher’s exact test, a statistical association between variations in the cagA and vacA alleles was identified (P=0.0007), and by using log linear modeling, this variation was shown to affect the severity of disease outcome (P=0.027). Additionally, we present evidence that variation within the m region of VacA is associated with disease outcome (P=0.011) and the distribution of vacA alleles across sex (P=0.008). In this population, the majority of H. pylori strains carry the vacA s1/i1/m1 allele and the CagA EPIYA-ABD allele. These facts may contribute to the high incidence of gastric maladies. [This work was supported by a National Research Foundation of Korea grant funded by the South Korean Government (grant 2009-0073957).] 1* 2 1 Helicobacter pylori causes diseases ranging from gastritis to gastric cancer. Geographically, areas with high incidences of H. pylori infection often overlap with areas with high incidences of gastric cancer, which remains one of the leading causes of cancerrelated deaths worldwide. Strains of H. pylori that carry the cytotoxin-associated gene A (cagA) are much more likely to be associated with the development of gastric cancer. Moreover, particular C-terminal polymorphisms in CagA vary by geography and have been suggested to influence disease development. We conducted a large-scale molecular epidemiologic analysis of Korean strains and herein report a statistical link between the East Asian CagA EPIYA-ABD genotype and the development of gastric cancer. Characterization of a subset of the Korean isolates showed that all strains from cancer patients expressed and delivered phosphorylatable CagA to host cells, whereas the presence of the cagA gene did not strictly correlate to expression and delivery of CagA in all noncancer strains. [This work was supported by a Korea Research Foundation grant, which was funded by the Korean government (MOEHRD) (grant KRF-2006-311-E00083).] D012 D010 After Contamination of Bacteria for Soil's Quality Improvement, DGGE and Pyrosequencing Are Used to Check Difference of Soil 1* 1 2 Hyun Jung Kim , Jaisoo Kim , Sung-Kee Kim , and Sang-Seob Lee1 1 Department of Biological Engineering, Kyonggi University of Korea, 2GyeonggiDo Agricultural Research & Extension Services Denaturing gradient gel electrophoresis (DGGE) is to study the diversity of soil bacteria in natural and contaminated soils. DGGE has been widely used to investigate several patterns of distribution of soil bacteria. Two primer sets gave a single band when used with soil bacteria and complex fingerprints. DGGE fingerprints were then used to compare the soil bacteria diversity in samples obtained at different soil bacteria with bacteria contamination soil samples. DGGE bands were each other species so the sequence analysis compare with other molecular technique such as phyrosequencing. We planted cabbages in control soil and contaminated soil, and we obsevered. as a result, growth of cabbage in contaminated soil by bacteria is the best. The DGGE results were compared with non-contaminated soil sample and contaminated by some bacteria such as photosynthesis bacteria, isolated from fecal, food and there mixture sample. In conclusion the DGGE profiles analysis was some different pattern each samples. Contaminated by photosynthesis bacteria sample’s had a few band compared with other samples. And soil sample of mixtured bacteria had a many band more than other samples. In Vitro Screening of Antiviral Activity of Natural Plants Against Pseudorabies Virus Jooyea Hwang*, Byung-Jae So, Hee Jung Hwang, Gyeong-Im Choe, Cheong-Up Choi, and Jae-Myung Kim National Veterinary Research & Quarantine Service MIFAFF Medicinal plants are traditionally used for the treatment of various illness including cancers, septicemia, and infectious diseases. Eight medicinal plants used in traditional oriental medicine were screened for potential antiviral activity against pseudorabies virus (PRV). The natural plants were extracted with polarities of solvents using 80% methanol, ethyl acetate, n-butanol, and water. The antiviral activity was determined by means of the activity of serial dilution extracts to inhibit the produced cytopathogenic effect (CPE). These results demonstrated that the extracts of Taraxacum mongolicum, Canavalia gladiata, Lonicerae flos, Hovenia dulcis, and Zanthoxylum piperitum have antiviral effects against PRV at completely non-toxic concentration ranges from 12.5 to 50μg/ml. Furthermore, the extracts of methanol and aqueous were more effective on the suppression of virus infection than ethyl acetate and n-butanol extracts. There preliminary results suggest that the examined natural plant extracts might be good candidates in control of PRV infections. [This work was supported by the grants from NVRQS of MIFAFF.] 171 www.msk.or.kr D013 D015 Activation of Lymphocytes and Macrophage Functions by Ethyl Acetate Fraction of Rhus verniciflua Stokes Extracts Analysis of Quorum Sensing-Dependent Extracellular Proteases of Pseudomonas aeruginosa for Their Involvement in Infectivity and Virulence Jae-Myung Kim*, Gyeong-Im Choe, Jooyea Hwang, Hee Jung Hwang, Hyun-Ok Ku, Cheong-Up Choi, and Byung-Jae So National Veterinary Research & Quarantine Service Ha-Young Park*, Soo-Kyoung Kim, Yusang Choi, Changwan Ha, Su-Jin Park, Su-jin Im, and Joon-Hee Lee Department of Pharmacy, College of Pharmacy, Pusan National University Rhus verniciflua stokes (RVS) have been used as a traditional food and medicine to enhance immune response against infectious agents and to treat cancers. In this study, the methanol extract of RVS and its successive n-butanol, ethyl acetate and aqueous extracts were assessed by in vitro neutrophil migration, spleen lymphocyte proliferation and nitric oxide (NO) production. Thus we investigated the effects of fractions of RVS on macrophage function including phagocytosis and cytokine production. The ethyl acetate fraction of RVS stimulated spleen lymphocyte proliferation, NO production, neutrophil migration, macrophage phagocytic activity and inhibited the viability of B16F10 mouse melanoma cells. In addition, the ethyl acetate fraction induced production of macrophage related cytokines such as TNF-α, IL-6 and IL1α. These results suggest that RVS ethyl acetate fraction examined here, exhibit immunostimulatory activity, which implicates that RVS ethyl acetate fraction may serve as a potential source of natural immunostimulants for treatment of some animal diseases. Pseudomonas aeruginosa is gram-negative bacteria and a typical opportunistic pathogen. It provokes cystic fibrosis, microbial keratitis, and burn wound infections. The virulence of P. aeruginosa is mainly mediated by quorum sensing (QS), a bacterial cell-to-cell communication in bacteria. Especially, QS of P. aeruginosa regulates several extracellular proteases that are important in its pathogenicity. To investigate whether the QS-dependent virulence could be mediated by these extracellular proteases, we selected 8 candidate extracellular proteases, protease IV, LasA, LasB, PA2939, PA1249, PA0355, PA4171, PA3535 that are expressed in QS-dependent manner, based on the microarray database. We cloned and overexpressed them in P. aeruginosa and examined any change of virulence by using insect and nematode infection model systems, where cell free culture supernatants from the protease-overexpressing P. aeruginosa strains were collected and injected into Tenebrio moliter, an insect to observe their immune response and the protease-overexpressing P. aeruginosa strains were fed to Caenorhabditis elegans, a nematode to see the killing or decrease of multiplication of C. elegans. [This work was supported by the grants from NVRQS of MIFAFF.] D014 D016 A Diagnostic Method Based on TaqMan-PCR for the Detection and Differentiation of Recombinant Vaccine and Wild-Type Pseudorabies Viruses Quorum Sensing Modulation in Pseudomonas aeruginosa by Signals Produced by Burkholderia vietnamiensis Jae-Myung Kim*, Hee Jung Hwang, Jooyea Hwang, Gyeong-Im Choe, Sang-Ho Cha, Cheong-Up Choi, and Byung-Jae So National Veterinary Research & Quarantine Service Changwan Ha*, Yusang Choi, Soo-Kyoung Kim, Ha-Young Park, Su-Jin Im, and Joon-Hee Lee Department of Pharmacy, College of Pharmacy, Pusan National University Psuedorabies, also known as Aujesky’s disease, is a disease caused by the Pseudorabies virus (PRV). Thus, recombinant Pseudorabies vaccine containing porcine interleukin-2 (IL-2) was developed and it is necessary to establish a detection and differentiation of vaccine and field strains of PRV. In this study, TaqMan real-time PCR assay was designed for detection and differentiation of Pseudorabies recombinant vaccine virus, from some field viruses, by comparing the amplification of two pairs of primers corresponding to glycoprotein B genes and porcine interteukin-2, respectively. The assay demonstrated good analytical specificity, sensitivity and reproducibility with coefficient of variation (CV%) ranging from 0.15% to 1.29% and from 0.046% to 0.466% for intra- and inter-assay variability respectively. The newly developed real-time PCR assays could be detect vaccine viruses from livers, brains and spleens of mouse inoculated PRV vaccine viruses. Pseudomonas aeruginosa is an opportunistic pathogen causing various infections in insects, plants, and human. Quorum sensing (QS) is a bacterial cell-to-cell communication mechanism using small diffusible molecules. P. aeruginosa uses acyl-homoserine lactones for QS that are common QS signals used by most gram (-) bacteria. The genome of P. aeruginosa codes for signal synthases and receptors, so called LuxI-R homologues, LasI-R, RhlI-R, and QscR. QscR was originally characterized as an orphan QS receptor that has no cognate signal synthase gene, but recently found to share the same signal molecule, N-3-oxododecanoyl-HSL with LasR. Burkholderia is another important pathogenic group including complex species, so called Burkholderia cepacia complex (BCC). In general, BCC strains have CepR-I system for QS, but B. vietnamiensis, a BCC strain has an additional QS system, BviI-R, producing N-octanoyl-HSL, N-hexanoyl-HSL, Ndecanoyl-HSL, N-dodecanoyl-HSL, and N-3-oxodecanoylHSL. In this study, we show that signals produced by B. vietnamiensis can modulate QS regulation of P. aeruginosa through the selective activation of QscR, and as consequence, influence on virulence of P. aeruginosa. [This work was supported by the grants from NVRQS of MIFAFF.] 172 Poster Sessions D017 D019 Modulation of Quorum Sensing and Virulence by an Unknown Function Operon, PA4350~PA4351 in Pseudomonas aeruginosa Analysis of Quorum Sensing Signal Production of Pseudomonas aeruginosa Clinical Isolates from Korean Patients Yusang Choi*, Soo-Kyoung Kim, Changwan Ha, Ha-Young Park, Su-Jin Im, and Joon-Hee Lee Department of Pharmacy, College of Pharmacy, Pusan National University Kyung-Ju Jung*, Yusang Choi, Changwan Ha, Ha-Young Park, Su-Jin Im, and Joon-Hee Lee Department of Pharmacy, College of Pharmacy, Pusan National University Quorum sensing (QS) is a bacterial communication strategy that enables bacteria to bear group behaviors. QS in P. aeruginosa influences the properties related to its infectivity, such as virulence, motility and biofilm formation. P. aeruginosa has three well-defined QS systems, LasR-I, RhlR-I and QscR. LasI and RhlI synthesize N-3-oxododecanoyl-HSL and N-butanoyl-HSL that bind to their cognate receptors, LasR, QscR, and RhlR. The signal-receptor complexes activate the transcription of their target genes. An unknown function operon, PA4350~1 was found to influence the activities of LasR and QscR differentially. When PA4350~1 was overexpressed, both LasR and QscR were significantly inhibited, but to a different extent, where QscR was more strongly inhibited than LasR. The PA4350~1 overexpression significantly alleviated the P. aeruginosa virulence in insect and nematode infection models, and influenced on the motility and biofilm formation, which are crucial properties for the P. aeruginosa infectivity. We suggest that the function of PA4350~1 operon can reduce the virulence of P. aeruginosa through the modulation of QS regulation. Pseudomonas aeruginosa is an opportunistic pathogen causing various infections on lung, urinary tract, eyes, and burn wound sites. P. aeruginosa possesses three well-defined quorumsensing (QS) systems, LasR-I and RhlR-I, and QscR. LasR-I consists of a signal receptor, LasR and a signal synthase, LasI that synthesizes 3-oxo-C12-HSL. RhlI-R comprises a signal receptor, RhlR and a signal synthase, RhlI that produces C4HSL. The third LasR-RhlR homologue, QscR has no cognate signal synthase gene, but shares 3-oxo-C12-HSL for its signal with LasR. To investigate the importance of QS in Pseudomonas infection in Korea, we isolated clinical strains from Korean patients and QS signal was detected by signal diffusion assay on solid plates containing X-gal for colorimetric detection. Our result showed that most clinical isolates produced QS signals at the similar level to the wild type PAO1, but significantly lower level production was detected in about 5% of them. Here we also present some additional tests for virulence factor production, biofilm formation, and motility in the clinical isolates. D018 Development of Inhibitors Against Virulence of Pseudomonas aeruginosa Soo-Kyoung Kim*, Yusang Choi, Ha-Young Park, Changwan Ha, Su-Jin Im, and Joon-Hee Lee Department of Pharmacy, College of Pharmacy, Pusan National University Pseudomonas aeruginosa is an opportunistic pathogen that mainly relies on quorum sensing (QS) and biofilm formation for its virulence. As central control of virulence, QS regulates the expression of many genes related to virulence factor production and biofilm enhances the persistence against challenge by host immunity and antibiotic medication. In P. aeruginosa, QS is mediated by two small diffusible aclyhomoserine lactones (acyl-HSLs), N-3-oxododecanoyl-HSL (3OC12-HSL) and N-butyl-HSL (C4-HSL) which are synthesized by two signal synthases, LasI and RhlI, respectively. These signals are recognized by three QS signal receptors, LasR, QscR, and RhlR. To control the virulence of P. aeruginosa, we tried to develop inhibitors against QS and biofilm formation. A series of QS signal analogues were synthesized based on in silico modeling analysis of QS receptor-ligand bindings and screened for anti-QS and antibiofilm activities. Some had a significant inhibition on either QS or biofilm formation, or both. To test the potential for an anti-Pseudomonas agent we investigated whether these compounds could alleviate the virulence of P. aeruginosa. D020 Temperature Dependent Secretome Analysis of Bacillus anthracis Sterns With CO2 and Plasmid Cues Sudipto Shahid*, Ji Hyun Park, Sang Hoon Kim, Kyoung Hwa Jung, and Young Gyu Chai Division of Molecular and Life Sciences, Hanyang University Analysis of secreted proteins in extracellular environment by the anthrax causative bug, Bacillus anthracis drew the key interest for the identification of potential novel virulence factors. Differential culture condition, presence or absence of CO2, chromosomal and plasmid regulators modulates the secreted proteins and were identified. Temperature is one of the host-related signals affecting transcription of the toxin and capsule genes. In our study a comparative proteomic approach was employed to analyze the secreted proteins (secretomes) of two strains of Bacillus anthracis in response to different temperature. In minimal medium under high CO2 tension, different temperature was introduced. A unique pattern of protein abundance was revealed when extracellular proteins were subjected for 2-DE. Computer assisted image analysis followed by 2-DE facilitated the identification of unique, upor down- regulated proteins. Total 41 proteins encoded on chromosome or pXO1 were identified by peptide mass fingerprinting. Several proteins considered as putative virulence factors were observed represents the insights of temperature and plasmid cues in Bacillus anthracis secretome. 173 www.msk.or.kr D021 D023 Lactobacillus sakei Probio 65: A Functional Probiotic for Atopic Eczema-Dermatits Syndrome 1* 1 1 Yong-Ha Park , Jeongheui Lim , Byeong Joo Seo , 1 2 1 V. J Rejish Kumar , Young-Mi Jung , Hongik Kim , 3 2 2 4 Yoonhwa Jeong , Han-Ki Lee , Insun Lee , Kook Hee Kang , 5 6 5 Sung-Il Woo , Nam-Shik Kim , and Youn-Soo Hahn 1 Department of Applied Microbiology and Biotechnology, Yeungnam University, 2Institute of Probionic, Korea Research Institute of Bioscience and Biotechnology, 3Department of Food Science and Nutrition, Dankook University, 4Department of Food Science and Biotechnology, Sungkyunkwan University, 5Department of Pediatrics, College of Medicine and Medical Research Institute, Chungbuk National University, 6 Department of Preventive Medicine, College of Medicine and Medical Research Institute, Chungbuk National University Lactobacillus sakei Probio 65 was isolated from kimchi, a traditional Korean fermented food. This strain was resistant to gastric acidity, bile, and several antibiotics and possessed antimicrobial activity against a range of pathogenic microorganisms. To investigate whether the probiotic activity of L. sakei probio 65 was effective for treating allergic dermatitis, the organism was supplied to mice triggered by allergen (1chloro-2,4-dinitrobenzene). Mice that received L. sakei Probio 65 showed a more rapid recovery compared to control mice, as assessed by visual evaluation of the severity of allergic dermatitis and levels of immunoglobulin (Ig) E and interleukin (IL)-4. Probio 65 exhibited good probiotic properties in vitro and in mice and was effective in reducing allergen-induced skin inflammation through the regulation of both elevated IgE and IL-4 in sensitized mice. Supplementation of L. sakei Probio 65 in children with atopic eczema-dermatitis syndrome (AEDS) resulted in substantial clinical improvement and a reduction in severity of AEDS evidenced by a significant decrease in chemokine levels. Evaluation of Antimicrobial Effect of Ursolic Acid Using Antimicrobial Model System for Screening of Anti-Caries Agent Chun Sung Kim1*, Jae-Yoon Park2, and Joong-Ki Kook1 Department of Oral Biochemistry, School of Dentistry, Chosun University, 2Department of Biochemistry and Molecular Biology, Medical School, Chosun University 1 The purpose of this study was to determine the antimicrobial activity of ursolic acid against mutans streptococci isolated from oral cavity of Koreans. The antimicrobial activity was evaluated by minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill curves on mutans streptococci (MS). We were tested by MTT assay in human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (PLG). MIC90 values of ursolic acid for Streptococcus mutans, Streptococcus sobrinus and Streptococcus downei isolated from Korean were 2 ug/ml, 4 ug/ml and 8 ug/ml, respectively. Ursolic acid had bactericidal effect on MS. Cytotoxicity values were 16 ug/ml on HGF and PGL for ursolic acid. The results indicate that ursolic acid have antimicrobial activity at mutans streptococci and can be useful in the development of oral hygiene products for the prevention of dental caries. [This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea. (grant number: A091074)] [Supported by grants from Ministry of Health & Welfare and Yeungnam University] D022 D024 Antimicrobial Effects of Methanol Extracts of Phellodendric cortex, Coptidis rhizoma and Galla rhois on Mutans Streptococci Isolated from Koreans Development of Prevotella intermedia-Specific PCR Primers Based on the P. intermedia-Specific DNA Probe Chun Sung Kim1*, Jae-Yoon Park2, and Joong-Ki Kook1 1 Department of Oral Biochemistry, School of Dentistry, Chosun University, 2Department of Biochemistry and Molecular Biology, Medical School, Chosun University Min Jung Kim1*, Jae-Yoon Park2, and Joong-Ki Kook1 1 Department of Oral Biochemistry, School of Dentistry, Chosun University, 2Department of Biochemistry and Molecular Biology, Medical School, Chosun University The purpose of this study was to determine the antimicrobial activity of methanol-extracted Phellodendirc cortex (P. cortex), Copitidis Rhizoma (C. Rhizoma) and Galla rhois (G. rhois) against mutans streptococci (MS) isolated from Koreans. The antimicrobial activity was evaluated by minimal bactericidal concentration (MBC) against clinical strains of MS as well as type strains of Streptococcus mutans and Streptococcus sobrinus and time kill-curve assay on type strains of S. mutans and S. sobrinus. MBC90 values of P. cortex and C. Rhizoma were 1 mg/ml against clinical strains of MS isolated from Korean. MBC90 values of G. rhois for S. mutans and S. sobrinus isolated from Korean were 2 mg/ml and 1 mg/ml, respectively. Methanol-extracted P. cortex, C. Rhizoma, and G. rhois had bactericidal effect on MST. The results suggest that methanol-extracted P. cortex, C. Rhizoma, and G. rhois could be useful in developing of oral hygiene products. The purpose of this study is to develop the Prevotella intermedia-specific PCR primers based on the P. intermediaspecific DNA probe. The P. intermedia-specific DNA probe was screened by inverted dot blot hybridization and confirmed by Southern blot hybridization. The nucleotide sequences of species-specific DNA probes were determined by chain termination method. The specificity of the PCR primers was tested against 6 clinical isolates of P. intermedia, 10 clinical isolates of Prevotella nigrescens, and 20 type strains of oral bacteria. The sensitivity of PCR primers was determined by testing serial dilutions of the purified genomic DNA of P. intermedia ATCC 49046. The data showed that the two sets of PCR primers, Pig27-F2/Pig27-R2 and Pig27-F5/Pig27-R5 had the species-specificity for the P. intermedia ATCC 49046. The detection limits of four primer sets were 0.4 pg-4 pg of the purified genomic DNA of P. intermedia ATCC 49046. These results suggested that the two sets of PCR primer, Pig27F2/Pig27-R2 and Pig27-F5/Pig27-R5, could be useful in the development of PCR kit for the epidemiological studies and bacteriological diagnosis or prognosis for the endontitis and periodontitis. [This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea. (grant number: A091074)] 174 Poster Sessions D025 Differential Gene Expression in Two Phase Variants of Streptococcus pneumoniae Sungkyoung Lee*, Jaehoon Lee, Yeonho Kang, Jaeyon Yu, and Songmee Bae Division of Bacterial Respiratory Infections, Center for Infectious Diseases, National Institute of Health, Korea Centers for Disease Control and Prevention Pneumococcal invasive diseases such as pneumonia, bacteremia, and meningitis can be the result of infection of the bacteria from upper respiratory tract or lung to blood or cerebrospinal fluid. Pneumococcal phase variation related with invasion to host has been described, from transparent phase for colonization to opaque phase for invasion. To gain more understanding on invasive pneumococcal pathogenesis, we analyzed the transcriptome profiles of S. pneumoniae in opaque phase to transparent phase by microarray analysis. Each opaque and transparent colony of S. pneumoniae D39 was collected as uniform, respectively and used for microarray analysis. The 2,240 oligo probes were designed on the basis of full genome data of S. pneumonaie D39. Total 148 genes with altered transcription of opaque to transparent variants were sorted by criteria of a >2-fold change in levels of expression and a P value of <0.05. At least 20 genes including lacA, lacB, lacC, aga, bgaA, and bgaC, related with carbohydrate metabolism, were highly upregulated in opaque phase. Results suggest that some of these genes are involved in pneumococcal pathogenesis. D027 Antiviral Activity Against Human Influenza Virus H1N1 (PR8) From Aqueous Extract of Fritillaria thunbergii Nguyen Dinh Van*, Jae-Yu Choi, Youn-Dong Cho, Jang-Hyun Lee, Hyun-Dong Paik, and Young Bong Kim College of Animal Bioscience and Technology, Konkuk University More than fifty extract samples from different Korean traditional herbal remedies were screened for the antiviral activity against the human influenza virus A/Puerto Rico/8/34 H1N1 (PR8) and toxicity by the in vitro system on MDCK cells. The medical dried plants were extracted by two methods, using methanol solvent at room temperature or boiling in water. The extracted solutions were filtered, evaporated and then evaluated the antiviral ability against H1N1 (PR8). Among the different potential herbal remedies, Fritillaria thunbergii (F. thunbergii) extract from the boiling water method showed meaningful antiviral ability with high selectivity index (SI) value, nearly 64, compared with Tamiflu? (Oseltamivir, Roche) with 96 SI. To confirm the antiviral activity of F. thunbergii extract, we tested it in ovo system. From this research; our findings indicated that F. thunbergii has a good inhibition effect on replication of the human influenza virus H1N1 with low toxicity. This new compound is expected to be a new drug candidate for anti-influenza therapeutics medicines in the future. [Supported by a grant from Korea CDC.] D026 D028 Global Transcriptome Analysis of Streptococcus pneumoniae Response to Hydrogen Peroxide The Impact of Storage Duration on Bacillus anthracis H9401 Spores Songmee Bae*, Sungkyoung Lee, Jaehoon Lee, Jaeyon Yu, and Yeonho Kang Division of Bacterial Respiratory Infections, Center for Infectious Diseases, National Institute of Health, Korea Centers for Disease Control and Prevention Hyun-Jung Kim*, Jeong-Hoon Chun, Hong-Mi Kim, Gi-Eun Rhie, Cheon-Kwon Yoo, and Hee-Bok Oh Division of High-Risk Pathogen Research, Center for Infectious Disease, National Institute of Health, KCDC Oxidative stress is a crucial stimulus for pathogenic bacteria because pathogens are exposed to reactive oxygen species produced by the immune response of the host during infection. We designed S. pneumoniae D39 microarray chips and investigated the dynamics of gene expression profiles during the response of S. pneumonia to oxidative stress by 2mM hydrogen peroxide. As a result, 128 and 186 genes showed significant increases and decreases, respectively, based on criteria of a greater than 2-fold change in mRNA levels and a P value of less than 0.05. From the analysis of their cellular role of total 314 genes by EMPAS program, the data indicate that the oxidative response includes the induction of genes involved in virulence, repair systems, and carbohydrate metabolisms, etc. Particularly, expression of genes encoding chaperone such as dnaK, dnaJ, and grpE and a gene encoding an alcohol dehydrogenase were upregulated upon early 5 min exposure of hydrogen peroxide. Herein, we showed the transcriptome profiles of the cellular response of S. pneumoniae to hydrogen-peroxide induced oxidative stress. [Supported by a grant from Korea CDC.] We has been producing and storing Bacillus anthracis H9401 strain spores in support of vaccine and therapeutic testing programs against anthrax for over ten years. Spore suspensions are either stored at 2-8°C and ≤-70°C in sterile water with 1% phenol, 1% pheonol-10% glycerol, 10% glycerol, 70% ethanol or 0.1% gelatin-PBS buffer. The goal of this research is to determine the most appropriate long-term storage conditions of Bacillus anthracis spore suspensions by examining the impact of storage duration, media, and temperature on spore characteristics. To date, we have studied spores placed into storage at regular intervals beginning August 2007 through July 2009. Enumeration of spore stored at 2-8°C in all storage medias were consistently within ± 0.09 log of initial concentrations(CFU/ml). Five spore lots stored for 24 months have been tested via guinea pig lethal dose (LD50) with i.m. injection and results indicated no change in the LD50 value at 2-8°C. These initial results led to design and implementation of a two year controlled study to examine the impact of storage media, temperature, and duration on viable spore counts and virulence. [Supported by grants from KCDC] 175 www.msk.or.kr D029 D031 Immune Responses of Human Papillomavirus 16/18 Bivalent DNA Vaccine; AcHERV-HP16L1 and AcHERV-HP18L1 Quorum Sensing of Pseudomonas aeruginosa Is Abnormally Controlled Under Anaerobic Growth Condition Hee-Jung Lee1*, Yoon-Ki Hur1, Jae-Yu Choi1, Hee-Jung Cho2, Yu-Kyung Oh2, and Young Bong Kim1 1 Department of Ainmal Biotechnology, Konkuk Univeristy, 2 School of Pharmacy, Seoul National University YongJin Park*, Mi Young Yoon, Kang-Mu Lee, and Sang Sun Yoon Department of Microbiology, College of Medicine, Yonsei University Vaccination against the most common oncogenic human papillomavirus (HPV) types, HPV 16 and HPV 18, could prevent development of up to 70% of cervical cancers worldwide. We developed a novel DNA vaccine for HPV; a recombinant baculovirus bearing human endogenous retrovirus (HERV) envelope protein, which cannot replicate in mammals, was used as a nano carrier for HPV-L1 DNA vaccines (AcHERV-HP16L1, AcHERV-HP18L1). For in vivo test, mice were injected intramuscularly with 107 particles of the constructs, with two boosts at 2-week intervals. Compared with Cervarix?, or Gardasil? (1/20 of human dose), the AcHERV-HP16/18L1 immunized mice showed similar high levels of humoral immunity in IgG/IgA and elicited strong HPV16/18L1-specific CD8+ T cell immunity. Therefore, we suggest that AcHERV-HPV16L1 and AcHERV-HP18L1 vaccine can be further developed as a potential DNA vaccine which can provide both humoral and cell-mediated immune responses. Pseudomonas aeruginosa causes chronic airway infections in cystic fibrosis. Recently, abnormally thickened mucus layer lining the patient airway was proved to be anaerobic. But, how virulence features of this pathogen are modulated upon anaerobic growth is largely unknown. Secretion of elastase, a virulence factor whose production is controlled by LasI-R quorum sensing, was highly suppressed under the anaerobic growth condition. qRT-PCR analysis showed that transcription of elastase-coding lasB gene was completely abrogated in anaerobically grown PAO1 suggesting that repressed elastase secretion is due to the regulatory modulation that occurs at the transcriptional level. These results suggest that a certain anaerobiosis-induced factor may bind specifically to lasB promoter to suppress its transcription. Together, our data suggest that anaerobiosis-induced metabolic shift causes a modulation of QS machinery in P.aeruginosa. D030 [This study was supported by Yonsei University College of Medicine, Intramural Grant Support for Starting Faculty, 62009-0055 to SSY and National Research Foundation of Korea funded by the Ministry of Education, Science and Technology, 7-2009-0524 to SSY.] D032 Evaluation of the Immune Response and Protection Against Botulinum Neurotoxin Type A in ToxoidImmunized Mice Antibiotic Resistant Characteristics and Identification of Bacteria Isolated from Diarrheic Feces in Korean Native Beef Calves Jeong-Hee Kim*, Na-Ri Shin, YunJeong Kim, Gi-eun Rhie, and Cheon-Kwon Yoo Division of High-risk Pathogen Research, Center for Infectious Diseases, National Institute of Health Yun-Jung Lee*, Jae-Won Park, Sang-Buem Cho, and Soo-Ki Kim Department of Animal Sciences and Environment, Konkuk University Botulism occurs by the intoxication with botulinum neurotoxin, resulting in flaccid paralysis. Botulinum neurotoxoid is a major component which has been classically used for protection against botulism. In this study, we examined immune responses against botulinum neurotoxin type A (BoNT/A) in mouse model. Two mouse strains, BALB/c and C57BL/6, were immunized three times with BoNT/A toxoid and we have challenged with BoNT/A. BoNT/A challenge into mice showed that complete protection against BoNT/A. In order to know the aspect of immune response in challenged mice, we measured IgG subclass in sera and, in vitro, spleen cell cytokine secretion in response to BoNT/A 4 weeks after last vaccination. In mouse sera, IgG1 antibodies were predominantly developed by BoNT/A vaccination in both strains. In cytokine assay, IL-4, IL-5, IL-10 and IL-13, Th2-type cytokines, were detected, and Th1-type cytokines, IL-2 and IL12, were also detected in spleen cell culture supernatants. But TNF-α and IFN-γ were not detected. These data suggest that the spleen cells from BoNT/A-vaccinated mice might be a mixed Th1/Th2 cells although Th2 response is predominant against BoNT/A toxoid in sera. Diarrhea in new born calves is serious disease which induces high mortality, delayed growth and economical loss in farm. Escherichia coli is an important causative factor in diarrhea. Bacterial infection and virulence is highly related to quorum sensing (QS). QS has been regarded as cell-to-cell communication and virulence of pathogenic bacteria. Therefore, understanding of QS is a promising way to prevent disease. In this study, bacterial strains related to QS were screened from the fecal specimen of Korean native calves that showed the symptom of diarrhea. Eleven strains were identified as E. coli using 16S rRNA gene analysis and their phenotype and Caenohabditis elegans killing activity were investigated. In swimming activity, 2201, 2310, 2312 and 2318 showed low activity and 2205, 2247, 2292, 2298, 2337, 2341 and 2387 were high in activity. In swarming activity, 2201, 2205, 2292, 2298, 2310, 2312 and 2318 were low and 2247, 2337, 2341 and 2387 were high. In protease activity, only 2292, 2337 and 2341 showed activity. In biofilm production, all of isolates showed similar activity. In C. elegans killing assay, 4 to 9 days of survival were detected. 176 Poster Sessions D033 D035 Screening of Burkholderia sp. Diguanylate Cyclase/ Phosphodiesterase Mutant Using Caenorhabditis elegans Model and Analysis of Its Phenotypes * Yun-Jung Kim , Yun-Jung Lee, Sang-Buem Cho, and Soo-Ki Kim Department of Animal Sciences and Environment, Konkuk University Quorum sensing(QS) controls various mechanisms in bacteria such as the expression of virulence factor, biofilm formation and luminescence. Similar to QS, cyclic di GMP also controls these mechanisms, however its function shows opposite way to QS and a negative feedback control between QS and c-di-GMP also has been reported. c-di-GMP is produced by diguanylate cyclase(GGDEF) and degraded by phosphodiesterase(EAL). Recently, it has been reported that GGDEF and EAL control the concentration of c-di-GMP and depending upon its concentration, bacterial motility, biofilm formation, virulence and extracellular saccharide production are controlled. Especially, high virulence activity was found at low concentration of c-diGMP. In this study, virulence and QS of Burkholderia sp. were investigated by transposon mutagenesis and C. elegans killing assay. Through the screening of mutants, the strain that showed lower C.elegans killing activity than wild type was isolated and the mutagenesis was detected at protein coding region which produce RNaseII stabilizer including GGDEF and EAL in Burkholderia sp. 383 chromosome 2. Subsequently, various phenotypes of isolated strain were investigated. D034 Proteomic Analysis of Secretory Proteins from the Streptococcus pneumoniae BAA-255 Chi Won Choi*, Sang Oh Kwon, Sung Ho Yun, and Seung Il Kim Division of Life Science, Korea Basic Science Institute Streptococcus pneumoniae is an important human pathogen that causes a variety of diseases, such as pneumonia, bacteremia, meningitis, otitis media, and sinusitis, in both adults and children. Especially, the morbidity and mortality rates associated with S. pneumoniae remain very high worldwide. Because of their clinical importance, genome sequencing of more than 11 stains were completed and available. However, proteomic studies of these stains were not advanced up to now. In the present study, supernatant of secreted proteins of S. pneumoniae BAA-255 were enriched with ammonium sulfate precipitation and 1-DE LC-MS/MS was performed to identify secretory proteins of S. pneumoniae. 48 proteins (21.5% of total identified proteins) were analyzed to have the signal peptide. 8 cell wall and 6 extracellular proteins were included in the identified proteins. Among the identified secretory proteins, PrtA, AmiA, ZmpB, NanA, Gsp781, and GlnP were known virulence factors. Our result suggests that secretome analysis of S. pneumoniae is useful approach to identify the virulence factors or biomarkers for diagnosis and drug targets. [Supported by grant from Korea Basic Science Institute KMeP] D036 Screening of a Virulence Gene in Burkholderia sp. by Using Caenorhabditis elegans Model Role of National Culture Collection for Pathogens (NCCP) As a Central Pathogen Bank in Korea Young-Hee Lee*, Yun-Jung Kim, Yun-Jung Lee, Sang-Buem Cho, and Soo-Ki Kim Department of Animal Sciences and Environment, Konkuk University Kyenam Lee, Juhee Heo, Kyung-Tae Jung, Hae-Seul Jeong, Won-Sun You, So-Jung Yoon, Cheon-Kwon Yoo, and Kwang-Jun Lee Division of High-risk Pathogen Research, Center for Infectious Disease, National Institute of Health, KCDC, Korea Burkholderia sp., a gram negative bacteria, has been regarded as a pathogenic bacteria causing respiratory disorder in animal and it is also occasionally causative to human. Especially, the infection in the patient of cystic fibrosis is critical because it reduced pulmonary function and increase mortality. Recently, Caenorhabditis elegans has been used for the research of bacterial virulence because various pathogenic bacteria or fungi showed killing activity against C. elegans. In this study, to construct transposon library, the conjugation between E. coli that carrying pRL27 and Burkholderia sp. was performed. To search virulence gene of Burkholderia sp., replica with five thousands Tn mutants was performed on 96-well plate containing both of NGM (kanamycin 25 ug/mL, ampicillin 50 ug/mL) and five to ten of C. elegans which is on L4 larval stage. One hundred six mutants that showed lower killing activity than wild type were found. Subsequent killing assay was performed for the verification. Finally, nineteen mutants that lost virulence were isolated. The phenotypes such as biofilm formation, swimming/swarming activity and protease were investigated with isolated mutants. National Culture Collection for Pathogens (NCCP) is a national pathogenic resource bank whose function focuses on the deposition, preservation, development and distribution of high quality pathogens including high-risk pathogens. NCCP was established in 1972 by KNIH, joined the WFCC in 2004. According to the international criteria, NCCP was officially certified for ISO 9001 in 2009 and strictly managed to become the global leader. NCCP has established the network, 3 of specialized pathogen banks in KNIH for pathogens isolated from infectious diseases patients designated by law, 2 of collaborative pathogen banks for special pathogens difficult to collect, and 3 of regional pathogen banks for pathogens isolated from clinical specimens with clinical information. NCCP has provided financial support as well as standard operation protocols for management of unit banks and quality control of pathogens, and established the network between central and unit banks. Informational network was constructed and operated by BIMS. NCCP works hard to provide high quality services to the whole research community and to connect with other resource centers to play the role of a central pathogen bank. 177 www.msk.or.kr E001 E003 Characterization of a Novel β-Mannanase from Paenibacillus sp. HP-8 Isolated from the Gut of Moechotypa diphysis Enzymatic Characteristics of a Modular Xylanase (XylE) from Cellulosimicrobium sp. AG-56 Overproduced in Escherichia coli BL21 Su-Jin Ham1*, Do Young Kim1, Lan Hee Lee1, Hyun Ju Lee1, Kyung Sook Bae2, Kwang-Hee Son1, and Ho-Yong Park1 1 Industrial Bio-materials Research Center, Korea Research Institute of Bioscience and Biotechnology, 2Biological Resources Center, Korea Research Institute of Bioscience and Biotechnology Hyun Ju Lee*, Do Young Kim, Su-Jin Ham, Lan Hee Lee, Kwang-Hee Son, and Ho-Yong Park Industrial Bio-materials Research Center, Korea Research Institute of Bioscience and Biotechnology Paenibacillus sp. HP-8 isolated from the gut of a longicorn beetle, Moechotypa diphysis, secreted a β-mannanase (ManH) a molecular mass of approximately 32.0 kDa when cultivated on a M9 medium containing 0.5% locust bean gum. In contrast to other well-known mannanases, ManH bound weakly to a cation exchanger such as CM-Sepharose. The ManH contained an amino acid sequence of APSFAVGADFSYVPG at its Nterminus, which did not show any high sequence identity with other similar enzymes. The maximum hydrolysis activity of ManH toward locust bean gum was observed at pH 7.5 and 55°C where the enzyme retained 50% of its original activity for 25 min. The enzyme was completely inhibited in the presence of 1 mM Hg2+ and 5 mM N-bromosuccinimide. In addition, partial inhibition (>20% of its original activity) of ManH was observed when incubated with some metal ions Ni2+, Fe2+, Ca2+, Zn2+, Cu2+, and Sn2+. The specific activity of ManH toward locust bean gum was determined to be 7,804 IU/mg, indicating that it is a highly active β-mannanase. E002 The novel modular xylanase (XylE) consisting of an Nterminal catalytic GH10 domain, fibronectin type 3 (Fn3) domain, and C-terminal carbohydrate-binding module 2 (CBM 2) from Cellulosimicrobium sp. AG-56 that was generated by the mutation of T440A was biochemically characterized and the enzymatic properties of XylE were compared to its processed forms. The maximum catalytic activity of XylE toward birchwood xylan was observed at pH 6.5 and 40°C. The enzyme retained >80% of its original activity at the range of pH 6.0-7.0 when preincubated for 1 h at 4°C. XylE was thermolabile because the enzyme lost >30% of its activity even at 35°C after the preincubation of 1 h. The binding affinities of XylE to various types of insoluble carbohydrate polymers clearly suggested that Fn3 and/or CBM 2 participated in substrate-binding because the Fn3-CBM 2-lacking XylE did not bind to glucose-based polysaccharides. E004 Characterization of a Novel β-Mannanase from Cellulosimicrobium sp. AG-56 Isolated from the Gut of Eisenia fetida Acid Tolerance Response (ATR) was not Induced in Leuconostoc mesenteroides ATCC 8293 by PreAdaptation in Mild Acid Condition Su-Jin Ham1*, Do Young Kim1, Hyun Ju Lee1, Kyung Sook Bae2, Kwang-Hee Son1, and Ho-Yong Park1 1 Industrial Bio-materials Research Center, Korea Research Institute of Bioscience and Biotechnology, 2Biological Resources Center, Korea Research Institute of Bioscience and Biotechnology Ji Eun Kim* and Nam Soo Han Department of Food Science and Technology, Research Center for Bioresource and Health, Chungbuk National University The gut bacterium of Eisenia fetida, Cellulosimicrobium sp. AG-56 was able to efficiently degrade mannans as well as xylan polymers. This organism produced a highly active βmannanase (ManA) with a molecular mass of approximately 37 kDa when cultivated on a M9 medium containing 0.5% locust bean gum as a sole carbon substrate. The highest catalytic activity of ManA toward locust bean gum was measured at 65°C in glycine-NaOH buffer (pH 9.0). At 65°C, the half-life of the enzyme was evaluated to be approximately 10 min. ManA was completely inactivated in the presence of 1 mM Hg2+ and 5 mM N-bromosuccinimide but not affected considerably by other divalent cations and chemical reagents such as Fe2+, Mn2+, Cu2+, Co2+, EDTA, sodium azide, and Triton X-100. The enzyme could decompose mannosic polysaccharides such as locust bean gum and guar gum but not degrade CMC, birchwood xylan, starch, and p-nitrophenolsugar derivatives. The molecular characteristics of the enzyme (ManA) will be also presented. 178 Poster Sessions Leuconostoc mesenteroides is commercially important lactic acid bacterium currently used as a starter culture for kimchi. However it has been tackled due to the acid sensitivity of this species. Induced acid tolerance defines a condition whereby, during exposure to mild acidic conditions, bacteria acquire the ability to survive lethal acid concentrations. This inducible mechanism is referred to as the acid tolerance response (ATR) This study was carried out to investigate the induction effect of pre-adaptation of L. mesenteroides ATCC 8293 in mild acid condition (pH 5.0) for 1, 2, and 3h for resistance to normally lethal intensity of acidic stress (pH 4.0). The survival of acidadapted and non-adapted cells of L. mesenteroides in phosphate buffer solution (PBS pH 4.0) revealed that acid adaptation has no influence on the increased tolerance of L. mesenteroides in PBS (pH 4.0).When compared the initial CFU/ml, the non-adapted cells and acid adapted cells were both decreased approximately 100 and 10000 fold subsequently during the incubation. This result has shown that acid tolerance is not induced by acid pre-adaptation and instead, pre-acid exposure may damage L. mesenteroides. E005 E007 Bacterial Gamma-Glutamyltranspeptidase as a Novel Bone-Resorbing Factor The Involvement of Bacteriophytochromes in Quorum Sensing in Rhodobacter sphaeroides 2.4.1 Jinmoon Kim*, Ho Gil Choi, Minyoung Kim, Eun Jung Bak, and Jeong-Heon Cha BK21 Project, Yonsei University, College of Dentistry Hae-Seon Kim1,2*, Ki-Hoon Yang1,2, Kwang-Eun Jung1,2, and Jeong-Il Oh1,2 1 Department of Microbiology, 2Pusan National University γ-Glutamyl Transpeptidase(GGT) was recently identified as a bone-resorbing factor. GGT is widely distributed in living organisms, and Bacillus subtilis GGT exhibits high similarity to the mammalian GGTs. B. subtilis and periodontitis-related Fusobacterium nucleatum GGTs were examined to determine if they could induce osteoclastogenesis. Recombinant GGTs (rGGTs) of B. subtilis and F. nucleatum purified in E. coli were treated to the co-cultures of osteoblast and bone marrow macrophages. All rGGTs stimulated osteoclastogenesis and expression of RANKL. Also, large subunit of B. subtilis rGGT induced osteoclastogenesis much more than its small subunit. In addition, osteoclastogenesis induced by rGGT was blocked by OPG. We present that bacterial GGT is able to induce osteoclastogenesis via RANKL-dependent pathway and osteoclastogenic activity of B. subtilis GGT is mostly located in its large subunit. We suggest that GGT of periodontopathogens can cause bone destruction in periodontitis. Bacteriophytochrome (Bph) is a red/farred-responsive photoreceptor in bacteria. Bphs were identified to regulate photosystem synthesis in many photosynthetic bacteria. Rhodobacter sphaeroides 2.4.1, one of the purple non-sulfur photosynthetic bacteria, has two Bphs (BphG1, BphG2). Unlike most Bphs in photosynthetic bacteria, BphG1 and BphG2 contain the GGDEF and EAL domains. They are involved in synthesis and degradation of c-di-GMP which is a second messenger used in bacterial signal transduction, especially quorum sensing. The expression of cerI and cerR encoding the autoinducer synthase and its transcriptional activator, respectively, in the wild-type strain of R. sphaeroides was increased in a cell-density dependent manner and reached the maximum level in stationary phase. In contrast, their expression in the BphG12 double mutant was fully derepressed even in the low cell density and remained constant regardless of the cell density. The secretion of exopolysaccharide in the BphG12 double mutant was substantially increased when compared with WT strain. On the basis of these results, we suggest that Bphs might be involved in the regulation of quorum sensing in R. sphaeroides. [This research was supported by Basic Science Research Program through the National Research foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (R13-2003-013-04002-0).] E006 E008 Inhibitory Effect of Wogonin on LPS-Induced Osteoclastogenesis Tartrate/Succinate Antiporter and Its Regulation in Escherichia coli Jinmoon Kim1,2*, Sungil Jang1,2, Eun Jung Bak1,2, Minyoung Kim1,2, Won-Yoon Chung1,2, Jeong-Heon Cha1,2, and Yun-Jung Yoo1,2 1 Department of Oral Biology, BK21 Project, Oral Science Research Center, and Research Center for Orofacial Hard Tissue Regeneration, Yonsei University College of Dentistry, 2 Department of Applied Life Science, The Graduate School, Yonsei University Ok Bin Kim1,2* and Gottfried Unden2 1 Department of Life Science, Ewha Womans University, 2 Institute of Microbiology and Wine Research, University of Mainz, Germany To evaluate the inhibitory activity of wogonin against LPSinduced bone resorption, we treated LPS and/or wogonin to the co-cultures of osteoblasts and pre-osteoclasts and mouse calvaria, and measured osteoclast differentiation and level of RANKL, OPG and PGE2. Wogonin inhibited LPS-induced osteoclastogenesis in co-cultures and LPS-injected mouse calvaria. In osteoblasts, the up-regulation of RANKL and the down-regulation of OPG by LPS were inhibited by wogonin. Wogonin and NS-398, a COX-2 inhibitor, suppressed LPSstimulated PGE2 production in osteoblasts. NS-398 inhibited the effect of LPS on RANKL and OPG expression in osteoblasts. These results suggest that wogonin acts as an inhibitor of LPS induced osteoclastogenesis through downregulation of RANKL and up-regulation of OPG via blockage of PGE2 production. Thus, wogonin has the potential for use as a therapeutic agent in bacteria-induced bone resorption. [This research was supported by Basic Science Research Program through the National Research foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (R13-2003-013-04002-0).] E. coli fermentates L-tartrate by the use of L-tartrate/succinate antiporter TtdT and L-tartrate dehydratase TtdAB. The transporter TtdT is required for the uptake of L-tartrate. The growth by L-tartrate and degradation of L-tartrate are destroyed by the deletion of ttdT. The gene ttdT is located downstream of the ttdA and ttdB genes, encoding the L-tartrate dehydratase, and the three genes form an operon ttdABT. The transport modus of the secondary carrier TtdT and its relation to the general C4-dicarboxylate carriers were studied. TtdT catalyze L-tartrate or succinate uptake and specific heterologous L-tartrate/succinate antiport, preferentially in the direction of L-tartrate uptake and succinate efflux. The Dcu carriers do not support L-tartrate transport. Expression of the ttdABT operon was stimulated by the LysR-type regulator TtdR in the presence of L- and mesotartrate, and repressed by O2 and nitrate. Expression of ttdR was repressed by O2, nitrate and glucose, and positively regulated by TtdR and DcuS. Purified TtdR bound to the ttdRttdA promoter region specifically. [Supported by grants from DFG and Innovationsstiftung Rheinland-Pfalz] 179 www.msk.or.kr E009 E011 Generation of Phlebia tremellosa Transformants Generating Two Lignin Degrading Enzymes with a New Expression Vector Analysis of Pyranose Oxidase Expression Under Degradation Conditions of Diethylphthalate in Phlebia tremellosa Hyun-Woo Kum1*, Sun-Hwa Ryu2, Sung-Suk Lee2, and Hyoung T Choi1 1 Department of Biochemistry, Kangwon National University, 2 Division of Forest Bioenergy, Korea Forest Research Institute Baek Joong Kim*, Hyang Soon Lim, Hye Won Kim, and Hyoung Tae Choi Molecular Microbiology Lab, Department of Biochemistry, Kangwon National University A white rot basidiomycete Phlebia tremellosa secretes laccase and peroxidase which are involved in the degradation polymeric lignin and many kinds of endocrine disrupting chemicals (EDCs). P. tremellosa does not secrete manganese peroxidase (MnP) in various liquid media which is also a major component of the lignin degrading enzymes. In order to get high MnP and Laccase producing transformants of P. tremellosa, an expression vector (pBARPTLac-TVMnP) has been constructed using Trametes versicolor MnP cDNA and Plebia Tremellosa Laccase genomic DNA. Genetic transformation of P. tremellosa was successfully carried out using the restriction enzyme mediated integration (REMI). Integration of transforming plasmid has been confirmed using PCR with bargene specific primers and laccase, MnP specific primers. The expression of the introduced MnP and laccase under degradation conditions for endocrine disrupting chemicals was analyzed. Explosives, insecticides and plasticizers are degraded very slowly in nature, and show harmful effects by disturbing endocrine system even at low concentrations. The sex hormone system is the first target of these chemicals. White rot fungi, degrading lignin, can degrade various recalcitrant compounds including the endocrine disrupting chemicals (EDCs). Lignin peroxidase (Lip) and manganese peroxidase (MnP) are involved in lignin degradation as well as EDC degradation. They both require H2O2 for their degrading reactions. Pyranose oxidase is in charge of suppling H2O2in white rot fungi. We have isolated a pyranose oxidase cDNA from EDC-degrading culture of Phlebia tremellosa using the RACE-PCR technique. In order to find out the relationship between expression of pyranose oxidase and degradation EDCs by P. tremellosa, the fungus was grown in a minimal medium with or without diethylphthalate (DEP), and then the pyranose oxidase expression was compared by the RT-PCR. E010 E012 Expression of a Chitinase of Coprinellus congregates Using Acid Inducible Promoter Cloning of a Chitinase 2 Gene from Coprinellus congregatus Yu Ri Kang* and Hyoung Tae Choi Molecular Microbiology Lab, Department of Biochemistry, Kangwon National University Hye Won Kim*, Sun Uk Bak, and Hyoung Tae Choi Molecular Microbiology Lab, Department of Biochemistry, Kangwon National University Coprinellus congregatus highly expresses chitinase at matured mushroom stage. When the chitinase cDNA was induced in the budding yeast, the transformed yeast cells stopped their growth, and it was almost impossible to get large amounts of chitinase protein. For chitinase protein production, we have constructed an acid inducible expression system using the acidic laccase promoter which was isolated from C. congregatus. Acidic laccase (lac2) is only expressed under acidic culture conditions, and we have constructed an expression vector using the acidic laccase promoter and chitinase cDNA. This construct had been transformed to C. congregatus in order to get the expression the chitinase. The successful integration of the expression vector in the transformants was confirmed by PCR with vector-specific primers. Two monokaryotic strains having the constructs were mated to get dikaryons which showed acid induction of lac2. We expect the expression of the chitinase protein in the supernatant of acidic liquid medium (pH 4.1). We will perform the protein purification and its biochemical characterization. Fungal cell walls consist of various glucans and chitin. Two monokaryotic strains of C. congregatus, Cc16 and Cc44, were mated and cultured in 25°C for 5day to isolate RNA. A chitinase cDNA from the hyphal cells of C. congregatus was successfully isolated using the rapid amplification of cDNA ends (RACE)-PCR technique. The deduced amino acid sequence of the cDNA has the conserved catalytic domain as in other fungal chitinase family 18. It showed higher expression at the matured mushroom stage than the hyphal growth stage, and the new chitinase cDNA was designated as chitinase 2 gene (chi2). We have also performed cloning of the promoter region of chitinase 2 from C. congregatus using DNA walking and RACE-PCR techniques. We have successfully cloned the full-length chitinase gene involved in the autolysis of mushroom in C. congregatus. To figure out the function of the gene, we will construct of knock-out mutant of these gene. 180 Poster Sessions E013 E015 Functional Expression of Trametes versicolor Cellobiohydrolase and Phlebia tremellosa Laccase in a Brewing Yeast Novel Deblocking Aminopeptidases of Hyperthermophilic Archaeon Thermococcus onnurineus NA1 Sun Jue Park , Hye Young Kim, and Hyoung Tae Choi Molecular Microbiology Lab, Department of Biochemistry, Kangwon National University Yeol Gyun Lee1*, Sung Gyun Kang2, Jung-Hyun Lee2, Jong-Soon Choi1, Seung Il Kim1, and Young-Ho Chung1 1 Division of Life Science, Korea Basic Science Institute, 2Marine Biotechnology Center, Korea Ocean Research & Development Institute White rot basidiomycetes Phlebia tremellosa and Trametes versicolor have enzyme system for the degradations of polymeric lignin, cellulose and hemicellulose. In T. versicolor, the cellobiohydrolase (Cbh) separates one cellulose chain at a time from cellulose and hydrolyses it, then Cbh releases cellobiose from the ends of cellulose molecules. In order to get high cellobiohydrolase and laccase producing transformants of yeast, two expression vectors (YIPGTVcbh, YIPGPTlac) have constructed using T. versicolor cellobiohydrolase cDNA and P. trmellosa laccase cDNA. We have used glyceraldehyde-3phosphate dehydrogenase promoter (GPD promoter) to get constitutive expression. We have transformed the expression vectors to yeast in order to get transformants having increased degrading activities of lignin and cellulose. Transformation of yeast was successfully carried out using two expression vectors (YIPGTVcbh, YIPGPTlac), and we have confirmed an activity and an expression of two enzymes of yeast transfomants. We will perform one-step ethanol production from acid hydrolysate of wood using the transformants. It has been reported that one of the hyperthermostable deblocking aminopeptidase (DAP) from Thermococcus onnurineus NA1 (TNA1_DAP) exhibits hydrolytic activity toward short peptides and acyl-peptides. In the genome database of T. onnurineus NA1, three new open reading frames homologous to the DAP of T.onnurineus NA1 were found. The deduced amino acid sequences were similar to DAPs of pyrococcus furiosus and pyrococcus horikoshii, a member of peptidase family M42.The three new genes for the proteins were cloned and overexpressed in Escherichia coli. Molecular masses assessed by SDS-PAGE were 38kDa, 37kDa and 36kDa respectively, and exhibited aminopeptidase activity, including deblocking activity. One of the purified DAPs showed the optimum activity at 90°C and its half-life (t1/2) was 2.5 hr. It was confirmed that T. onnurineus NA1 has four similar aminopeptidases with deblocking activity and that these enzymes appear to play important roles in hydrolyzing small and acyl-peptides in T.onnurineus cells. * [Supported by the Marine and Extreme Genome Research Center Program of the Ministry of Land, Transport and Maritime Affairs] E016 E014 Medium Optimization for the Production of Nattokinase by Bacillus subtilis NK7-1 Using Response Surface Methodology 1* 2 1 Hee-Jong Yang , Moon Chang Kim , Nack-Shick Choi , Seong-Yoep Jeong1, Keug-Hyun Ahn1, Chan-Sun Park1, Yong-Il Hwang2, Byung-Dae Yoon1, and Min-Soo Kim1 1 Institute Bioindustry Research Center, Korea Research Institute of Bioscience and Biotechnology, 2Department of Food Science and Biotechnology, Kyungnam University Bacillus subtilis NK7-1 producing a potent fibrinolytic enzyme was isolated from Chungkook-Jang a traditional fermented food in Korea. Nattokinase is a potent fibrinolytic enzyme that is considered to be a promising agent with the potential for fighting cardiovascular diseases. Response surface methodology (RSM) and statistical analysis system (SAS) 9.1 program were employed to optimize a fermentation medium for the production of nattokinase by B. subtilis NK7-1. The three variables involved in this study were beef extraction, peptone, and initial pH. Using this methodology, the optimum values of the critical components for maximum production of nattokinase for concentration of beef extraction, peptone, and initial pH were 0.4%, 0.28%, and 8.84, respectively. Analysis of variance (ANOVA) showed a high coefficient of determination (R2) value of 0.9717. Using the optimized condition, nattokinase production by B. subtilis NK7-1 was obtained at 24 h-cultivation. Inhibition of Bacillus cereus by Bacteriocin from Lactococcus lactis ET-45 in Fermented Kimchi Seong-Yoep Jeong1,2*, Chan-Sun Park1, Nack-Shick Choi1, Hee-Jong Yang1, Da-i Jung1, Keug-Hyun Ahn1, Dae-Ook Kang2, Byoung-Dae Yoon1, and Min-Soo Kim1 1 Jeonbuk Branch Institute Bioindustry Research Center, Korea Research Institute of Bioscience and Biotechnology, 2Department of Biochemistry and Health Science, Changwon National University Bacteriocin producting lactic acid bacteria were isolated from Kimchi. Strain ET-45 was identified as L. lactis subsp. lactis based on sugar fermentation pattern test using API 50 CHL. The 16S rDNA sequence of strain ET-45 showed 99% identity to those of reference strain of L. lactis. The bacteriocin exhibited inhibitory activity against Bacillus cereus, Leuconostoc mesenterides, Leuconostoc carnosum, Leuconostoc lactis, Leuconostoc mesenteroides subsp. Cremoris, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus viridesceus, Pediococcus dextrinicus, and Enterococcus faceium. Optimal production of the bacteriocin was at pH 7.5 and 30°C for 18 ~24 h. Sucrose and protease peptone are essential for bacteriocin production as carbon source and nitrogen source, respectively. Antibacterial activity of the bacteriocin was completely disappeared by proteinase K, which indicates it is proteinous nature. The bacteriocin was fully stable at 121°C for 60 min. Solvents such as chloroform, ethanol, acetone, acetonitrile, hexane, isopropanal did not effect on the activity. The molecular weight of bacteriocin was estimated to be about 1.3 ~ 2.0 kDa by Tricine-SDS-PAGE. 181 www.msk.or.kr E017 E019 Biological Activity of Extracts from Hypholoma sublateritium Insights into SUMO Function under Oxidative Stress in Saccharomyces cerevisiae Sunga Choi1*, Jung-A song1, Donghyun Kim1, Gilhwa Jeong2, Jihong Kim3, and Jongwoon Choi2 1 Department of Life Science, Hallym University, 2Forest Research Institute of Gangwon-do, 3Department of Forest Management, Kangwon National University Gyu-bum Lim* and Won-Ki Huh School of Biological Sciences, and Research Center for Functional Cellulomics, Institute of Microbiology, Seoul National University This study was first carried out to investigate the biological activities of Naerypholoma sublateritiumi. The major chemical components of N. sublateritiumi were analyzed. There were not large differences in chemical contents of carbohydrates, fatty acid and ash content: however, total protein contents were 19.3%, relatively higher. As a result of comparing with antilipid peroxidation of each extract, the effects were highest in hexane and dichloromethan extracts. For analysis of antiproliferation activity, we used five cancer cell lines, MDA-MB 231 and MCF7 (Breast cancer cells), HT29 (Colon cancer cell), HeLa (cervical cancer cell) and SKOV 3 (ovarian cancer cell). Dichloromethan and ethyl acetate extracts showed an effective decrease of cancer cells viability in time- and concentrationdependent manner. These results of the present study show that the ability of N. sublateritiumi extracts might be a promising agent for performing clinically useful chemotherapy against diverse ailments as well as cancer. [This paper consisted by assistances of a technology development assignment scientific forest(과제번호: S120909L110120).] Since the discovery of ubiquitin in the mid-1970s, an entire family of small proteins related to ubiquitin has been defined. Among them, SUMO (small ubiquitin-like modifier) is highly conserved in eukaryotic cells and a budding yeast Saccharomyces cerevisiae has a single SUMO protein which has also been termed Smt3. Although the function of Smt3 has not been defined clearly, it is expected to control the activity or localization of target proteins under various environmental conditions. To examine this possibility, we performed a genome-wide analysis of in vivo protein sumoylation in S. cerevisiae using the bimolecular fluorescence complementation (BiFC) assay under oxidative stress. 5,911 yeast strains expressing C-terminally VN-tagged proteins (representing ~95% of the yeast proteome) were mated with a strain expressing N-terminally VC-tagged Smt3 and all the resulting diploid cells were analyzed by the BiFC assay in the presence or absence of hydrogen peroxide. Through this analysis, we identified several proteins showing interesting changes in the BiFC signal pattern. Based on the obtained results, we suggest a potential role of SUMO under oxidative stress. E020 E018 Expression of amyX and ytlR Genes that Play Important Role in Glycogen Metabolism of B. subtilis * Tianshi Wang , Jung-Hoon Choi, and Jung-Wan Kim Department of Biology, University of Incheon B. subtilis, an endospore forming bacterium, undergoes alternative life cycle by environmental cues. Pullulanase plays important role in glycogen accumulation, which is likely to be closely related to determination of cell’s destiny. Its gene is located in a gene cluster of ytlP-ytlQ-ytlR-amyX-ytmP with no known function. YtlR shares 50% homology with a transcriptional regulator of Lactobacillus reuteri, suggesting that it might be a novel regulator. The ytlR mutation caused decrease of sporulation efficiency and increase of glycogen accumulation. The expression of the genes was monitored using PamyX or PytlR-lacZ fusion in B. subtilis. Both promoters were induced mostly at the beginning of stationary phase and reached the highest level during endospore formation in both 2XSG and defined media. The promoters were induced earlier and higher levels in the presence of β-cyclodextrin (CD), fructose, or sucrose. The promoters were expressed at low level in the presence of maltodextrin or glycerol and at the highest level in the presence of β-CD. The results imply that the two genes are expressed coordinately and might be under the control of various regulators. Characteristic of PR Homologues from Marine Bacterioplanktonic SAR116 and SAR11 Clades Ah Reum Choi1*, Hyun-Myung Oh2, Ki Young Lee2, Jang-Cheon Cho2, and Kwang-Hwan Jung1 1 Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, 2Division of Biology and Ocean Sciences, Inha University Proteorhodopsins are light-dependent proton pumps. Proteorhodopsin (PR), retinal-containing integral membrane proteins, was originally detected in the uncultured marine γproteobacterial SAR86 group. Recently, PRs are discovered diverse ocean sea water, such as Monterey Bay, Hawaii Ocean, Palmer station in South Pole, Mediterranean Sea, Red Sea, Sargasso Sea, and Pacific Ocean. Here, from Western Pacific Ocean Margin a bacterioplankton designated IMCC1322 was successfully grow in tubes, it was identified as a member of uncultured SAR116 clade of α-proteobacterial lineages and two bacteria from SAR11 group. From SAR116 and SAR11, we isolated the PRs by arbitary primed PCR. The PR genes encode a protein of 261 a. a., 262 a. a, and 232 a. a, respectively. The genes were functionally expressed in E. coli and bound all-trans retinal to form a pigment (λmax=519 nm, 557 nm, 535 nm at pH 7). In PR of SAR116, titration of Asp105 occurred with a pKa of 5.6. In first PR of SAR11 (9062), one of Asp102 occurred with a pKa of 9.3. It showed a light-driven proton pumping activity and the rates of those photocycle are also fast similar to GPR. [Supported by grant from KOSEF R01-2008-000-20731-0] 182 Poster Sessions E021 E023 A Comprehensive In Vivo Analysis of Ras Interactome in Saccharomyces cerevisiae Using Bimolecular Fluorescence Complementation Assay Photoregulation of ASR (Anabaena Sensory Rhodopsin) Through ASRT (Anabaena Sensory Rhodopsin Transducer) Dae-Gwan Yi*, Eun-Bin Yang, and Won-Ki Huh School of Biological Sciences, and Research Center for Functional Cellulomics, Institute of Microbiology, Seoul National University So Young Kim* and Kwang-Hwan Jung Department of Life science and Interdisciplinary Program of integrated Biotechnology, Sogang University Activation of diverse cell-surface receptors can stimulate the convergent signals that lead to the activation of Ras. To identify novel Ras effectors, we performed a genome-wide in vivo analysis of Ras interactome in Saccharomyces cerevisiae using the bimolecular fluorescence complementation (BiFC) assay. 5,911 yeast strains expressing C-terminally VN-tagged proteins (~95% of the yeast proteome) were constructed and mated with a strain expressing N-terminally VC-tagged Ras2, the major Ras protein in S. cerevisiae. Through analysis of all the resulting diploid cells with the BiFC assay, we identified several novel candidates showing positive interaction signals with Ras2. The proteins interacting with Ras2 were found to be involved in several biological processes, including transport, lipid metabolic process, and RNA metabolic process. We also acquired the interactome of constitutively active or inactive mutant form of Ras2. We hope the analysis of these putative Ras interactors will provide deeper understanding of the Rasmediated signaling pathways and their cellular functions. Anabaena Sensory rhodopsin (ASR) from the fresh-water cyanobacterium Anabaena sp. PCC7120 has a sensory function and it is believed to play a role as a photoreceptor for phycobilin expression. Its function appears to involve modulation of a soluble cytoplasmic transducer (ASRT). ASRT is a soluble protein and transmitted a light signal to cytoplasmic part through physical interaction with ASR. We confirmed that ASR interacts with ASRT through in vitro binding assay. ASRT was also bound to a promoter region of cpc, pec, kai ABC and asr operon in gel shift assay and CHIP assay. It seems that ASRT regulates the photochemical property of ASR. In this study, we measured the photoconversion rate of ASR without ASRT and with tetramer and monomer formed ASRT. In the presence of ASRT, the rate is faster than in the absence of ASRT. And we are trying to understand the binding affinity between ASR and ASRT or ASRT and promoter region with each monomer and tetramer of ASRT protein. We confirmed the binding affinity of ASR and promoter region with in vivo beta-galactosidase assay and ITC (Isothermal Calorimetry). [Supported by 21C Frontier Microbial Genomics and Application Center Program] E022 E024 Glycogen Accumulation and Characterization of Debranching Enzyme in Vibrio vulnificus 1* 1 2 Ah-Reum Han , Yeon Ju Lee , Jong-Tae Park , and Jung-Wan Kim1 1 Department of Biology, University of Incheon, 2 Department of Food Science, Iowa State University, USA V. vulnificus is a gram-negative halophilic marine pathogen, experiencing alternative life cycle between marine environment and bodies of host. Glycogen is major energy source for Vibrio and other microorganisms. It is also related to pathogenicity. V.vulnificus accumulated glycogen 10X more than B.subtilis and E.coli when 1% maltodextrin was supplemented to the media. Glycogen accumulation was dependent on media and carbon source. Interestingly, glycogen synthesized by V.vulnificus had shorter side chains compared to glycogen from B. subtilis and E. coli. Analysis of V.vulnificus genome revealed that constitution of genes for glycogen metabolism is unique, with two glycogen phosphorylase genes and three putative debranching enzyme genes. One of debranching enzyme genes, VV2_1226, that have been annotated as type II secretory pathway protein of 661 amino acids with a predicted molecular mass of 76,560 daltons was cloned on pET28a and over-expressed in E. coli BL21 with IPTG induction. The protein purified using Ni-NTA column hydrolyzed glycogen most efficiently but pullulan and amylopectin only poorly. The optimal pH and temperature for the enzyme was 6.5 and 35°C, respectively. The Existence of FD Blh Dioxygenase You Sun Kim1*, Soon-Kyeong Kwon2,3, Jihyun F. Kim2,4, and Kwang-Hwan Jung5 1 Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, 2Korea Research Institute of Bioscience and Biotechnology, 3University of Science and Technology, 4Korea University of Science and Technology, 5 Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University The microbial rhodopsins are typically seven transmembrane proteins that use a retinal as a chromophore attached to conserved lysine residue. A variety of rhodopsins was discovered in many habitats. Proteorhodopsin (PR) functions as a light-driven proton pump, which was found in uncultured marine bacteria of SAR86. We obtained two flavobacterial strains called IMCC1997 and Donghaeana dokdonensis from the surface of sea water in Korea East Sea. Interestingly, IMCC1997 has one PR and D. dokdonensis two types of microbial rhodopsins that co-exist in the genome. There is a gene which encodes Blh protein (bacteriorhodopsin-related protein-like homolog protein) in D. dokdonensis that has been proposed to catalyze or regulate conversion of β-carotene to retinal to make rhodopsins in prokaryotes and is located next to the PR gene but is transcribed differently in Dokdonia sp. MED134 and Polaribacter sp. MED 152. DD blh showed enhanced activity compared to PR blh since we could detect the formation of rhodopsin as pink color even though its activity is a little bit lower than that mouse dioxygenase. [Supported by KOSEF R01-2008-000-20731-0] 183 www.msk.or.kr E025 E027 Expression and Characterization of ARⅡ(Acetabularia RhodopsinⅡ) Comparative Study of Microbial Rhopdopsins Between Arctic and Antarctic Ocean Se Jun Kim* and Kwang-Hwan Jung Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University Byung Hoon Jung*, Jae Young Jung, and Kwang-Hwan Jung Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University The microbial rhodopsins are typically seven transmembrane proteins that use retinal as a chromophore absorbs light energy for ion transport or photoreceptor functions. Acetabularia acetabulum is green algae, which has photosynthetic activity. The life cycle of A. acetabulum has distinct 3 developmental stages: juvenile, adult, and reproductive. Juvenile and adult stages are temporally sequential, but physiologically and morphologically distinct. It has been discovered bacteriorhodopsin (BR)-like protein and that was called Acetabularia rhodopsin I (ARI). ARI is intronless and encodes a protein of 246 amino acids with molecular weight of 27 kDa, which function was characterized to be a proton-pump stimulated by green light. Recently, it was revealed that new rhodopsin-like proteins exist in genomes of A.acetabulum which is called new rhodopsin Acetabularia rhodopsin II (ARII). We expressed the ARII-mistic fusion in E. coli and purified only ARII by NiNTA affinity chromatography. We measured an absorption spectra (λmax=532nm) and will study further about its pKa, photocycle, and pumping activity. Proteorhodopsin (PR) is discovered from marine bacteria and is 7-transmembrane light-harvest protein that has all-trans retinal as a chromophore. PR has a proton pumping activity from inside to outside of the cell using light energy. Generally, PR is classified into two groups by the maximum absorption wavelength. One is the BPR which absorbs blue around 500 nm wavelength and the other is the GPR which absorbs green (530 nm) region of visible spectrum. Previously, we have isolated and characterized 18 GPR homologs from the sea near Svalbard, Norway. We have confirmed the positive-correlation between proton pumping function and photochemical reaction rate. In this study, we isolated the conserved regions of rhodopsin by PCR from the sea near King George Island and compared with Arctic microbial rhodopsins. We also isolated a single full sequence of Antarctic rhodopsin and several piece of conserved regions between helix C and F. We are now characterizing Antarctic rhodopsin and also trying to express in E. coli to characterize the photochemical properties. [Supported by KOSEF R01-2008-000-20731-0] E026 [Supported by KOSEF R01-2008-000-20731-0] E028 Purification Method for Donghaeana dokdonensis Rhodopsin I Photochemistry of Acetabularia RhodopsinⅠand of its Important Mutants Sehwan Kim1*, Soon-Kyeong Kwon2,3, Jihyun F. Kim3,4, and Kwang-Hwan Jung1 1 Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, 2Korea Research Institute of Bioscience and Biotechnology, 3Korea University of Science and Technology, 4Korea Research Institute of Bioscience and Biotechnology Keon Ah Lee* and Kwang-Hwan Jung Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University Rhodopsins are a family of membrane-embedded photoactive retinylidene proteins. Rhodopsins have several functions as an ion pump, sensory receptor and ion channel. Donghaeana dokdonensis Rhodopsin I (DDRI) is the one of rhodopsin that is isolated from the Korea East Sea. It has a typical proton acceptor and donor which mediate proton translocation from intracellular side to extracellular region when it absorbs light energy, and its in vitro proton pumping activity has been demonstrated. It has biophysical features which are very similar to proteorhodopsin (PR). In this study, we focused on how to get high yield of pure DDRI. We transformed DDRI to E. coli UT5600 cell for rhodopsin expression. DDRI expressed cells lyses by sonicator and proteins are solubilized with dodecyl maltoside. Since DDRI has six-histidine at c-terminal, we use Ni-NTA resin for the purification. We tried to remove non-specific binding with pre-treatment of imidazole when its binding and repeated twice of Ni-NTA purification. It seems that the purity is suitable to get a crystal of DDRI. [Supported by 21C Frontier Microbial Genomics and Application Center Program] 184 Poster Sessions Microbial rhodopsins are retinal-binding proteins known as ion pumps and photosensory receptors. Acetabularia rhodopsin I (ARI) is from Acetabularia acetabulum, a giant unicellular green alga (kindly provided by Professor Dina Mandoli). ARI fused with MISTIC was expressed in E. coli UT5600. Several properties of ARI were measured; the absorption spectra, pKa of the acceptor residue, the amount of protons pumped outward, light-induced difference spectra, and half-life of several intermediates. Unmisticated ARI showed pink color and its absorption maximum was around 516 nm. The pKa of ARI was measured and tested the proton pumping outward activity at pH 7.5. Two of photointermediates of ARI have been detected including K (or L)-like and M-like that have absorption maximum at 584 and 396 nm, respectively. We could also detect the mRNA of Acetabularia rhodopsin in the A. acetabulum whole cell by RT-PCR. We also constructed 4 mutants, D89N, D89E, D100N, and D100E (D89 and D100 are expected to be acceptor and donor residues, respectively) mutants and spectroscopic properties of these were also measured. [Supported by the 21C Frontier Microbial Genomics and Application Program] E029 E031 Transcriptional Regulation of Aldehyde Reductase YqhD of E. coli by YqhC, an AraC-Type Regulator, Involving Its 5’-UTR Region * Junghoon Lee and Chankyu Park Korea Advanced Institute of Science and Technology YqhD is an aldehyde reductase detoxifying glyoxal (GO), a reactive α-oxoaldehyde accumulated by a stress associated with glucose metabolism. YqhD was previously characterized as an enzyme, while its transcriptional regulation mechanism is unknown. Glyoxal-resistant mutants isolated from yqhC gene, over-expresses YqhD protein, implying YqhC functioning as a transcriptional regulator for YqhD. We observed that the level of YqhD transcript was dramatically increased in the glyoxalresistant mutant. By screening for mutants down-regulating YqhD, we obtained point mutations in the yqhD promoter and 5’-UTR region. In order to test whether these mutants alter transcriptional regulation, we carried out a real-time PCR experiment. The promoter and 5’-UTR mutations lowered the level of yqhD transcript, suggesting that the YqhC protein and the RNA secondary structure in 5’-UTR affect YqhD transcription. Furthermore, gel-shift assay revealed that the YqhC protein binds to the yqhD promoter region that contains AraCbinding consensus sequence. [Supported by the 21C Frontier Microbial Genomics and Application Center Program.] E030 Rapamycin-Induced Abundance Changes in the Proteome of Budding Yeast Chun-Shik Shik1*, Yeon-Ji Chang1, Hun-Goo Lee2, and Won-Ki Huh1 1 School of Biological Sciences, Seoul National University, 2 Department of Molecular Biology and Biochemistry, Rutgers University The target of rapamycin (TOR) signaling pathway conserved from yeast to human plays critical roles in regulation of eukaryotic cell growth. However, due to the functional diversity of TOR pathway, we do not know yet some key effectors of the pathway. To find unknown effectors of TOR signaling pathway, we took advantage of a green fluorescent protein (GFP)-tagged collection of budding yeast Saccharomyces cerevisiae. We analyzed protein abundance changes by measuring the GFP fluorescence intensity of 4156 GFP-tagged yeast strains under inhibition of TOR pathway. Our proteomic analysis argues that 83 proteins are decreased whereas 32 proteins are increased by treatment of rapamycin, a specific inhibitor of TOR complex 1 (TORC1). We suggest that the 115 proteins indentified in this study may be directly or indirectly involved in TOR signaling and can serve as candidates for further investigation of the effectors of TOR pathway. [Supported by grants from the 21C Frontier Functional Proteomics Project and the 21C Frontier Microbial Genomics and Application Center Program.] E032 Dephosphorylated NPr of The Nitrogen-Metabolic PTS Regulates Lipid A Biosynthesis by Direct Interaction with LpxD 1* 1 1,2 Hyun-jin Kim , Chang-Ro Lee , and Yeong-Jae Seok Department of Biological Sciences and Institute of Microbiology, Seoul National University, 2Department of Biophysics and Chemical Biology, Seoul National University 1 Bacterial phosphoenolpyruvate-dependent phosphotransferase systems (PTS) play multiple roles in addition to sugar transport. Recent studies revealed that enzyme IIANtr of the nitrogenmetabolic PTS regulates the intracellular concentration of K+ by direct interaction with TrkA and KdpD. In this study, we show that NPr of the nitrogen-metabolic PTS directly interacts with and regulates Escherichia coli LpxD which catalyzes biosynthesis of lipid A of the lipopolysaccharide (LPS) layer and therefore is essential for growth. LpxD showed a preferential interaction with unphosphorylated NPr. Mutations in lipid A biosynthetic genes such as lpxD are known to confer hypersensitivity to hydrophobic antibiotics such as rifampin; in contrast, a ptsO (encoding NPr) deletion mutant showed increased resistance to rifampin and increased LPS biosynthesis. Taken together, our data show that unphosphorylated NPr decreases LPS biosynthesis by inhibiting LpxD activity. [Supported by grants from the 21C Frontier Microbial Genomics and Applications Center Program, Ministry of Science & Technology (Grant MG08-0201-1-0)] Quantitative Proteomic Analysis of Ribosomal Protein L35b Mutant of Saccharomyces cerevisiae Yong Bhum Song1*, Min A Jhun2, Taesung Park3, and Won-Ki Huh1 1 School of Biological Sciences, and Research Center for Functional Cellulomics, Institute of Microbiology, Seoul National University, 2Bioinformatics Program, Seoul National University, 3Department of Statistics, Seoul National University Recent studies have revealed that in higher eukaryotes, several ribosomal proteins are involved in some pathological events or developmental defects, indicating that ribosomal proteins perform unconventional functions other than protein biosynthesis. To obtain an insight into the novel roles of ribosomal proteins, we aimed to analyze the changes in proteome expression in ribosomal protein mutants by using S. cerevisiae as a model system. We introduced the rpl35bΔ mutation into the 4,159 green fluorescent protein (GFP)-tagged yeast strains by using the synthetic genetic array (SGA) method, and performed quantitative proteomic analysis by using a multilabel microplate reader and flow cytometer. We identified 22 upregulated and 20 downregulated proteins in the rpl35bΔ mutant. These proteins were primarily classified into the Gene Ontology (GO) categories of cellular biosynthetic process, translation, protein or nucleotide metabolic process, cell wall organization and biogenesis, and hyperosmotic response. Our results show that a ribosomal protein mutation can lead to perturbation in the expression of several proteins, including some other ribosomal proteins. 185 www.msk.or.kr E033 E035 In Escherichia coli, the Regulation of RppH in Peptidoglycan Synthesis Pathway Fractionation of Components for Atopy Treatment from Fermented Products of Herbs Miri Kim1*, Seung-Hyon Cho1, Hyun-Jin Kim1, Chang-Ro Lee1, and Yeong-Jae Seok1,2 1 Laboratory of Macromolecular Interactions, Department of Biological Sciences and Institute of Microbiology, Seoul National University, 2Department of Biophysics and Chemical Biology, Seoul National University Hye Young Kim1*, Sun Wook Park1, Doo Il Rung1, Sung Gu Lee2, and Hyoung Tae Choi1 1 Department of Biochemistry, Kangwon National University, 2 Mushville Regulation of cellular mRNAs level is important for metabolite and homeostasis in the cell to regulate protein level in various environmental conditions. In prokaryotes, though 5’ end of mRNA is not modified, it retains the 5’ triphosphate to stabilize, likely to the cap structure in eukaryotes. RppH (NudH/YgdP) is a prokaryotic functional homology of a decapping enzyme in eukaryotic cells to remove 5’ triphosphate to generate a 5’ monophosphate mRNA which is a substrate for the endocuclease RNase E. Moreover, RppH, belonging to the Nudix superfamily of enzymes which hydrolyze Nucleoside diphosphates linked to other moiety X, is well known for hydolysis diadenosine oligophosphates (alarmone) in vitro. It was reported that RppH of hydrolyse activity regulates endogenous alarmone levels in stress. Here we show that RppH strongly interacts with DapF, diaminopimelate epimerase, which converts LL-DAP into meso-DAP. Meso-DAP(diaminopimelate) plays a important role as a precursor in lysine synthesis pathway and peptidoglycan synthesis pathway. Thus, the function of NudH regulating dapF activity is meaningful to study in addition to that of NudH pyrophosphohydrolase and hydrolase activity E034 Potassium Mediates Escherichia coli Enzyme IIA Ntr -dependent Regulation of Sigma Factor Selectivity Chang-Ro Lee1*, Seung-Hyon Cho1, Hyun-Jin Kim1, Miri Kim1, Alan Peterkofsky2, and Yeong-Jae Seok1,3 1 Laboratory of Macromolecular Interactions, Department of Biological Sciences and Institute of Microbiology, Seoul National University, 2Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, USA, 3Department of Biophysics and Chemical Biology, Seoul National University An Escherichia coli mutant devoid of enzyme IIANtr (EIIANtr) of the nitrogen PTS is extremely sensitive to leucinecontaining peptides due to decreased expression of acetohydroxy acid synthase. We report here that the leucine hypersensitivity of a ptsN (encoding EIIANtr) mutant was suppressed by deleting the rpoS gene, encoding the stationary phase σfactor. Despite intracellular levels of sigma factors comparable to the wild-type strain, most of the genes downregulated in a ptsN mutant are controlled by σ70, while all the up-regulated genes are controlled by σS, implying that the balance of sigma activities is modified by ptsN deletion. This change of sigma factor activity was found to be due to increased levels of K+. In vitro transcription assays showed that a σ70 controlled gene and a σS controlled gene were differentially affected by potassium concentration. Biochemical studies revealed that K+ is responsible for sigma factor competition by differentially influencing the binding of σ70 and σS to core RNA polymerase. Taken together, the data indicate that EIIANtr controls sigma factor selectivity by regulating the intracellular K+ level. 186 Poster Sessions Atopy is a very notorious disease because there are so many reasons for the disease, and that is the main reason for extremely hard to get healing. Since even many foods which are used in our normal life can cause the disease in young children, it is highly required to have good treatments with no side effects. Extracts of four different herbs and cell extract of Phellinus linteus were fermented with lactic acid bacteria and budding yeast for 2 months, and the fermented product showed positive healing effect against atopy when examined at the cell level experiment. In order to isolate the effective components, ultrafiltration with stepwise molecular weight cut-off filtration, and thin layer chromatography have been carried out. Here we present the possible protocols for the isolation of the effective chemicals from the fermented mixtures. We have isolated several chemical mixtures which show good effects, and these will be isolated into each single chemical to determine their structure. F003 F001 Molecular Characterization of FinR, a Novel RedoxSensing Transcriptional Regulator in Pseudomonas putida KT2440 * Jinki Yeom and Woojun Park Division of Environmental Science and Ecological Engineering, Korea University FinR is required for the induction of the fpr (ferredoxinNADP+ reductase) under superoxide stress conditions in P. putida. Many proteobacteria harbor FinR homologues in their genome as a putative LysR-type protein. When these conserved cysteines along with two other cysteine residues present in FinR were individually mutated to serines, the FinR remained active, unlike SoxR and OyxR in E. coli. The results of our in vitro DNA-binding assay with cellular extracts showed that FinR binds directly to the fpr promoter region. In order to identify the FinR functional domain for sensing superoxide stress, we employed random and site-directed mutagenesis of FinR. Interestingly, two mutants (L215P, D51A) appeared to be constitutively active, regardless of superoxide stress conditions. Ferrous iron depletion, ferric iron addition, and fdxA (ferredoxin) gene deletion also participate in the regulation of fpr. These data indicate that the FinR has unusual residues for redox sensing and that the redox-sensing mechanism of FinR differs from the well-known mechanisms of OxyR and SoxR. Cell-Free Culture Fluid-Mediated Expression in Corynebacterium glutamicum Hee-Sung Shin*, Yong-Jae Kim, In-Hwa Yoo, and Un-Hwan Ha Department of Biotechnology and Bioinformatics, Korea University Autoinducing expression is an environmental sensing system that allows bacteria to monitor their own population density. It is mediated by a diverse signaling molecule that is produced, released and detected by bacterium itself. However, there have been no reports on autoinduction-mediated expression in Corynebacterium glutamicum, an important gram-positive bacterium widely used for glutamine production. In this study, we report the identification of genetic loci, including acyltransferase, whose expression is under the control of cellfree culture fluid from C. glutamicum. It indicates that C. glutamicum possesses an autoinduction system, producing signaling molecules to the threshold stimulatory level, resulting in the induction of acyltransferase identified in this study. [Supported by grants from CJ Cheiljedang Corporation as a project of 21C Frontier Microbial Genomics and Applications Center.] [This work was supported by a grant from the NCRC (grant #: 20090091491) and KSEF (R01-2008-000-10697-0) program.] F002 NtrC-Sensed Nitrogen Availability Is Important for Oxidative Stress Defense in Pseudomonas putida KT2440 Jinki Yeom* and Woojun Park Division of Environmental Science and Ecological Engineering, Korea University The zwf gene is repressed by NtrC under nitrogen-limited condition. Previously, we demonstrated that induction of zwf-1 is required for protecting P. putida cells under oxidative stress, which could be possible probably because of derepression of HexR on the zwf-1 gene under oxidative stress. These findings led us investigate that NtrC still represses the zwf-1 under nitrogen-limited oxidative stress condition, which makes cells more sensitive under such condition. Interestingly, deletion of the ntrC gene significantly reduces growth rate, but renders cells more resistant to oxidative stress, under nitrogen limited condition in P. putida. The results of transcriptome analysis demonstrated that the derepression of several oxidative stress genes along with the zwf-1 gene might confer high resistance to oxidative stress in the ntrC mutant. Here, we presented that different sets of genes are involved in N-rich and N-limited oxidative stress conditions and NtrC-sensed nitrogen availability is the most important prerequisite for full cellular defense against oxidative stress in P. putida. F004 Metaproteomics in Microbial Ecology Jong-Shik Kim*, Jung-Hee Woo, Jun-Tae Kim, Nyun-Ho Park, and Choong-Gon Kim Gyeongbuk Institute for Marine Bioindustry New technologies are providing unprecedented knowledge into microbial community structure and functions. Even though nucleic acid based approaches do provide a lot of information, metaproteomics could provide a high-resolution representation of genotypic and phenotypic traits of distinct microbial communities. Analyzing the metagenome from different microbial ecosystems, metaproteomics has been applied to seawater, human guts, activated sludge, acid mine drainage biofilm and soil. Although these studies provide different approaches, they elucidated that metaproteomics could provide a link between microbial community structure, function, physiology, interaction, ecology and evolution. These approaches are reviewed here to help gain insights into the function of microbial community in ecosystems. [This work was supported by The NCRC (grant #: 20090091491 and KSEF grant (R01-2008-000-10697-0).)] 187 www.msk.or.kr F005 F007 A Tip-to-tip Interaction Between AcrA and TolC Plays an Essential Role for the Formation of Functional Bacterial Tripartite Efflux Pump AcrAB-TolC 1* 2 2 Hong-Man Kim , Yongbin Xu , Shunfu Piao , Se-Hoon Sim1, Minho Lee1, Nam-Chul Ha2, and Kangseok Lee1 1 Department of Life Science (BK21 program), Research Center for Biomolecules and Biosystems, Chung-Ang University, 2Department of Manufacturing Pharmacy, College of Pharmacy and Research Institute for Drug Development, Pusan National University Tripartite efflux pumps are involved in antibiotic resistance and toxic protein secretion. In this study, we investigated the assembly mechanism of the major multidrug efflux pump AcrAB-TolC. Site-directed mutational analyses revealed that the conserved residues located at the tip region of the α-barrel of the membrane fusion protein (MFP) AcrA play an essential role in the binding of an outer membrane factor (OMF) TolC, as well as in the action of tripartite efflux pumps. Genetic complementation experiments further confirmed that the AcrA hairpin tip region is functionally related to the TolC aperture tip region. In addition, we provided in vivo functional data showing that AcrA shares a TolC binding mode with the hexameric MacA. Our results demonstrate that a tip-to-tip interaction between AcrA and TolC is required for the formation of the functional tripartite multidrug efflux pump. This finding may offer a molecular basis for understanding the multidrug resistance of pathogenic bacteria. Synthesis of CMP-Neu5Ac Using Metabolically Engineered Escherichia coli Hwa Young Choi*, Seong kee Cho, Jeong Mi Park, and Nam Soo Han Chungbuk National University, Department of Food Science and Technology Cytidine 5'-monophosphate N-acetylneuraminic acid (CMPNeuAc) is an essential precursor for the synthesis of sialyloligosaccharides. In order to produce CMP-NeuAc, we employed a recombinant E. coli system which was engineered by gene transformation and gene knock-out techniques. The neuB gene was for the conversion of N-acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP) to Neu5Ac, and the neuA gene was for the production of CMP-Neu5Ac from cytidine triphosphate (CTP) and NeuAc. Additionally, neuC encodes an epimerase that catalyzes the formation of ManNAc from UDP-GlcNAc. We constructed expression vector using pETDuet™-1 for the continuous production of CMP-Neu5Ac. pETDuet™-1 was designed for the coexpression of two target genes, pET-BA, pET-BC, which were made by cloning from each of neuB, neuA, neuC. The NeuAc synthase, CMPneu5Ac synthase and UDP-GlcNAc 2 epimerase were successfully expressed in E. coli BL21 (DE3) star. We determined the molecular weight of neuB, neuA neuC on SDSPAGE and they were 38kDa, 48kDa and 44kDa, respectively. [This work was supported by grants from the 21C Frontier Microbial Genomics and Application Center Program of the Korean Ministry of Science and Technology and Seoul R&BD program (10550).] F006 F008 The Tip Region of the MacA Alpha-Hairpin Is Required for the Binding to TolC of the Escherichia coli MacAB-TolC Pump 1* 2 2 Yongbin Xu , Se-Hoon Sim , Saemee Song , Shunfu Piao1, Hong-Man Kim2, Xiao Ling Jin1, Nam-Chul Ha1, and Kangseok Lee2 1 Department of Manufacturing Pharmacy, College of Pharmacy and Research Institute for Drug Development, Pusan National University, 2Department of Life Science (BK21 program), Research Center for Biomolecules and Biosystems, Chung-Ang University The tripartite efflux pump MacAB-TolC found in gramnegative bacteria is involved in resistance to antibiotics. We previously reported the funnel-like hexameric structure of the adaptor protein MacA to be physiologically relevant. In this study, we investigated the role of the tip region of its α-hairpin, which forms a cogwheel structure in the funnel-like shape of the MacA hexamer. Mutational and biochemical analyses revealed that the conserved residues located at the tip region of the α-hairpin of MacA play an essential role in the binding of TolC. Our findings offer a molecular basis for understanding the drug resistance of pathogenic bacteria. [This work was supported by grants from the 21C Frontier Microbial Genomics and Application Center Program of the Korean Ministry of Science and Technology and Seoul R&BD program (10543).] 188 Poster Sessions Identification of Differentially Expressed Genes in Flammulina velutipes with Anti-Tyrosinase Activity Jae-Yong Cho* and Sang-Yoon Kim Department of Pharmaceutical Engineering, College of Health Science, Sangji University It was previously shown that fruiting bodies of the mushroom, Flammulina velutipes, exert anti-tyrosinase activity by producing a triacylglycerol characterized as 1′,3′-dilinolenoyl-2′-linoleoylglycerol (LnLLn). In this study, we provide evidence that the mycelia of F. velutipes grown on glucose, but not on glycerol, exhibits anti-tyrosinase activity. To identify genes involved in the rate-limiting step(s) in the biosynthesis of LnLLn by F. velutipes, a RT-PCR method that involves annealing control primers (ACPs) was employed. By using 120 ACPs, a total of 84 differentially expressed genes (DEGs) in F. velutipes mycelia with anti-tyrosinase activity were cloned and sequenced. Basic Local Alignment Search Tool (BLAST) searches revealed that 72 of the genes have known sequence homology. Of these, the genes involved in the modification of fatty acids and their assembly into triacylglycerol (TAG) were selected and further quantified by real-time RT-PCR. [This research was supported by a grant (Code # 20070401034021) from Biogreen 21 Program, Rural Development Administration, Republic of Korea, and in part by the Sangji University Research Fund 2009.] F009 F011 SigR-RsrA System Is Conserved in Actinomycetes as Specific Thiol-Oxidative Stress Sensor Characterization of A-Type Scaffold Proteins of FeS Assembly System in Schizosaccharomyces pombe Yoo-Bok Cho1*, Yong-Gyun Jung1, Min-Sik Kim1, Seok-Hyun Hong1, and Jung-Hye Roe1,2 1 School of Biological Sciences, Seoul National University, 2 Institute of Microbiology, Seoul National University Su-Jin Jung*, Kyung-Chang Lee, and Jung-Hye Roe Lab. of Molecular Microbiology, School of Biological Sciences and Institute of Microbiology, Seoul National University Zinc-containing anti-sigma factors (ZAS) are widespread bacterial proteins that regulate their cognate sigma factors in response to a variety of environment stresses or stimuli. They have characteristic, conserved, HX3CX2C motif that is responsible for zinc co-ordination. RsrA from Streptomyces coelicolor (ScoRsrA) is one of the best characterized ZAS proteins and is known as a disulfide stress sensor. However, it is not well known of the signature pattern that makes it a distinct disulfide sensor. We constructed simple validation system to determine the thiol-oxidative stress sensitivity of the anti-sigma factors from the fact that SigR autoregulates its expression upon thiol-oxidative stress through RsrA. Comparing conserved amino acid profiles of the confirmed sensitive ZAS proteins with those of insensitive ones, we identified a region that is important for thiol-oxidative stress sensing. Importance of each residue within this region needs be confirmed experimentally. Inspection of gene database to predict the presence of putative redox-sensing ZAS factors has been done. F010 Nisin-Controlled Gene Expression System in Leuconostoc mesenteroides ATCC 8293 Seungkee Cho1*, Hyun-Ju Eom2, and Nam Soo Han1 1 Department of Food Science and Technology, Chungbuk National University, 2Department of viticulture & Enology, University of California, USA Lactic acid bacteria (LAB) have been used successfully to express a wide variety of recombinant proteins, ranging from flavor-active proteins to antibiotic peptides and oral vaccines. The nisin-controlled expression (NICE) system is the most common of the systems for production of heterologous proteins in LAB. The Nisin-Controlled gene Expression system (the NICE system) is an efficient and promising gene expression system based on the autoregulation mechanism of nisin biosynthesis in the Lactococcus lactis. In the NICE system, the membrane-located histidine kinase NisK senses the inducing signal nisin and autophosphorylates, then transfers phosphorous group to intracellular response regulator protein NisR which activates nisA promoter to express the downstream gene(s).The plasmid pJH24 is a popular vector for nisin-inducible expression of heterologous genes in lactic acid bacteria, and it was successfully expressed in Leuconostoc mesenteroides ATCC 8293. Iron-sulfur (Fe/S) cluster proteins are ubiquitous and play critical roles in diverse cellular processes such as enzyme reaction, respiration, DNA replication, and gene regulation. The process of forming Fe-S clusters is best studied in Escherichia coli and Saccharomyces cerevisiae, but not much in Schizosaccharomyces pombe. Especially we investigated the A-type scaffold proteins which is E.coli IscA homologues (Isa1 and Isa2). The isa1 (SPCC645.03c) and isa2 (SPBC3B9.17) genes have been initially found in S. pombe to suppress growth defects of Δgrx5 mutant that lacks mitochondrial monothiol glutaredoxin. Each of these genes is essential for the growth of S.pombe, in contrast to S.cerevisiae where even the double mutant is viable. Since Grx5 is involved in Fe-S assembly in mitochondria, we investigated interaction of Isa1 and Isa2 with other Fe-S assembly proteins by in situ fluorescence complementation analysis. We found that Isa1 and Isa2 interact with several components of Fe-S assembly system and several other Fe-S containing proteins in mitochondria. F012 Conservation of Genes with Redox-Responsive Promoters as Direct Targets of Sigma Factor SigR for Thiol-Reactive Stress Response in Actinomycetes Ji-Sun Yoo1*, Min-sik Kim1, Yoo-Bok Cho1, Joo-Hong Park1, Gi-Baeg Nam1, Yong-Gyun Jung1, Yann Dufour2, Tim Donohue2, and Jung-Hye Roe1 1 Laboratory of Molecular Microbiology, School of Biological Sciences, Seoul National University, 2Department of Bacteriology, University of Wisconsin, USA In Streptomyces coelicolor a specific sigma-antisigma pair SigR-RsrA modulates response toward thiol-oxidative stresses, which are sensed through reactive cysteines in RsrA. In this study we performed a genome-wide screening of direct target genes of SigR by ChIP-chip analysis. Upon diamide treatment, significantly elevated binding of SigR was observed in the promoter regions of 79 genes that transcribes up to 122 genes when considering operon structures. These direct target genes of SigR encode known and predicted proteins for thiol homeostasis, sulfur metabolism, modulation of ribosome functions, proteolysis, transcriptional regulation, transporters, oxidorectases, co-factor biosynthesis, stress response, DNA damage repair, etc. The consensus SigR binding promoter sequence was used to predict target genes in 47 genomes of actinomycetes that contain SigR-RsrA orthologues, such as corynebacteria, mycobacteria, streptomycetes, rhodococci, etc. A large portion of the predicted SigR regulon members were conserved throughout 47 bacterial species and some were limited more to the genus level. Conserved functions toward thiol-reactive stresses among actinomycetes system are found. 189 www.msk.or.kr F013 F015 The Construction of Gene Disruption and Shuttle Vectors Based on Antibiotic Selection for Sulfolobus, the Model Organism of Crenarchaeota * Sungmin Hwang , Kyoung-Hwa Choi, and Jaeho Cha Department of Microbiology, College of Natural Sciences, Pusan National University Sulfolobus acidocaldarius, the first isolated hyperthermophile, is one of the best studied species of thermoacidophilic organism. It can be grown under aerobic and heterotrophic conditions at 77°C and pH 3. To establish facile selection of mutants and effective expression of heterologous genes, Sulfolobus target gene disruption vector and EscherichiaSulfolobus shuttle vector were constructed. In the gene disruption analysis in S. acidocaldarius, the gene disruption vector was constructed by substitution of the gene of interest with a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene,resistant to thermostable antibiotic simvastatin, and it was designed to overexpress by the promoter of major heat shock chaperonin tf55α. In order to prevent the possibility of homologous recombination between host genomic DNA and endogenous vector DNA, the HMG-CoA reductase and tf55α genes were adopted from S. solfataricus P2. For Escherichia-Sulfolobus shuttle vector, the ori from Sulfolobus infected virus was used to replicate autonomously. In addition, tf55α promoter and HMG-CoA reductase gene of S. solfataricus P2 were used as a strong promoter and selection marker, respectively. F014 Glyoxal Detoxification via NADPH Dependent Reductases of E. coli Changhan Lee*, Insook Kim, and Chankyu Park Department of Biological Science, Korea Advanced Institute of Science and Technology Glyoxal and methylglyoxal are reactive carbonyl compounds and accumulated in vivo through various pathways, such as glycation, lipid peroxidation and DNA oxidation. Previously, we studied the involvement of aldo-ketoreductases (AKRs) in methylglyoxal detoxification. We also reported that YqhD, NADPH dependent aldehyde reductase, is crucial in glyoxal detoxification. Here, we assessed the role of various reductases including AKRs (YqhE, YafB and YghZ) that are likely to be involved in GO metabolism. Enzyme activities of the reductases, including YqhD, were assessed by measuring oxidation rate of NADPH. In addition, glyoxal sensitivity of the mutant strains was compared by measuring inhibitory concentrations. Expression levels of the genes induced by GO were measured by real-time RT-PCR. The results imply that GO can efficiently be detoxified by several different types of NADPH-dependent redutases, i.e. AKRs and YqhD. [Supported by the 21C Frontier Microbial Genomics and Application Center Program] F016 Construction of High Sensitive Detection System for Endocrine Disruptors with Yeast n-Alkane-Assimilating Yarrowia lipolytica The Expression of Sterigmatocystin Genes in Aspergillus nidulans Is Controlled by flbF, a Gene Required for Asexual Development Eun-Min Cho* and Chi-Yong Eom Seoul Center, Korea Basic Science Institute Yong Jin Kim*, Yeong Man Yu, Sang Eun An, Sun-Ho Kim, and Pil Jae Maeng Department of Microbiology & Molecular Biology, College of Biological Sciences and Biotechnology, Chungnam National University To construct a highly sensitive detection system for endocrine disruptors (EDs), we have compared the activity of promoters with the n-alkane-inducible cytochrome P450 gene (ALK1), isocitrate lyase gene (ICL1), ribosomal protein S7 gene (RPS7), and the translation elongation factor-1gene (TEF1) for heterologous gene in Yarrowia lipolytica. The promoters were introduced into the upstream of lacZ or hERα reporter gene, respectively, and the activity was evaluated by β-galactosidase assay by lacZ or western blot analysis by hERα. The expression analysis revealed that the ALK1 and ICL1 promoter were induced by n-decane and by EtOH, respectively. The constitutive promoter of RPS7 and TEF1 showed significantly high level of expression in the presence of glucose and glycerol, respectively. Particularly, the TEF1 promoter showed the highest β-galactosidase activity and a significant signal by western blotting with the anti-estrogen receptor compared with the other promoters. Secondary metabolites, or biochemical indicators of fungal development, are of intense interest to humankind due to their pharmaceutical and/or toxic properties. Here, we present that flbF, which encodes encode a potential transcription factor with a C2H2 zinc finger DNA binding motif, bipartite nuclear localization signal, and glutamine rich region, a gene shown to control the Aspergillus nidulans asexual development, also controls secondary metabolism. We generated a flbF deletion mutant and analyzed the phenotype. As a result, flbF deletion mutant showing delayed in the asexual development, suggesting that the flbF gene is required for asexual development in A.nidulans, also accumulation of brown pigment on media, suggesting that the flbF gene is involved in secondary metabolism. Specifically, flbF regulates the expression of genes implicated in the asexual development and synthesis of the mycotoxin sterigmatocystin. [This work was supported by grant from Korea Research Foundation] 190 Poster Sessions F017 F019 Glyoxal-Induced DNA Damage Involves the Recombinational Repair System Jihong Kim*, Changhan Lee, and Chankyu Park Department of Biological Science, Korea Advanced Institute of Science and Technology Glyoxal (GO) is a toxic and mutagenic aldehyde compound, causing DNA damage by modifying guanine and cytosine or by disrupting sugar-phosphodiester backbone. GO-sensitive mutants were isolated from Escherichia coli MG1655 strain by disrupting gene randomly with TnphoA-132 transposon. The mutations were mapped and found in recA as well as recC gene, by inverse-PCR and DNA sequencing. RecA is central to the SOS-response, an error-prone DNA repair system. RecC is a subunit of exonuclease that is required for the homologous recombination after double strand break. The isolation of such mutants indicates that the SOS repair system is somehow involved in repairing DNA damage induced by GO. We also tested whether other DNA repair systems play a role in GOinduced DNA damage, such as the nucleotide excision repair (uvrB), mismatch repair (mutS), base excision repair (mutM, mutY), and SOS repair (umuC, dinB, recF) systems. Effects of other repair systems were much lower than that of RecAC, confirming the major role of RecA-mediated SOS repair in GO-induced cell survival as well as GO-induced mutagenesis. [Supported by the 21C Frontier Microbial Genomics and Application Center Program.] Cloning, Sequencing, and Phylogenetic Analyses of a Carbon Monoxide Dehydrogenase Gene Cluster in Terrabacter carboxydivorans Jae Ho Lee*, Sae Woong Park, and Young Min Kim Department of Biology, Yonsei University Carboxydobacteria are a group of bacteria which are capable of growing aerobically on carbon monoxide as a sole source of carbon and energy by using carbon monoxide dehydrogenases (CO-DH) as a key enzyme for the oxidation of carbon monoxide (CO). Terrabacter carboxydivorans, one of the carboxydobacteria, is able to grow aerobically on CO of low concentration as a sole source of carbon and energy. It has been known that CODH in T. carboxydivorans has no immunological interactions with those of other carboxydobacteria. This imply that CO-DH in T. carboxydivorans might be divided into a new group of CO-DHs. To identify this hypothesis, the three structural genes of CO-DH in T. carboxydivorans were cloned, sequenced, and analyzed. The genes were clustered in the transcriptional order cutB-cutC-cutA. The cloned cutB, cutC, and cutA genes had open reading frames of 894, 552, and 2,433 nucleotides, and encoded 297, 183, and 810 amino acids, respectively. The mean identities in the deduced amino acid sequences of the CutA, CutB, and CutC of T. carboxydivorans with those of other carboxydobacteria were 31.75%, 34%, and 48%, respecttively. The phylogenetic trees of the CutA, CutB, and CutC of T. carboxydivorans showed that CO-DH in T. carboxydivorans is a new group of CO-DH that has not been reported before. [This study was supported by a grant from NRF [20090071521].] F020 F018 Cadmium Regulates Expression of SML1 Which Is a Rnr Inhibitor on the Post-Transcriptional Level in Saccharomyces cerevisiae * In-Joon Baek and Cheol-Won Yun School of Life Sciences and Biotechnology, Korea University To understand the toxic mechanism of cadmium in living organisms, we used Saccharomyces cerevisiae model system and screened yeast mutants which are related to cadmium. Δsml1, Δrnr3, Δwtm1, and Δwtm2 strains were found that resistant to cadmium. In plate assay, Δwtm1, Δwtm2 and Δrnr3 strains were resistant to cadmium and expression level of RNR3 was down-regulated in Δwtm1 and Δwtm2 strains. In northern blot, there was no change in SML1 expression in WT, Δrnr3, Δrnr1, Δwtm1, Δwtm2, Δdun1,and including deletion strains of ESCRT subunits whether cadmium treated or not. It is well known that sml1p is phosphorylated and degradation by dun1p in DNA stress condition. However there were remarkably increased translational level of SML1 in response to cadmium stress in above strains. In plate assay, many deletion strains of ESCRT subunits were sensitive to cadmium stress. To find degradation pathway of sml1p, we performed western blot with MG-132, proteasome inhibitor, in Δpdr5 strain that limit the efflux of protease inhibitor. Sml1p was not degradation by DNA damage agents. These results imply that cadmium has a different toxic mechanism from other DNA damage agents such as MMS and HU. Comparative Transcriptome Analysis of the Heat Shock Response in Hansenular polymorpha Hye-Yun Moon1,2*, Min jeong Shon3, Ohsuk Kwon3, Jeong-Yoon Kim2, and Hyun Ah Kang1 1 Department of Life Science, Chung-Ang University, 2 Department of Microbiology, Chungnam National University, 3 Korea Research Institute of Bioscience and Biotechnology Hansenula polymorpha is a thermotolerant methylotrophic yeast that can grow up to 48°C. We carried out the transcriptome analysis of two H. polymorpha strains, DL1 and NCYC495, and observed more extensive change of expression profiles in the DL1 strain with less thermotolerance than in the NYCY495 under heat stress condition. Bioinformatics analysis revealed the presence of heat shock elements in the promoter region of commonly up-regulated genes in the both H. polymorpha strains upon heat stress. Interestingly, some sets of genes were differentially regulated for each strain of H. polymorpha, implying that the detailed heat tolerance mechanism of two strains might be significantly different. Moreover, comparative transcriptome analysis showed that the expression of a set of genes related to stress-response and cellwall integrity was already higher in the NCYC495 than in the DL1 strain even at normal growth temperature, reflecting the thicker cell wall structure and the higher tolerance to heat shock of the NCYC495 strain. [Supported bygrants from 21C Frontier Microbial Genomics and Application Center Program and from NRF.] 191 www.msk.or.kr F021 F023 Global Expression Profile Analysis of Ethanol and Heat Stress Responses in Saccharomyces cerevisiae Identification of Putative Proteins Interacting With Mating Type Proteins in Gibberella zeae Jinho Choo*, Hye Yun Moon, Seon Ah Cheon, and Hyun Ah Kang Department of Life Science, Chung-Ang University Eun Ji Cho1*, Hee-Kyoung Kim1, Yin-Won Lee2, and Sung-Hwan Yun1 1 Department of Medical Biotechnology, Soonchunhyang University, 2Department of Agricultural Biotechnology, Seoul National University For the development of bioethanol production process based on lignocellulosic biomass, the yeast strains with increased resistance to heat and ethanol stress are highly desired. In the present study, we analyzed the change of global gene expression profiles of Saccharomyces cerevisiae under three conditions, such as 8% (v/v) ethanol stress, 39°C heat stress, and the combined stress of ethanol and heat (8% ethanol at 39°C). Extensive alteration of gene expression was observed at single stress condition of heat or ethanol. The commonly upregulated genes were mostly related to energy and cell stress defense, whereas the commonly down-regulated genes were involved in DNA processing, transcription, and protein synthesis. Unexpectedly, the extent of gene expression change was shown to be much less under the double stress condition compared to under the single stress condition. We combined the expression profiles with metabolic pathways to investigate the common and unique features of metabolic remodeling in the ethanol and heat shock responses, which would provide new information applicable to the construction of multi stresstolerant yeast strains. [Supported by a grant from NRF.] F022 Gibberlla zeae is a self-ferile ascomycete with ubiquitous geographic distribution, causing serious diseases in cereal crops. We have focused on the mating-type (MAT) genemediated regulatory network which governs the sexual developmental pathway in G. zeae. To identify putative proteins that interact with MAT proteins, we have employed a yeast two-hybrid assay using 4 different MAT gene products as baits. So far, screening the fungal cDNA library obtained from sexual development stage with MAT1-1-3 or MAT1-2 bait revealed 17 positive cDNA clones, for examples, those encoding GTP cyclohydrolase 2 and MAT1-1-1. The former is known to be involved in folic acid metabolism, and the latter is a gene product encoded by MAT1-1 locus. In particular, the identification of MAT1-1-1 from the prey library indicates that both MAT1-1-3 and MAT1-1-1 proteins may undergo heterodimerization for the transcriptional regulation of sexual development-related genes in G. zeae. A more detailed description of the genes obtained in this study will be presented. [This work was supported by a grant (2009-0075256) from NRF.] F024 Effect of PyrR on the Expression of pyrH in Corynebacterium glutamicum Acyl Carrier Protein IacP Contribute to Salmonella typhimurium Invasion of Epithelial Cell Jae-Hyung Jo* and Hyune Hwan Lee Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies Jeong Seon Eom*, Jin Seok Kim, Jung Im Jang, and Yong Keun Park School of Life Science and Biotechnology, Korea University The UMP kinase of Corynebacterium glutamicum, encoded by pyrH, is the key enzyme in the biosynthesis of pyrimidine nucleotides. It catalyzes the conversion of UMP to UDP. It was reported that the expression of pyrH in Bacillus subtilus is controlled by several factors such as UMP, UDP, UTP and specially by the attenuation of the transcript of pyrH by binding of PyrR. However, very little is known in the mechanism of regulation of pyrH in Corynebacterium glutamicum. Previously, we reported that the direct binding of PyrR to the pyrH promoter regulates the expression instead of attenuation in C. glutamicum. To further reveal the regulation of pyrH by PyrR, pyrR was knocked-out and the effect on the expression of pyrH was studied by real time PCR. As result, the level of pyrH expression in pyrR knocked-out mutant is two times higher than the wild type. This result is the evidence of the regulation of pyrH expression PyrR in C. glutamicum. Salmonella Pathogenicity Island 1(SPI-1) encodes a type III secretion system(T3SS) that translocates effector proteins into intestinal cells to promote invasion through actin cytoskeletal rearrangements. The iacP gene belongs to the SPI-1 and is able to be categorized into ACP family by sequence similarity, however its function is poorly understood. We investigated the role of IacP in Salmonella-induced invasion. Immunofluorescence microscopy of actin revealed that iacP mutant partially impaired the actin rearrangement. The inositol polyphosphatase SopB is involved in invasion, Akt activation, biogenesis of Salmonella-containing vacuole (SCV). Secretion of SopB in iacP mutant was reduced and translocation and membrane localization of SopB into INT407 cells were also decreased. Furthermore, decreased secretion of SopB resulted in inability of Akt activation and SCV maturation. And, we identified the flagellar subunit which was upregulated in iacP mutant from microarray data analysis and over secretion of the flagellar subunit were also confirmed by LC/MS. These results suggest that S.typhimurium IacP could modification of the target protein to reinforce the invasion into host cell. 192 Poster Sessions F025 F027 N-Terminal Signal of InvE is Required for Type III Secretion of Salmonella Translocon SipB and SipD Fission Yeast Homolog of Tho1p Is Involved in mRNA Export Jin Seok Kim*, Jung Im Jang, Jeong Seon Eom, Dae Woo Kang, and Yong Keun Park School of Life Sciences and Biotechnology, Korea University Ye-Seul Cho*, Cha-Yeon Kim, Ae-Rhee Chae, and Jin Ho Yoon School of Biological Sciences and Chemistry, Basic Science Research Institute, Sungshin Women’s University Type III secretion system (T3SS) is composed of more than 25 proteins, which constitute three major part to inject the virulence effector proteins into host cytoplasm ; basal body embedded in bacterial membrane, polymerized needle, and translocon connecting T3SS needle to host membrane. Secretion of Salmonella translocon proteins, SipB, SipC and SipD were regulated by InvE which is broadly localized in cytoplasm and membrane, but not secreted into culture supernatant. InvE has two coiled-coil domains in N-terminus and C-terminus respectively and C-terminus coiled-coil domain is important for SipB secretion. We show here that secretion of SipB and SipD were blocked when N-terminus 30 amino acids were deleted in InvE, indicating that InvE N-terminus take SipB and SipD into T3SS. The fact that N-terminal signal is characteristics of many type III secreted proteins suggested that InvE has ancestral secretion signal in N-terminus recognized by T3SS membrane protein. [Supported by grants from KHIDI-A090891] F026 In eukaryotes, nuclear export of mRNA takes place through the nuclear pore complex (NPC) embedded in the nuclear envelope and several soluble transport factors are involved in this process. We constructed deletion mutants of fission yeast Schizosaccharomyces pombe gene that encodes a protein homologous to Tho1p in budding yeast Saccharomyces cerevisiae, which is a conserved RNA binding nuclear protein that specifically binds to transcribed chromatin in a THO- and RNA-dependent manner and genetically interacts with the shuttling hnRNP Nab2. The fission yeast tho1 gene encodes an 245 amino-acid protein with predicted molecular weight of 26.7 kDa. The tho1 gene is not essential for vegetative growth. The accumulation of poly(A)+ RNA in the nucleus is exhibited when expression of tho1 is repressed or overexpressed. And functional Tho1-GFP signal was detected in nucleus. These results suggest that tho1 in fission yeast is involved in nuclear export of RNA. [Supported by grants from Sungshin Women’s University] F028 The Organization of Gene Cluster in the Length of 55 kb for the Biosynthesis of a Polyether Antibiotic, Laidlomycin, from Streptomyces sp. KCTC 10631BP Isolation of Synthetic Lethal Mutants with rsm1 Null Allele That Is Involved in mRNA Export in Fission Yeast Jae Yoon Hwang*, Nguyen Phan Kieu Hanh, and Doo Hyun Nam Faculty of Pharmacy, Yeungnam University Yun-Seon Park*, DongGeRaMi Moon, and Jin Ho Yoon School of Biological Sciences and Chemistry, Basic Science Research Institute, Sungshin Women’s University Laidlomycin, a polyether antibiotics produced by Streptomyces sp. KCTC 10631BP, shows strong antimicrobial activity against methicilin-resistant Staphylococus aureus (MRSA) and vancomycin-resistant enterococci (VRE). A 708 bp PCR fragment of epoxidase gene was used as a probe to screen the cosmid library of S. sp. KCTC 10631BP constructed in SuperCos-1 vector. Among cosmid clones, pSLD-36 gave a strong signal by colony hybridization and was found to contain some essential genes like type I polyketide synthase (PKS), epoxidase and epoxide hydrolase genes involved in the biosynthesis of polyether antibiotic, laidlomycin. By chromosome walking using enoyl reductase and epoxide hydolase I probes, another cosmid clone named pSLD-BI5 encoding the downstream region of PKS gene was further screened. The full length of the sequenced laidlomycin biosynthetic gene cluster was around 55kb. The putative gene cluster was highly homologous with the biosynthetic gene cluster for a typical type I PKS polyether antibiotic, monensin of S. cinnamonensis. The organization of the cloned gene cluster will be discussed in this presentation. In order to identity the genes in fission yeast Schizosaccharomyces pombe that are functionally involved in mRNA export, mutants that showed growth retardation or synthetic lethality with the rsm1 null allele were isolated. The rsm1 null mutant is not essential for growth but it showed a mild accumulation of poly(A)+ RNA in the nucleus. For this screening, we used the rsm1 null mutant harboring the pREP81X-rsm1 vector, where the expression of rsm1 is repressed in the presence of thiamine. This strain was mutagenized with EMS and approximately 320,000 colonies were analyzed. The mutant colonies that showed growth defects only in the presence of thiamine were screened at 27°C. Ten mutants were finally isolated in this screening and tentatively named as SLrsm1 through SLrsm10. Vectors containing the genes known to be involved in mRNA export were transformed into theses SLrsm mutant cells and checked whether these genes could complement the growth defects of these mutants in the presence of thiamine. These results suggest that SLrsms isolated in fission yeast interacted genetically with rsm1 and were defective in mRNA export. [Supported by grants from KRF] 193 www.msk.or.kr F029 F031 The Function of Novel Non-Coding RNA Against Innate Immune System in Salmonella typhimurium Isolation and Characterization of a HypoxiaSensitive Mutant UV7 in Aspergillus nidulans Sin Yeon Kim*, Yong Heon Lee, and Yong Keun Park School of Life Sciences and Biotechnology, Korea University Chinbayar Bat-Ochir* and Suhn-Kee Chae Department of Biochemistry and Center for Fungal Pathogenesis, Paichai University Salmonella are bacterial pathogens that have been well studied on virulence mechanisms, pathogenesis, and many fundamental pathways. While these investigations have focused on protein functions, Salmonella have also the remarked mechanism for RNA-mediated regulation. Bacterial non-coding RNAs were transcribed in response to various stresses. We have searched for a non-coding RNA as being part of stress responses including pH, nitrosative stress and oxidative stress. Reactive oxyzen species (ROS), reactive nitrogen species (RNS), and an acidic stress function as strong antimicrobial substances in macrophage innate immunity. In this study, we chose a mutant that shows susceptibility to oxidative stress by transposon mutagenesis. We show that the non-coding RNA is increased at oxidative stress and stable throughout the time course like other regulatory small RNA. Also, we are investigating target gene related to mechanism of the novel transcript from microarray. Our results revealed that this novel intergenic transcript may promote intracellular survival within the macrophage and mouse in vivo. Pathogenic microorganisms must overcome numerous obstacles to successfully colonize in a host after their infection. One of these barriers is hypoxia. Several hypoxia-sensitive mutants in Aspergillus nidulans were obtained by UV mutagenesis in our lab. One of those mutants UV7 showed a strong hypoxia-sensitive phenotype and high sensitivity to itraconazole. A genomic DNA fragment complementing the hypoxia-sensitivity of UV7 was isolated. One of ORFs within the cloned genomic DNA complemented UV7. This gene was named hosB (hypoxia-sensitive) and has 1,035 bp of ORF encoding a polypeptide of 244 amino acids. A mutation UV7 was identified by DNA sequencing of hosB PCR products. This indicated that the hosB gene was not an intergenic suppressor. One putative conserved domain of unknown function was found in HosB. A point mutation resulted in the 68th tyrosine into aspartic acid was occurred in this domain. HosB had transmembrane domains and was suggested to be localized in ER membrane. Null mutant of hosB showed the hypoxia-sensitive phenotype as UV7 which was complemented by overexpressing of hosB under the inducible promoter niiA. [Supported by Grants from NRF] F030 F032 Functional Analysis of the hitB Gene Required for Hypoxic Growth in Aspergillus nidulans Transcriptomic Response of the Methylotrophic Yeast Hansenula polymorpha to Hydrogen Peroxide Jun-Yong Kwak* and Suhn-Kee Chae Department of Biochemistry and Center for Fungal Pathogenesis, Paichai University Eun Hye Kim1,2*, Min Jee Kim1, Doo-Byoung Oh1, Jeong-Yoon Kim2, Hyun Ah Kang3, and Ohsuk Kwon1 1 Integrative Omics Research Center, Korea Research Institute of Bioscience and Biotechnology 2Department of Microbiology, Chungnam National University, 3Department of Life Science, Chung-Ang University Sterol-regulatory element binding protein (SREBP) is an ER membrane tethered transcription factor that controls synthesis of cholesterol and fatty acid in mammalian cells. Previously, we showed that HitA an A. nidulans SREBP homolog was shown to be essential for hypoxic growth. In this study, the hitB gene encoding a polypeptide showing similarities to the N-termini but lacking the transmembrane regions and the Ctermini found in HitA and SREBPs was analyzed. hitB null mutants failed to grow in hypoxic condition in similarly shown to hitA null mutants. The N-terminus of HitA or HitA complemented the hypoxia-sensitive phenotype of ∆hitB, while the hypoxia-sensitive phenotype of ∆hitA was not complemented by HitB in hypoxia. We further confirmed direct binding of HitA to the promoter of hitB by the ChIP assay, while HitB failed to bind to the hitA promoter. HitB interacted with the HitA N-terminus to form heterodimer as well as with HitB in in vitro resin binding assay. HitB was localized to the nucleus based on the observation of HitB-RFP fusion proteins with a fluorescence microscope. [Supported by Grants from NRF] In the methylotrophic yeast Hansenula polymorpha, methanol is first oxidized to formaldehyde by a peroxisomal alcohol oxidase, generating high levels of hydrogen peroxide. H. polymorpha can tolerate oxidative stress, but the global oxidative stress response has not been elucidated. In this study, the genome-wide expression profiles of H. polymorpha in response to exposure to H2O2 were analyzed. About 50 genes were either up-regulated or down-regulated more than twofolds in the presence of H2O2. The majority of the up-regulated genes were involved in the oxidative stress response and general stress response. On the other hand, the majority of the down-regulated genes were involved in glucose utilization, cell wall metabolism, and ribosomal protein synthesis. When compared to the previously reported H2O2-induced transcriptmic responses of S. cerevisiae and S. pombe, similar subsets of genes were also differently expressed in H. polymorpha. However, the number of differently expressed genes was much smaller clearly reflecting its relatively high tolerance to oxidative stress. [Supported by KRIBB Research Initiative Program grant and Korea Research Foundation grant (No. 2009-0075186).] 194 Poster Sessions F033 F035 Transcriptome Analysis of Xylose Metabolism in the Theromotolerant Methylotrophic Yeast Hansenula polymorpha Characterization of the CpxAR Two-Component Signal Transduction System of the Capnophilic Rumen Bacterium Mannheimia succiniciproducens Oh Cheol Kim1,2*, Surisa Suwannarangsee1, Doo-Byoung Oh1, Jeong-Yoon Kim2, Hyun Ah Kang3, and Ohsuk Kwon1 1 Integrative Omics Research Center, Korea Research Institute of Bioscience and Biotechnology, 2Department of Microbiology, Chungnam National University, 3Department of Life Science, Chung-Ang University Seulgi Yun1,2*, Eun-Gyeong Lee1, Won Seok Jung1, Doo-Byoung Oh1, Sang Yup Lee3, and Ohsuk Kwon1,2 The thermotolerant methylotrophic yeast Hansenula polymorpha is naturally capable of alcoholic fermentation of xylose, a pentose sugar abundant in lignocellulosic biomass, even at high temperature up to 48°C. In the present study, the transcriptomes of H. polymorpha grown on xylose were compared with those of glucose-grown cells both under aerobic and microaerobic conditions. About two percent of the 5,848 H. polymorpha genes were either up-regulated or down-regulated more than two-fold during growth on xylose. The majority of the up-regulated genes were involved in metabolism. Some genes involved in xylose metabolism such as TAL2, XYL1 and GRE3 were also upregulated, even though their induction levels were only about three folds. On the other hand, the majority of the downregulation genes were involved in metabolism and cellular transport. Interestingly, some genes involved in glycolysis and ethanol fermentation were also repressed during growth on xylose, suggesting that these genes might be good targets for engineering H. polymorpha to improve xylose fermentation. [Supported by KRIBB Research Initiative Program grant and Korea Research Foundation grant (No. 2009-0075186).] 1 Integrative Omics Research Center, Korea Research Institute of Bioscience and Biotechnology, 2University of Science & Technology, 3 Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering, Center for Ultramicrochemical Process Systems, and Department of BioSystems, BioProcess Engineering Research Center and Bioinformatics Research Center, Korea Advanced Institute of Science and Technology The cpxR and cpxA genes of Mannheimia succiniciproducens encode, respectively, proteins having about 70% and 50% of amino acids sequence homology with CpxR response regulator and CpxA sensor kinase of the bacterial Cpx two-component system involved in envelop stress response. The purified N-terminally truncated CpxA which was deleted for its transmembrane domain was able to autophosphorylate and transphosphorylate CpxR, demonstrating that these two proteins are a functional sensor kinase and a response regulator, respectively. We constructed a cpxR mutant strain and examined its phenotype under various growth conditions. The cpxR mutant exhibited growth retardation in the presence of 0.2 mM H2O2 or 0.2 M NaCl. In addition, when putative targets were searched by employing transcriptome analysis to overexpression of CpxR, many genes involved in cell wall biosynthesis were differentially expressed. Our data clearly indicate that CpxAR system of M. succiniciproducens might be involved in the envelop stress signaling. [Supported by the Genome-based Integrated Bioprocess Project grant of the Ministry of Education, Science and Technology (MOEST) through the Korea Research Foundation (KRF).] F034 Transcriptomic Response of Escherichia coli Host to Fosmid Metagenome Library Mun-Kyoung Min1*, Min Jee Kim1, Jae Jun Song2, Doo-Byoung Oh1, and Ohsuk Kwon1 1 Integrative Omics Research Center, Korea Research Institute of Bioscience and Biotechnology, 2Microbial Fusion Technology Research Center, Korea Research Institute of Bioscience and Biotechnology To explore the effect of metagenomic DNA on the genomewide expression in Escherichia coli, we compared the transcriptome profiles of E. coli transformed with the metagenomic DNA libraries to those of untransformed E. coli. When a fosmid library was transformed into E. coli EPI300 strain, 40 and 60 genes were up-regulated and down-regulated by at least twofold, respectively. The majority of up-regulated genes upon fosmid transformation were functionally grouped into those responsible for transport and binding proteins. In contrast, the majority of down-regulated genes were functionally categorized into energy metabolism. Further, when we compared changes in transcriptome profiles of E. coli upon transformation with different fosmid metagenomic libraries, similar genes were shown to be differentially expressed in both transformants. Thus, our transcriptome data will provide useful information to understand the genome-wide response of E. coli to metagenome library and to develop an efficient host system for metagenome cloning and functional expression for activity-based screening. [Supported by Bio R&D Programs of the Korea Research Foundation (KRF).] F036 Characterization of the NarPQ Two-Component Signal Transduction System of the Capnophilic Rumen Bacterium Mannheimia succiniciproducens Eun-Gyeong Lee1,2*, Ju-young Kim1, Doo-Byoung Oh1, Seon-Won Kim2, Sang Yup Lee3, and Ohsuk Kwon1 1 Integrative Omics Research Center, Korea Research Institute of Bioscience and Biotechnology, 2Division of Applied Life Science (BK21), EBNCRC and PMBBRC, Gyeonsang National University, 3Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering, Center for Ultramicrochemical Process Systems, and Department of BioSystems, BioProcess Engineering Research Center and Bioinformatics Research Center, Korea Advanced Institute of Science and Technology The putative narP and narQ genes of Mannheimia succiniciproducens encoding, respectively, proteins having about 90% and 70% of amino acids sequence homology with NarP response regulator and NarQ sensor kinase were identified. To determine its target operons, we analyzed the genome-wide transcriptome profiles of M. succiniciproducens in response to overexpression of NarP response regulator. Our data showed that about 20 genes and 150 genes were respectively up-regulated and down-regulated more than two-folds. Interestingly, the expression of the putative napF gene encoding periplasmic nitrate reductase was most strongly induced. A transcriptional fusion Φ(napF’-lacZ) was constructed between the promoter of napF and lacZ gene and introduced into an M. succiniciproducens lacZ mutant strain to analyze its expression under various culture conditions. The expression of Φ(napF’-lacZ) was strongly induced in the presence of nitrate, suggesting that the expression of napF gene is regulated by a nitrate dependent manner. [Supported by the Genome-based Integrated Bioprocess Project grant of the Ministry of Education, Science and Technology (MOEST) through the Korea Research Foundation (KRF).] 195 www.msk.or.kr F037 F038 Characterization of Plasmid pSY1 in Sphingobium chungbukense DJ77 Nucleotide Sequence and Secondary Structure of 23S rRNA from Sphingobium chungbukense DJ77 Mun-Sik Shin1, Kyung-Bae Min2, Ji-Eun Yu2, Hye-Jin Bae2 and Young-Chang Kim1,2,3* 1 Department of Synthetic Biology, Chungbuk National University, 2 Department of Microbiology, Chungbuk National University, 3 Biotechnology Research Institute, Chungbuk National University Mun-Sik Shin1, Won-Ho Lee2, Joo-Han Gwak2, Woo-Yeoung Jang2, and Young-Chang Kim1,2,3* 1 Department of Synthetic Biology, Chungbuk National University, 2 Department of Microbiology, Chungbuk National University, 3 Biotechnology Research Institute, Chungbuk National University Sphingobium chungbukense DJ77, isolated in our laboratory, is an aerobic gram-negative bacterial strain. This strain is able not only to degrade aromatic hydrocarbons, such as phenanthrene, anthracene, biphenyl, salicylate, p-hydroxybenzoate and benzoate, but also to produce exopolysaccharides, which possess an increasing importance in the food industry. This study determined the complete nucleotide sequence of plasmid pSY1 from S. chungbukense DJ77. It was 402,272 bp long with a G+C content of 61.81%. The 362 open reading frames (ORFs) were found. We predicted these ORFs would encode proteins associated with plasmid replication, transcription, conjugation of genes, plasmid stability/partition, hypothetical protein, and some other functions. Besides these genes, the plasmid contained Phn genes for aromatic hydrocarbon degradation. No other plasmid homologous to pSY1 in overall nucleotide sequence or gene organization could be found in the NCBI database. 196 Poster Sessions Sphingobium chungbukense DJ77 is a Gram-negative bacterium that is a very interesting organism due to its capacities to degrade monocyclic and polycyclic aromatic compounds, synthesizing glycosphingolipids as components of the cell envelope, and producing exopolysaccharides as extracellular polymers. Three 23S ribosomal RNA genes from the Sphingobium chungbukense DJ77 have been sequenced. All the three sequences are the same. The secondary structure of the 2796base-long RNA was proposed. We made the secondary structure of the 23S rRNA based on E. coli model and found eight specific regions. We found the variable regions in Sphingomonads, NovoSphingobium aromaticivorns, Sphingomonas wittichii, Sphingopyxis alaskensis, and Sphingobium chungbukense DJ77. G001 G003 Solvent/Detergent Process for the Manufacture of Porcine Acellular Dermal Matrix (ADM): Efficacy of Decellularization and Virus Inactivation Identification of a Multicopy Suppressor Gene Complementing the Arginine-Auxotrophic argJ Mutation in Corynebacterium glutamicum Jung Eun Bae1*, Dong Hyuck Lee1, Jeong Im Lee1, Eun Kyo Jeong1, Jae Il Lee1, Da Mi Choi2, Jin Young Kim2, Jae Hyoung Ahn2, and In Seop Kim1 1 Department of Biological Sciences, Hannam University, 2 Hans Daedeok R&D Center, Hans Biomed Corp. Jae-Yong Cho* and Gui-Hye Hwang Department of Pharmaceutical Engineering, College of Health Science, Sangji University Human ADM has been used in the management of fullthickness injuries as well as full-thickness burns. Human ADM is currently produced by decellularization from human cadaver skin in special buffered solutions. It retains intact basement membrane complex, collagen bundle architectures and the dermal vasculatures. However human viral contamination of donated skin as well as finding suitable donors are major obstacles to produce human ADM in a timely manner. To address these shortcomings, the manufacturing process for acellular xenograft dermal matrix using porcine skin has been developed. The most critical process for porcine ADM is to remove porcine immunogenic cells while maintaining the integrity of the nonimmunogenic components of xenograft dermis. Decellularization process using a solvent/detergent combination (0.1% TnBP/2% deoxycholic acid) has been developed. This process is also effective in inactivating porcine enveloped viruses such as pseudorabies virus, porcine epidemic diarrhea virus, and porcine rotavirus. [Supported by Business for Cooperative R&D between Industry, Academy, and Research Institute funded Korea Small and Medium Business Administration in 2008-2009] G002 We recently proposed a metabolic engineering strategy for Lornithine production based on the hypothesis that an increased intracellular supply of N-acetylglutamate may further enhance L-ornithine production in a well-defined recombinant strain of Corynebacterium glutamicum. In the present work, an argJ– deficient arginine auxotrophic mutant of C. glutamicum is suppressed by a different locus of C. glutamicum ATCC13032. Overexpression of the NCgl1469 open reading frame (ORF), exhibiting N-acetylglutamate synthase (NAGS) activity, was able to complement the C. glutamicum arginine-auxotrophic argJ strain and showed the increased NAGS activity from 0.03 to 0.17 units mg-1 protein. Additionally, overexpression of the NCgl1469 ORF resulted in a 39% increase in excreted Lornithine. These results indicate that the intracellular supply of N-acetylglutamate is a rate-limiting step during L-ornithine production in C. glutamicum. [This research was supported by the Advanced R&D Supporting Business between Industry and University funded by the Small and Medium Business Administration, Republic of Korea, and in part by the Sangji University Research Fund 2009.] G004 Hydrogen Peroxide Treatment as an Effective Measure for Enhancing Viral Safety of Allo Bone Transplants Genetically Engineered Salmonella typhimurium as a Drug Delivery System for Interferon-Gamma Induced Therapy Against Melanoma Jeong Im Lee1*, Jung Eun Bae1, Eun Kyo Jeong1, Jae Il Lee1, Dong-Joo Yu1, Seon Hyun Jeon2, Ji-Hwa Chae2, Jung Sun Jeong1, and In Seop Kim1 1 Department of Biological Sciences, Hannam University, 2Hans Daedeok R&D Center, Hans Biomed Corp. Won Suck Yoon1,2*, EunJae Kim3, Ji-hyeon Choi3, and YongKeun Park3 1 School of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea, 2Office of Research, Korea University, Seoul, Republic of Korea, 3School of Life Sciences and Biotechnology, Korea University Human demineralized bone matrix (DBM) has been clinically used as it is osteoinductive. The ability to remove and/or inactivate known and potential viral contaminants during the manufacturing process of DBM has become an important parameter in assessing the safety of the products. In order to increase the safety of DBM, 3% hydrogen peroxide treatment process has been applied. Viral clearance validation method to evaluate efficacy of 3% hydrogen peroxide was developed and then the effect of the treatment in inactivating viruses was kinetically determined. A variety of experimental model viruses for human pathogenic viruses, including the human immunodeficiency virus (HIV), hepatitis A virus (HAV), bovine herpes virus (BHV), bovine viral diarrhea virus (BVDV), and porcine parvovirus (PPV) were selected for this study. All the enveloped viruses such as HIV, BHV, and BVDV were completely inactivated to undetectable levels in 1 hour treatment. Non-enveloped viruses such as HAV and PPV were also very sensitive with the log reduction values of 1.58 and 4.21, respectively. Salmonella has been used experimentally as an anticancer agent because it shows selective growth in tumors. In this study, we exploited genetically-engineered Salmonella typhimurium expressing interferon-gamma (IFN-γ) as a tumoricidal agent to enhance its therapeutic efficacy. IFN-γ was fused to SipB and recombinant IFN-γ was produced and secreted into culture supernatants. Attenuated S. typhimurium expressing recombinant IFN-γ, invaded and induced the lysis of tumor cells, and activated NK cells. Furthermore, when it was subcutaneously administered into mice bearing melanomas, S. typhimurim expressing IFN-γ, significantly inhibited tumor growth by 80% (eight of ten mice were cured of cancer) and prolonged the survival of the mice without inducing permanent immunity against tumors. These results suggest that tumor-targeted therapy using Salmonella expressing IFN-γ has potential for the treatment of cancer. [Supported by MKE and KOTEF through the Human Resource Training Project for Strategic Technology] 197 www.msk.or.kr G005 G007 A Novel Method for the Detection of Viral Protein NS5B Using a Streptavidin-Tagged RNA Oligonucleotide Exploration of Biocatalytic Potential of Epoxide Hydrolases from Marine Bacteria by Whole Genome Analysis Changhyun Roh* and Sung-Kee Jo Korea Atomic Energy Research Institute Jung-Hee Woo1*, Young-Ok Hwang2, Ji-Hyun Kang2, Jangcheon Cho3, Jarone Pinhassi4, Sung Gyun Kang2, and Sang-Jin Kim2 1 Gyeongbuk Institute for Marine Bio-Industry, 2From the Marine Biotechnology Research Centre, Korea Ocean Research & Development Institute, 3Department of oceanography, Inha University, 4Marine Microbiology, Department of Pure and Applied Natural Sciences, University of Kalmar, Sweden The monitoring of viral protein NS5B has been considerable interest of in developing simple and reliable methods for detection the hepatitis C virus (HCV) for applications in diagnostic medicine. At present, a variety of assay methods have been developed for the qualitative and quantitative detection of HCV. Generally, antibody is the most common reagent in enzyme-linked immunosorbent assay and western blotting for the detection of such specific proteins in samples. The detection limit using antibody is approximately μmol level. To use antibody bring problem such as being temperaturesensitive, specific reactions condition and requiring secondary antibody conjugated with enzyme and fluorescent dye. To overcome these bottlenecks, aptamers are introduced as a substitute for antibody in the application of biosensors for detection and measurement of biological or environmental molecules. In this study, we designed a streptavidin-biotin conjugation method, that is, the RNA oligonucleotide sensor system that could quantify viral protein in solution with streptavidin-tagged aptamer. The detection level was fmol level with low affinity interactions in a novel designed system. Marine organisms, in particular, represent great phylogenetic diversity, making them reservoirs of unique genetic information and important natural resources for possible development. As analysis of various genomic databases, one or more putative epoxide hydrolase genes of sequenced organism were identified about 10% of marine source. Multiple sequence alignments of epoxide hydrolase and putative epoxide hydrolase genes can be classified into three groups with subgroup. The known, putative epoxide hydrolases from marine sources were tested for activity towards various epoxide substrates using a GC analysis assay. Using this assay, it appeared that of the 11 EHases that were tested, 11 were active with one or more epoxides. These results demonstrate that a couple of epoxide hydrolases have the possible application as an industrial biocatalyst for the production of enantipure epoxides or vicinal diols. [This work was supported by the Marine and Extreme Genome Research Center Program, Ministry of Land, Transport and Maritime Affairs, Republic of Korea.] G006 G008 Peptide Directed Synthesis of Silica Coated Gold Nanocables Screening of an Enantioselective Epoxide Hydrolase from PAH-Degrading Bacteria Jungok Kim1*, Nosang V. Myung2, and Hor-Gil Hur1 1 Department of Environmental Science and Engineering and International Environmental Research Center, Gwangju Institute of Science and Technology, 2Department of Chemical and Environmental Engineering and Center for Nanoscale Science and Engineering, University of California at Riverside Jung-Hee Woo*, Tae-Hyung Kwon, Jong-Shik Kim, Nyun-Ho Park, Jun-Tae Kim, Sun-Mee Hong, and Choong-Gon Kim Gyeongbuk Institute for Marine Bio-Industry Here we report a biomimetic peptide directed method to synthesize one-dimensional co-shell nanocables by sequentially guide formation of silica insulating shell on metallic gold nanoribbons in aqueous and ambient conditions. In our previous study, we have shown that gold nanoribbon was fabricated by gold-synthesizing peptide, Midas-11, isolated from phage-displayed peptide library. Furthermore, the silicasynthesizing peptide Si#6-C, which was designed and modified from peptide R5 found in natural diatom, was used for binding onto gold nanoribbon surfaces and the production of silica shell. TEM, SEM, AFM, and fluorescence microscopic analysis clearly indicate the formation of evenly coated silica on gold nanoribbons regardless of the shapes of template cores and thiol group attached with peptide is necessary for the binding and forming amorphous silica coating. This is significant since it demonstrated the ability to fabricate functional nano-electronic component using a biomimetic method. [This work was supported by grants from the Research Center for Biomolecular Nanotechnology at GIST, Korea, and the 21C Frontier Microbial Genomics and Applications Center Program.] 198 Poster Sessions Enantioselective synthesis or hydrolysis is getting much more attention due to current concerns about mixed chirality of a lot of chemical, pesticides and medicine. To screen strains producing an epoxide hydrolase (EHase) which hydrolyzes (R) or (S)-epoxide preferentially, 7 strains of PAH-degrading bacteria isolated from oil-contaminated sediment and commercial gasoline primarily by the capability of living on styrene oxide were tested for EHase activity using gas chromatography (GC). Among those, one strain was selected by highly enantioselective hydrolysis of styrene oxide, confirmed by GC. The EHase from one strain preferentially hydrolysed the (R)epoxide of styrene oxide, with a value of 98% ee (enantiomeric excess). This study presents a first example which discovered an enantioselective epoxide hydrolase from PAH-degrading bacteria successfully. [This work was supported by the Marine and Extreme Genome Research Center Program, Ministry of Land, Transport and Maritime Affairs, Republic of Korea.] G009 A Cell-Free Protein Producing DNA-Polymer Hydrogel Young Hoon Roh*, Nokyoung Park, and Dan Luo Cornell University A Cell-Free Protein Producing DNA Hydrogel Proteins are important biomaterials and are generally produced in living cells. Here, we show a novel DNA hydrogel that is capable of producing functional proteins without any living cells. This protein-producing gel (termed ‘the P-gel system’ or ‘P-gel’) consists of genes as part of the gel scaffolding. This is the first time that a hydrogel has been used to produce proteins. The efficiency was about 300 times higher than current, solutionbased systems. In terms of volumetric yield, the P-gel produced up to 5 mg ml-1 of functional proteins. The mechanisms behind the high efficiency and yield include improved gene stability, higher local concentration and a faster enzyme turnover rate due to a closer proximity of genes. We have tested a total of 16 different P-gels and have successfully produced all 16 proteins including membrane and toxic proteins, demonstrating that the P-gel system can serve as a general protein production technology. G011 Enhanced Butyric Acid Production and Resistance to Sodium Butyrate by Clostridium tyrobutyricum Mutant Ki-Yeon Kim*, Youngsoon Um, and Byoung-In Sang Clean Energy Center, Korea Institute of Science and Technology To improve butyric acid production, Clostridium tyrobutyricum mutant LR7B4 was selected after repeated mutagenesis of the parent strain Clostridium tyrobutyricum ATCC 25755 with N-methyl-N’-nitro-N-nitrosoguanidine followed by an selective enrichment on sodium butyrate. The selected mutant was fivefold more resistant to sodium butyrate than the parent strain. In batch culture on 120 g of glucose per liter, the mutant produced more than 30 g/L butyric acid under pH control using NaOH in bioreactor. Compared with the parent strain, this mutated strain produced 14.3% more butyric acid and 18.5% less acetic acid in 96 hours under the same culture conditions. This result suggested that mutagenesis with N-methyl-N’-nitroN-nitrosoguanidine together with selective enrichment on sodium butyrate improved production of butyric acid from glucose in the fermentation. [Supported by NYSTAR Faculty Development Program Award, NYSTAR CAT grant, US National Science Foundation's CAREER award (grant number: 0547330) and a USDA NRI grant.] G010 Properties of Bacillus licheniformis B1 β-1,4Glucanase Overproduced in Escherichia coli Han Bok Kim*, Hye Jung Song, Hwang Yeon Kim, Jae Sung Hwang, and Cho Hee Lee Department of Biotechnology, The Research Institute for Basic Sciences, Hoseo University The Bacillsus licheniformis B1β-1,4-glucanase gene was overexpressed in Esherichia coli BL21. A soluble protein with a mass of 50 kDa was overproduced. A protein having a mass of 37 kDa was secreted from B. licheniformis. It is likely that the β-1,4-glucanase produced in E. coli contained the leader peptide and unprocessed carboxy-terminal region, but its processing occurred in the carboxy-terminal in Bacillus. The optimal temperature of β-1,4-glucanase was 40°C and the enzyme still had 76% maximal activity at 60°C. Although he optimal pH of the enzyme was 7, the enzyme retained considerable activities over the broad pH range. Acidic fungal cellulases are frequently used in food, detergent, pulp, textile industries. However, studies about neutral and alkaline cellulase are limited. The β-1,4-glucanase developed in this study might be useful for industrial applications in the fields of biofuel development. G012 Cloning and Expression of Indole Oxygenase Gene Derived from Corynebacterium glutamicum in Escherichia coli Sisi Patricia Efkaen* and Jinho Lee Department of Food Science & Biotechnology, Kyungsung University A putative gene derived from Corynebacterium glutamicum was confirmed for its indole oxygenase activity. This gene was cloned into the shuttle vector, pCES208 in Escherichia coli under the regulation of tac promoter, and designated as pCOX1. The complete open reading frame of this indole oxygenase was 1,413 bp long, which encodes a protein of 470 amino acids. Crude extract of E. coli W3110/pCOX1 was prepared and subjected to SDS-PAGE analysis. A band corresponding to molecular mass of about 54 kDa was appeared and this result correlated with the predicted molecular mass of the cloned indole oxygenase. The E. coli harboring pCOX1 showed blue color colony in LB plate. The pigment showing blue color was prepared from E. coli/pCOX1, and identified as indigo by experiments using UV spectrophotometer, TLC, and HPLC. The enzyme activity of the indole oxygenase from the whole cell of E. coli/pCOX1 cultured at LB medium with 0.1 M IPTG showed 2.347nmol/min/mg DCW (dry cell weight). Also, E.coli W3110 containing pCOX1, produced about 281mg/L of indigo after 48 hours cultivation in LB medium with 5 g/L of tryptophan. 199 www.msk.or.kr G013 Diversity of Polyketide Synthase Genes in Lichen Cladonia spp. Hyun-Ju Noh1,2*, Jin sung Lee1, Chae Haeng Park1, Eung-Soo Kim2, and Soon Gyu Hong1 1 Polar BioCenter, Korea Polar Research Institute, Korea Ocean Research and Development Institute, 2Department of Biological Engineering, Inha University Lichens are well known to produce a great variety of secondary metabolites including polyketides which have diverse biological roles and potential uses in pharmaceutical application. To attain a comprehensive understanding of polyketide synthase (PKS) gene diversity of lichen species from polar regions, forty-two Cladonia samples were selected from Chile, the Artic and the Antarctic regions. The βketosynthase (KS) domains of putative PKS genes were amplified and sequenced using degenerate primers. We obtained 25 KS sequence fragments from direct sequencing and 33 fragments from cloning. Phylogenetic analyses of 58 KS sequences have shown that 10 distinct PKS types were retrieved, of which all belonged to non-reducing (NR) PKS. Three and six types were included in NR clade I and clade II, respectively. The last type of PKS was related to NR type I and type II, but the specific relationship was not resolved well. The most abundant PKS type consisted with 30 sequences from 23 samples in NR clade II. The sample with the most diverse PKS types was included Cladonia furcata. It contained 8 types of PKS, two of which belonged to NR clade I and the other six types belonged to NR clade II. G014 Screening of Garlic Fermentative Bacteria Using Isolation Medium Containing Garlic Juices as Sole Carbon and Nitrogen Sources Su-Ok Kim1*, Jae-Won Park1, Mi-Young Won1, Sang-Buem Cho1, Chan-Gul Lee2, and Soo-Ki Kim1 1 Department of Animal Sciences and Environment, Konkuk University, 2Chodae F&P Co. Ltd. Historically, garlic (Allium sativium) has been regarded as protective food throughout the countries. The effectiveness of garlic in cancer, aging and cardio-vascular disease have been researched and free radical scavenging activity was regarded as the most promising characteristics of garlic. Sulfuric compounds in garlic are known as representative to antioxidant activity. However, their low stability during processing such as washing and packing are a problematic to manufacturing of garlic product. To ensure the stability of their antioxidant activity, fermentation has been investigated. In this study, the bacteria that involved in garlic fermentation were screened. Grinded fresh garlic juice was used as sole carbon and nitrogen sources in isolation medium. Sodium acetate also used as an ingredient to stabilize cytoplasmic pH. Finally grinded garlic juice, sodium acetate, dipotassium phosphate, magnesium sulfate and sodium chloride were included in isolation medium. From isolation medium, one hundred eight strains were isolated and thirty strains were selected using relative performance index including both of antioxidant activity and viability. 200 Poster Sessions G015 Biochemical Characterization of β-Glucosidase Produced by Phoma sp. Isolated from the Surface of Rotten Tangerine Peel Jung Youn Choi1,2*, Ah Reum Park1, Yong Jin Kim1, Chang-Jun Cha2, and Jeong-Jun Yoon1 1 Green Process R&D Department, Green Chemistry & Manufacturing System Division, Korea Institute of Industrial Technology, 2Department of Biotechnology, Chung-Ang University Recently, red seaweed attacks growing interests as 3rd generation biomass due to their notable characteristics e.g., no lignin, fast-growing rate, high contents of carbohydrates such as cellulose and galactan. However, cellulose and galactan should be degraded to mono sugar which will be used for the fermentation to produce bioethanol. In this study, three fungal strains with cellulose degrading activity were isolated. When these fungus grown on cellulose culture the β-glucosidase activity showed the highest. One of them was already identified to phoma sp. and β-glucosidase from this fungus purified to homogeneity. The purified β-glucosidase is a tetramer protein of 440 kDa molecular weight and exhibits optimal activity at 65°C, pH 4.5 The kinetic parameters, Km and Vmax values with ρ-Nitrophenyl-β-D-glucopyranoside (ρNPG) as a substrates are 0.21 mM and 113.87 μmol/min/mg, respectively; with cellobiose the corresponding values are 0.14 mM and 78.55 mmol/min/mg. The activity is stimulated by Mg2+, Zn2+ and inhibited by Fe2+, Cu2+. [This work was supported by a grant from Korea Institute of Energy Technology Evaluation and Planning, Ministry of Knowledge Economy, Republic of Korea] H003 H001 Chemoattractant-Mediated Rap1 Activation Requires GPCR/G Proteins in Dictyostelium Mannitol Accumulation in Fermentation of Kimchi Using Leuconostoc Starters and Their Mutants Taeck Jeon* and Injun Cha Department of Biology, College of Natural Sciences, Chosun University Gan-Erdene Otgonbayar*, Seung Kee Cho, Hwa Young Choi, and Nam Soo Han Chungbuk National University, Department of Food Science and Technology Rap1 is rapidly activated upon chemoattractant stimulation and plays an important role in cell adhesion and cytoskeleton reorganization during chemotaxis. Here, we demonstrate that G-protein coupled receptors and G-proteins are essential for chemoattractant-mediated Rap1 activation in Dictyostelium. The rapid Rap1 activation upon cAMP stimulation is absent in cells lacking chemoattractant cAMP receptors cAR1/cAR3 or a subunit of the heterotrimeric G-protein complex Gα2. Loss of guanylyl cyclases GCA/SGC or a cGMP-binding protein GbpC exhibits no effect on Rap1 activation kinetics. These results suggest that Rap1, a key regulator for the regulation of cytoskeletal reorganization during cell movement, is activated through GPCRs cAR1/cAR3 and Gα2 proteins in a way independent upon cGMP signaling pathway. F-actin polymerization is linked to Rap1 activation. In this study, we show that inhibition of F-actin assembly result in extension of Rap1 activation kinetics, suggesting that F-actin assembly is required for deactivation of Rap1 after chemoattractant stimulation. [Supported by NRF grant funded by the Korean Government (2009-0065992 and 2009-0070924)] D-Mannitol, a six-carbon sugar alcohol, is about half as sweet as sucrose and assumed to have several beneficial effects such as an antioxidant and a non-metabolizable sweetener. Among lactic acid bacteria only heterofermentative species are known to convert fructose into mannitol. Mannitol production by lactic acid bacteria in kimchi fermentation offers several important advantages. This study is composed of two parts, at first screening best mannitol producing wild type strains and their EMS mutants secondly application of the promising strains as starter culture for kimchi production. 8 heterofermentative lactic acid bacteria were compared as to their ability to convert fructose to mannitol. Leuconostoc mesenteroides 9135, Leuconostoc mesenteroides 8293, and Leuconostoc mesenteroides DRC strains were treated with EMS and were grown with fructose addition. The yield of mannitol from fructose was improved by 22%, 53%, 3% respectively. Leuconostoc mutants were applied to kimchi as starter with addition of 1%-3% fructose syrup and the mannitol accumulation in kimchi was measured. H002 H004 Understanding of Complex Nature of Evolution Process Through Integrated Analysis of Multiple Evolutionary Trees * Nutritional Requirements of Leuconostoc mesenteroides in a Chemically Defined Medium Yujin Kim1*, DongYup Lee2, and NamSoo Han1 Department of Food Science & Technology, Chungbuk National University, 2Department of Chemical & Biomolecular Engineering, National University of Singapore, Singapore Soon Ho Hong and Thuy Vu An Nguyen School of Chemical Engineering & Bioengineering, University of Ulsan 1 Evolution of living organism is combination of highly complicated processes involving modification of various features such as appearance, metabolism and sensing systems. In spite of complex process of evolution, traditional evolutionary analyses can only estimate single aspect of it. In this paper, three evolutionary trees were constructed based on twocomponent system contents, metabolic network contents and 16S rRNA sequences. Then integrated analyses of trees were carried out to understand various aspects of evolution process. The results showed that integrated analysis can give new insight into bacterial evolution study. Leuconostoc species are heterofermentative gram-positive bacteria that are commonly found in the fermented foods. They play key roles in industrial and food fermentations. For metabolic investigations, it is desirable to have a defined growth medium for these bacteria which provides reproducibility of chemical composition; avoids an unnecessary excess of the nutrients, facilitating the adjustment of their levels; meets the experimentally determined nutritional requirements of several strains. Therefore we developed a chemically defined medium(CDM) for the growth of Leuconostoc mesenteroides. The single-omission technique was applied to each component of complete CDM in order to determine the nutritional requirements. L. mesenteroides required glutamine, isoleucine, methionine, valine, leucine, cysteine as essential amino acids and threonine, phenylalanine, tyrosine, asparagine, lysine, serine as additional amino acids for the growth. Glucose, Mn, Mg, eight vitamins, adenine, uracil, and Tween 80 were also consumed for the growth. This medium is simple and well defined, and should be preferable to complex media for conducting future biochemical, physiological, and genetic studies. [This work was supported by the Korean Systems Biology Research Program (M10503020001-07N0302-00112) of Ministry of Science and Technology (MOST).] 201 www.msk.or.kr H005 H007 Molecular Characterization of Lignin Degradation Enzyme Genes for Pretreatment of Lignocellulosic Biomass * Sun-Hwa Ryu , Myung Kil Cho, Bo-Young Kim, Ji Yang, Mi-Hwa Choi, Myungkil Kim, and Sung-Suk Lee Division of Forest Bioenergy, Korea Forest Research Institute White rot fungi secret a large number of lignin enzymes such as laccase, Mn-peroxidase(MnP) and lignin-peroxidase for degradation of aromatic compounds in nature. Two laccase and six MnP genes were isolated from Polyporus brumalis. The expression of cDNAs was investigated with a view to understanding the physiological functions of laccase and MnP in relation to degradation of recalcitrant materials.The induction of gene expression was stimulated by treatment of DBP. Transcripts of pblac2, pbmnp2 and pbmnp4 were highly expressed in the both of medium, whereas pblac1, pbmnp1 and pbmnp5 genes were induced by DBP treatment. But the pattern of total laccase and MnP enzyme activities were not exactly coincided with that of the gene expressions. It can be information that each laccase and MnP may have different enzymatic properties and physiological functions in the mechanism of DBP degradation. The identification of cDNAs of laccase and MnP should contribute to the efficient production of lignin degradation enzyme and the utilization of basidiomycetous fungus for degrading lignin as well as many recalcitrant xenobiotics. H006 Identification of the Single-Nucleotide Polymorphisms of COI in Crabs of the East Sea of Korea Sun-Mee Hong*, Nyun-Ho Park, Dong-Goong Choi, So-Jung Kim, Jung-Hee Woo, and Choong-Gon Kim Department of Research and Development, Gyeongbuk Institute for Marine Bioindustry The aim of this study is to screen single nucleotide polymorphisms (SNPs) of mitochondrial gene, COI, in crab in the east sea of Korea. The cytochrome c oxidase (CO) plays important roles in oxidative phosphorylation regulation and oxygen sensing transfer. Defects involving genetic mutations altering CO functionality or structure can result in severe, often fatal metabolic disorder. In the present study, SNPs of Cytochrome c oxidase subunit I (COI) were identified with the techniques of sequence aligns and single-strand conformation polymorphism (SSCP) in snow crab of Uljin, Pohang, Youngduk and red crab in the east sea of Korea and Russia crab. In total, 12 SNPs were identified in the COI gene of the snow crab including Russian crab, and red crab was defined for the 1 SNPs. This work will afford reference for the further study on the association of COI with the adaptation to crab distribution in the east sea of Korea. [This study was supported by grant from MIFAFF.] H008 Characterization of Diverse Lipolytic Enzymes Derived from Marine Metagenomic Libraries Accuracy Evaluation of Pyrosequencing for Microbial 16S rRNA Metagenome Analysis Jeong Ho Jeon1,2*, Yun Jae Kim1, Hyun Sook Lee1,3, Sang-Jin Kim1,3, Sang Ho Choi2, Sung Gyun Kang1,3, and Jung-Hyun Lee1,3 1 Marine Biotechnology Research Center, Korea Ocean Research & Development Institute, 2Molecular Microbiology and Toxicology, Department of Agricultural Biotechnology, Seoul National University, 3Department of Marine Biotechnology, University of Science and Technology Suk-Hwan Yoon1*, Soyeon Ahn2, Ok-Sun Kim1, You-Seak Go3, Jeong-Sun Seo3,4, Taesung Park2, Jonathan M. Adams1, and Jongsik Chun1 1 School of Biological Sciences and Institute of Microbiology, Seoul National University, 2Department of Statistics, Seoul National University, 3Macrogen, Inc., World Medridian Center, 4 Genomic Medicine Institute, Medical Research Center, Seoul National University Metagenomic libraries were constructed using marine sediments from the deep sea of the Edison Seamount, the intertidal flat of Ganghwa Island and the seashore of the Arctic station. Seventeen positive clones were identified by screening for lipolytic activity on tributyrin plates. Based on the phylogenetic analysis, 13 enzymes were classified as belonging to Families I, IV, and V, Feruloyl esterase group or Patatin-like protein group, and 4 other enzymes were assigned to four new groups that have never described. In an attempt to express the genes with lipolytic activity as soluble proteins in Escherichia coli, we employed a combination of approaches such as removing the hydrophobic region in the N-terminus, co-expression of chaperone genes and low temperature induction. Using affinity chromatography, we obtained soluble proteins encoded by 14 genes. The optimum activities of the enzymes were determined in the temperature range of 20–45°C and the pH range of 7.5– 9.5. Cold-activity was observed for 7 enzymes. While 13 enzymes showed esterase activity by hydrolyzing short-chain fatty acid esters, EM3L4 showed lipase activity by hydrolyzing long-chain fatty acid esters. Pyrosequencing, one of the next generation sequencing methods, generates massive sequence data with low cost without clone library construction. At present, the most up-todate version, called 454 Genome Sequencer(GS) FLX Titanium system, generates nucleotide sequence data with read lengths of 400-500 bp on average. Use of pyrosequencing in microbial 16S rRNA metagenome analysis has been developed by several researchers and proved to be efficient in elucidation of microbial community structure even of very complex environments. However, the accuracy of the sequence data from 454 GS FLX Titanium system has not yet been assessed to date. We evaluated the accuracy of 454 GS FLX Titanium by massive sequencing of 16S rRNA amplicons from fourteen bacterial pure cultures. Sequencing by 454 GS FLX Titanium produced reads with 51-547 bp in length, and the average accuracy in raw data is 99.6%. In the analysis of comparisons by read lengths, the higher accuracy rate was obtained in longer read ranges. This study suggests that error rate of pyrosequencing method is not substantially high enough for hampering microbial 16S rRNA metagenome analysis based on PCR of rRNA. 202 Poster Sessions H009 H011 Agreement, Precision and Accuracy of Epifluorescence Microscopy Methods for Enumeration of Total Bacterial Numbers 1* 1 2 Eun Young Seo , Tae Seok Ahn , and Young Gun Zo Department of Environmental Science, Kangwon National University, 2Department of Biology, Kyungsung University 1 To assess interchangeability of estimates of bacterial abundance by epifluorescence microscopy methods, total bacterial numbers (TBNs) determined by widely accepted protocols were statistically compared. Bacteria in a set of distinctive samples were stained with acridine orange (AO), 4'-6diamidino-2-phenylindole (DAPI), and BacLight and enumerated by visual counting(VC) and image analysis (IA). Model II regression and Bland-Altman analysis proved general agreements between IA and VC methods, although IA counts tended to be lower than VC counts by 7% on a logarithmic scale. Distributions of cells and latex beads on polycarbonate filters were fitted to negative binomial models. The fitted models revealed higher precisions of TBNs by the IA method than those by the VC method. In pairwise comparisons of the staining methods, TBNs by AO and BacLight staining showed good agreement with each other, but DAPI staining had tendencies of underestimation. The TBN values estimated by AO and BacLight staining are relatively accurate and interchangeable for quantitative interpretation and that IA provides better precision than does VC. H010 Rap1 Regulation of Actin Cytoskeleton in Dictyostelium Taeck Jeon* and Injun Cha Department of Biology, College of Natural Sciences, Chosun University Chemoattractant-mediated Rap1 activation helps establish cell polarity by locally modulating cytoskeletons. Here, we demonstratethat Rap1 interacts with RacGEF1 in vitro and stimulates F-actin polymerization at the sites where Rap1 is activated upon chemoattractant stimulation. Live cell imaging using GFP-coronin, a reporter for F-actin, demonstrates that cells expressing constitutively active Rap1 (Rap1CA) exhibit a high level of F-actin uniformly distributed at the cortex including the posterior and lateral sides of the chemotaxing cell. Examination of the localization of a PH-domain containing PIP3 reporter, PhdA-GFP, and the activation of Akt/Pkb and other Ras proteins in Rap1CA cells reveals that activated Rap1 has no effect on the production of PIP3 or the activation of Akt/Pkb and Ras proteins in response to chemoattractant stimulation. In vitro binding assay using truncated RacGEF1 proteins shows that Rap1 interacts with the DH domain of RacGEF1, raising a possibility that Rap1 mediates F-actin assembly by interacting with RacGEFI. [This work was supported by National Research Foundation of Korea Grant funded by the Korean Government (20090070924).] H012 The Enhanced Half-Life of MxaJ Protein by Methanol Improves Methanol Dehydrogenase Activity Regulation of Actin Cytoskeleton by Cortexillin Through Arp2/3 Complex Hee Gon Kim1*, Wonduck Kim2, and Si Wouk Kim1 Department of Environmental Engineering, BK21 Team for Biohydrogen Production, 2Pioneer Research Center for Controlling of Harmful Algal Bloom, Chosun University Taeck Jeon* and Jisun Kang Department of Biology, College of Natural Sciences, Chosun University 1 A methanol dehydrogenase (MDH) is a key enzyme of methanol oxidation process in Methylophaga aminisulfidivorans MPT. Previously, two types of MDH (MDH I and II) in cell free extracts were purified from MPT cells grown on methanol. The purified MDH I was confirmed to be composed of two subunits (α, β MW = ~66, ~10 kDa) in a form of α2β2. In addition, it was found that MDH II contained an additional ~30 kDa protein (MxaJ), designated γ, in a form of α2β2γ and MDH II had 1.5 ~ 2.0 times higher activity than MDH I. In this study, to investigate effect of methanol on MDH stability, the half life of α and γ subunit of MDH in M. aminisulfidivorans MPT grown on methanol and fructose was compared. To obtain the half-life values, chloramphenicol was added into the medium for translation halt. The analysis of each MDH subunit analyzed by western blot indicates MxaJ protein from the methanol grown cells was more stable than that from fructose grown cells. The relative activity of MDH was proportional to the amount of MxaJ protein. Based on these experimental data, it appears that the γ subunit contributes to formation of active MDH, thereby increases stability of MDH. Cortexillin (Ctx) plays a critical role in the formation of cell shape and reorganization of actin cytoskeleton in Dictyostelium. Here, we demonstrate that Ctx mediates reorganization of actin cytoskeleton through Arp2/3 complex. Loss of Ctx leads to a delayed chemoattractant-mediated ArpD translocation kinetics to the cortex with a peak at 8 sec, 2-3 sec slower than in wild-type cells, indicating that proper translocation of Arp2/3 complex to the cortex requires Ctx. Reduced cell polarity and increased number of lateral pseudopodia in chemotaxing cells lacking Ctx suggest Ctx’s functions in controlling cell polarity and pseudopodia formation. In chemotaxing cells, CtxI is enriched in the lateral sides of the moving cells. Upon chemoattractant stimulation, there is a rapid release of CtxI from the cortex followed by a transient translocation to the cell cortex with a peak at -5 sec and a subsequent decrease to basal levels. Highly dynamic subcellular localization of CtxI implicates two more signaling components mediating the translocation of Ctx during chemotaxis. [This work was supported by National Research Foundation of Korea Grant funded by the Korean Government (20090065992).] 203 www.msk.or.kr H013 Infectious Disease Biomarker Database (IDBD) Kyung-Tae Jung, Kyenam Lee, Juhee Heo, Hae-Seul Jeong, So-Jung Yoon, Cheon-Kwon Yoo, and Kwang-Jun Lee Division of High-Risk Pathogen Research, Center for Infectious Disease, National Institute of Health, KCDC For the biotechnological research revitalization and diagnosis development of these infectious, We have constructed Infectious Disease Biomarker Database (IDBD) by knowledge-based web platform on useful biological contents of pathogen biomarkers. IDBD provides several omics information and global news of biomarkers as well as the description of pathogens and disease. It is a community annotation database utilizing collaborative Web 2.0 features, providing a convenient user interface to input and revise data online. It supports various types of data searches and application tools to analyse sequence and structure features of potential and validated biomarkers. Currently, IDBD integrates 710 biomarkers for 11 disease group, 79 infectious diseases and 79 pathogens. Researchers can effectively utilize this website as new information entry in the biotechnology field. The content in IDBD is open and freely accessible to the general public. (http://biomarker.cdc.go.kr) Also, we will link biomarker information with pathogenic resource of national culture collection for pathogen (NCCP) to distribution service. H014 Korea National Microorganisms Research Resource Center Sang Seob Lee Kyonggi University The Korea National Microbiological Research Resource Center is the core center of the twelve microorganism banks designated by the Ministry of Education, Science and Technology. The KNMRRC supports microorganism banks with necessary guidelines, standards, training for efficient operation of the banks. It also provides with an effective forum to solve common issues of the related banks. The ultimate goal of the KNMRRC is the followings: ①construction of standardized and integrated management system, ②construction of Core center and other organs network, ③Quality Control(QC) of microbial resources in the member banks, ④conservation of Resources in the member banks and the interrupted banks, ⑤education for professionals in the member banks, ⑥public Relations for raising people's awareness of the importance of microbiological resources. 204 Poster Sessions H015 Korea National Environmental Microorganisms Bank Sang Seob Lee Kyonggi University Korea National Environmental Microorganisms Bank(KEMB) has been established as a microbial and genetic resource center for environmental industries. The KEMB plays an essential role as follows: ①the collection and conservation of native environmental microorganisms and genetic resources, ②the construction of systematic management system for effective conservation and application of microbiological resources for environmental industries, ③the provision fundamental data for ecosystem research and microbial classification, and ④the development of biological treatment system for bioremedation of environmental pollutant and ecosystem restoration. There are about 14,000 strains of bacteria collected from environments, at this time. These collections including photosynthetic bacteria, oil-derived compounds removal bacteria, industrial waste water disposal bacteria, odor degrading bacteria and BOD/COD degrading bacteria are classified in accordance with scientific and functional characteristics, respectively. It is considered to promote academic and industrial activities by supplying basic materials for research and industrial applications, which accomplish the ecological recovery through constructing eco-friendly bioremediation system by supplying basic microbial resources. H016 Center for Fungal Genetic Resources (CFGR): Housing Plant Pathogenic Fungi For Educational and Research Purposes Yeo Kyoung Yoon* and Yong-Hwan Lee Center for Fungal Genetic Resources, Seoul National University Fungi are eukaryotic organisms, growing in a wide range of habitats. Fungi are significantly important in a variety of ways. They play an essential role in the decomposition of organic matter. They have been used as a source of food, and agents for fermentation of food products and for the production of various antibiotics and enzymes that are used in a field of research, industry, medicine, etc. In contrary, impact of many fungi on animals and plants is economically and socially detrimental. For example, Magnaporthe oryzae causes the most destructive disease, “rice blast”. Annual yield loss of rice by rice blast is equivalent to rice that could feed about 60 million people. The Center for Fungal Genetic Resources (CFGR) was established to collect, maintain and distribute genetic resources mainly from plant pathogenic fungi, which are important for both educational and research purposes. This will contribute to development of new strategies for management of crop diseases and of new components for improvement of our lives. CFGP possesses important fungal species; a total of 35,400 isolates from 52 species of fungi including 21,090 T-DNA transformants of rice blast fungus and anthracnose fungus. In addition to the biological materials, CFGP has developed user-friendly databases to maintain genetic information of fungal stocks and help to solve questions about fungal pathogenicity, population genetics, development, and evolution. Also, CFGP seeks strategies for sustainable and scientific plant quarantine to better protect our ecosystem from invasive microorganisms. H017 H019 Bank of Waterborne virus Culture Collection of Mushrooms Soon-Young Paik Department of Microbiology, College of Medicine, The Catholic University of Korea Cha Yoon Jeong, Jeong Hwa Kim, Jae Seong Lee, Hae Jin Cho, Nuhu Alam, Ajith Indrawansha Rathnayake, U Youn Lee, and Tae Soo Lee Department of Biology, University of Incheon The purpose of this bank is collecting and storing various waterborne virus isolates provoking severe infections in animal and human. This bank collects research information on various viruses and provides this information when it is needed. We provide various waterborne viruses, genomes and host cells to hospitals, universities, research institutes, and government institutes in the country as well as abroad. We also provide the identification services of waterborne viruses and the research data through on-line. Finally, it contributes to progress biological science and to improve public health. H018 The “Culture Collection of Mushrooms” (CCM) was founded in 2008 and succeeded to Culture Collection of Wild Mushroom Species which was established in 2002 at the Department of Biology, University of Incheon, Korea. The National Research Foundation of Korea (NRF) provides most of the financial assistance for operation of CCM. All of the mushroom cultures in CCM were preserved at 4°C refrigerator or deep freezer of -80°C. The cultures in agar slant stored at 4°C were transferred periodically at the interval of 6 months. The cultures stored in deep freezer were also transferred every 3 years. Since establishment of “Culture Collection of Mushrooms”, CCM has been played supporting role for the various research organizations by providing mushroom cultures and mushroom DNA. CCM, also provides useful information on mushroom researchers and industries. So far, the CCM holds 3,700 mushroom strains which can be used for basic and applied mushroom science research purposes. CCM has been a member of World Federation for Culture Collection (WFCC) since 2005. If anyone who are interested in mushroom cultures and DNA from CCM, please contact [email protected] or http://www.wildmush.or.kr H020 Phagebank Korea Bank for Pathogenic Viruses * KyoungEun Cha and Heejoon Myung Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies Bacteriophages are viruses growing on bacterial hosts. They are antagonistic to bacteria and first reported by Frederick Twort and Felix d'Herelle in 1915 and 1917, respectively. They are found in sea, air, land and even foods. It is assumed that 1030 to 1032 phages exist on earth and they play a role in maintenance of biological balance. Recently, new applications for phages are increasingly reported. As they are a part of useful biological resources, there are increasing demand for secure these resources. In response to these demands, this bank collects phages from environments as well as from working groups worldwide. Currently, 50 different phages are stocked. At the same time, we serve as a distributor for the collected phages. Ki-Joon Song Korea Bank for Pathogenic viruses Korea Bank for Pathogenic Viruses in Korea(KBPV; www.kbpv.co.kr) has been established in 2005 as a repository agent for the collection, management and distribution of the various pathogenic viruses that are essential to use for researches in biomedical sciences. The Institution operates in collaboration with The Institute for Viral Disease at Department of Microbiology, College of Medicine, Korea University, founded in 1973. The bank has unique viral collections such as Hantaan, Seoul, Muju, and Soochong, the etiologic agents of hemorrhagic fever with renal syndrome. To date, total of more than 43,000 materials(~100,000vials) from human and animal have been collected and maintained. We have provided a highly collaborative environment for researchers in various fields by providing valuable viral resources including consulting service. We also provide the educational program related to pathogenic viruses including biosafety training. Requestors of such agents are required to register with KBPV and to supply details of their laboratory facilities and safety management. More details about KBPV can be found at http://www.kbpv.co.kr. 205 www.msk.or.kr H021 H023 Plant Virus GenBank Myxobacteria Bank Ryu Ki Hyun Department of Horticultural Science, Seoul Women's University Jinwoo Chung and Kyungyun Cho* Myxobacteria Bank, Department of Biotechnology, Hoseo University Plant Virus GenBank (PVGB) is a nonprofit semi-governmental organization, one of the Korea National Research Resources Collections (KNRRC) for special research materials Banks program financially supported by the Ministry of Education, Science & Technology (MEST) dedicated to collection, identification, characterization, preservation, research development, distribution and deposition of plant virus research biomaterials established since 1999. PVGB is one of substructure of Korea National Microbiological Research Resources Collections (KNMRRC). PVGB retains a number of accessions and a wide range of collections of Plant Virus Biomaterials useful for Plant Virology and Biotech-related research areas. PVGB has moved to its current status on November in 2000 and has modern facilities and infrastructures for supporting broad research fields as well as Plant Virology Community. PVGB has been recognized as a member of World Federation for Culture Collection & World Data Center for Microorganisms (WFCC-WDCM) and ISBER since April of 2001 and June of 2007, respectively. Main objectives and contents of PVGB can be categorized as 7 topics as follow ; collection and development of Plant Virus Research Biomaterials such as infectious plant virus culture, plant viral cDNA clone, plant virus antiserum, biologically active full-length cDNA clone, viral cDNA library, virusinduced plant cDNA library, and diagnostic primers, preservation of Plant Virus Research Biomaterials, Distribution of Plant Virus Research Biomaterials to worldwide researchers to support their research fields and Safe Deposit from virologists, Development of New Plant Virology Techniques, i.e., molecular taxonomy of plant viruses, infectious cDNA clones, molecular indexing of virus variation, screening of virus resistance, virus-resistant transgenic plants, and risk assessment for living modified (LM) virus and LM plant systems, collection and support of Research Information of Plant Virology, and workshop and seminar for Plant Virology. H022 Culture Collection of Antimicrobial Resistant Microbes Yeonhee Lee*, Eunju Shin, Hakmi Lee, Kieun Lee, Hyunjin Hong, Hyeran Nam, Miyoung Lee, and Jihye Park Culture Collection of Antimicrobial Resistant Microbes, Department of Biology, Seoul Women’s University Antimicrobial resistance became a world-wide problem and this must be dealt via cooperation among microbiologist, biochemist, medical doctor, pharmacologist, agricultural scientists. Since Culture Collection of Antimicrobial Resistant Microbes was established in 1999, CCARM has been played a role as a connector among various research fields by providing the antimicrobial resistant microbes with known mechanism and information. CCARM collects, keeps, and preserves the resistant microbes in a systemic manner for constant supply of certified microbes and share the information with researchers in various fields. To date, CCARM has a collection of over 19,000 strains of bacteria and yeast from 87 genera and provides various information including international meeting, newest information related to resistance via homepage and newsletter. CCARM is now increasing the interaction and collaboration between culture collections through national and international network as a member of Clinical Laboratory Standards Institute since 2000, World Federation for Culture Collection & World Data Center for Microorganisms since 2003, International Society of Biological and Environmental Repositories since 2007, Korea National Research Resource Center since 2008, and Biological Repositories since 2009. 206 Poster Sessions Myxobacteria—gram-negative soil bacteria belonging to δproteobacteria—produce a wide variety of bioactive secondary metabolites and enzymes with unique action mechanisms. However, in comparison with other microorganisms such as actinomycetes, relatively few myxobacterial strains have been isolated; moreover, the biological activities of their metabolites and enzymes have not been studied extensively. Thus, myxobacteria are considered as one of the last microbial resources for identifying new medicines, enzymes, and biopolymers. In the light of this situation, Myxobacteria Bank has been appointed as one of the National Research Resources Banks by the Korean Science and Engineering Foundation in 2007. Currently, Myxobacteria Bank maintains a collection of 2,451 wild myxobacterial strains isolated in Korea and 488 culture extracts, 40 genomic DNA, and 48 clones of secondary metabolite biosynthetic genes obtained from these strains. The bank also has 34 myxobacteria type strains and genetic tools used to manipulate myxobacteria. H024 Lichen as a Novel Bioresources in Korea Jae-Seoun Hur and Young Jin Koh Korean Lichen Research Institute, Sunchon National University Lichens are symbiotic organisms composed of a fungus (mycobiont) and an alga (photobiont). They produce characteristic secondary metabolites, lichen substances, which seldom occur in other organisms. Lichen and their metabolites have many biological activities. In spite of the wide spectrum of biological activities shown by the lichens, they have long been neglected by mycologists and overlooked by agrochemical industry because of its slow growth in nature and difficulties in the artificial cultivation of organisms. Use of lichen-forming fungi can overcome the disadvantage of natural lichen extracts for industrialization of their metabolites because of their much faster growth and larger production of the metabolites in culture than the natural thalli. Korean Lichen and Allied Bioresources Center focuses on isolation, maintenance and distribution of lichen bioresources to research groups in universities, national institutes and industrial sectors. It also screens their biological activities, and investigates cultural conditions for large production of lichen substances. Chemical library of some lichen extracts is also available from the center. H025 Korea Marine Microalgae Culture Center Sung Bum Hur Department of Marine Bio-materials and Aquaculture, Pukyong National University Today microalgae are widely used in research and as educational materials. They are also commercialized in the industries of food, animal feed and environment. Microalgae exhibit a promising potential to be converted into pharmaceutical products and bio-fuel energy. For this reason, there are active, ongoing researches on microalgae with tremendous expectations of scientists The Korea Marine Microalgae Culture Center (KMMCC) was established with a financial support from Korea Science & Engineering Foundation. In 1995, the collection of microalgae strain has been increasing continuously since 1995, and its number reached to about 1,600 strains in 2010. The collection mainly consists of marine strains (83%) which are mostly isolated from Korean waters (89%). The major classes of the strains are Bacillariophyceae (57%), Chlorophyceae (20%), Dinophyceae (5%), Cyanophyceae (5%), Prasinophyceae (4%), etc. With respect to identification of the strains, about 52% and 44% of them are identified at the level of species and genus, respectively. In addition, 4% are unidentified, and about 56% of the strains are under axenic state. The culture strains of the KMMCC are introduced regarding information on sampling, culture, biological and chemical characteristics of each strain. The initial direction of the KMMCC focused on finding microalgal strains that had a good dietary value for larvae in aquaculture. Such strains used to be supplied to the hatchery. Our recent work, however, has shifted to collecting a wider range of diverse microalgae which are taxonomically different. In accordance with the KMMCC's progression on the strain collection, the demand for the strains in research fields has been expanding from the industry of aquaculture to biotechnology, environmental sciences, engineering, biological oceanography, etc. in Korea. Recently, the annual domestic request for the algal strains from academic and commercial organizations has increased up to nearly 100 requests for as many as 200 strains. Making a deposit with newly isolated microalgal strains in the KMMCC by other investigators is always possible if they agree to be open about distributing their strains to other researchers as well. The final decision of the strains to be deposited belongs to the KMMCC. We have diversity isolated more than 1,600 monospecific strains mainly from Korean coastal waters. Even though assigning a correct taxonomical position to the strains has always been our primary concern, many difficulties still exist in identifying them. H026 Helicobacter pylori Korean Type Culture Collection (HpKTCC) Collects and Distributes Clinical Isolates of H. pylori Hyung-Lyun Kang1,2, Myung-Je Cho1, Jae-Young Song1,2, Woo-Kon Lee1, Seung-Chul Baik1,2, Kon-Ho Lee1, Kwang-Ho Rhee1,2 1 Department of Microbiology, Gyeongsang National University School of Medicine, 2Helicobacter pylori Korean Type Culture Collection, Gyeongsang National University School of Medicine H. pylori that colonizes only in human gastric mucosa is one of the most common human pathogens and is the main cause of gastritis, peptic ulcer, and gastric cancer. Despite the clinical and commercial importance of H. pylori, many researchers have been blocked to investigate the diagnosis, treatment, and prevention of H. pylori infections because of difficulty in obtaining H. pylori isolates from patients. We have collected and characterized H. pylori isolates obtained from worldwide areas to allow researchers to access a variety of characterized H. pylori isolates. characterized H. pylori isolates. H. pylori KTCC contributes to promote the study for the diagnosis, treatment, and prevention of H. pylori infections by providing fundamental research materials to investigators. 207 www.msk.or.kr 2010 International Meeting of the Microbiological Society of Korea Author Index 209 www.msk.or.kr 210 Author Index A Abell, Guy Adams, Jonathan M. Ahn, Hye-Ran Ahn, Jae-Hyung Ahn, Jae Hyoung Ahn, Joong-Hoon Ahn, Keug-Hyun Ahn, Soyeon Ahn, Tae-Seok Alam, Nuhu Algire, Mikkel An, Dong-Shan An, Ji Eun An, Kwang-Deuk An, Sang Eun Anandham, Rangasamy Atomi, Haruyuki B002 H008 B039 B010 G001 B029 E014, E016 H008 B004, B006, H009 H019 S9-3 C026 A028 B032 F016 A042 S10-1, S10-3 B Bae, Dongryeoul C1-3 Bae, Hee Sun C021 Bae, Hye-Jin S9-5, F037 Bae, Jin-Woo S1-2, A001, A002, A005, A006, A007, A008, A010, A026, A027, A032, B002, B005, B033, B045, B048 Bae, Jung Eun G001, G002 Bae, Kyung Sook A009, A011, E001, E002 Bae, Songmee D025, D026 Baek, Hyun B039 Baek, In-Joon S8-2, F018 Bahk, Jang-Jun B040 Bahn, Young-Ho S6-1 Baik, Seung-Chul H026 Bak, Eun Jung E005, E006 Bak, Sun Uk E012 Bang, Duhee S9-2 Bat-Ochir, Chinbayar F031 Bok, Jung-Im A036 C Cha, Chang-Jun Cha, Injun Cha, Jaeho Cha, Jeong-Heon Cha, Ju-Hee Cha, KyoungEun Cha, Sang-Ho Cha, Sun-Shin Chae, Ae-Rhee Chae, Chang-Suk C025, G015 H001, H011 F013 D009, D011, E005, E006 C025 H018 D014 S10-4 F027 S6-5 Chae, Ji-Hwa Chae, Jong-Chan Chae, Suhn-Kee Chai, Young Gyu Chang, Ho-Won Chang, In-Ho Chang, Kyung-Soo Chang, Yeon-Ji Chang, Young-Hyo Chen, Wilfred Chen, Xiaoqiang Cheong, Chaejoon Cheong, Soon-Gee Cho, Eun-Jung Cho, Ahnna Cho, Eun-Min Cho, Eun Ji Cho, Ga Youn Cho, Hae Jin Cho, Hee-Jung Cho, Hyun-Hee Cho, Jae-Chang Cho, Jae-Yong Cho, Jang-Cheon Cho, Kyeung Hee Cho, Kyung-Ju Cho, Kyungyun Cho, Myung-Je Cho, Myung Kil Cho, Nam Hyuk Cho, Sang-Buem Cho, Seong Kee Cho, Seung-Hyon Cho, Seung Kee Cho, Su-Hee Cho, Ye-Seul Cho, Yong-Hyun Cho, Yoo-Bok Cho, You-Hee Cho, Youn-Dong Cho, Yun-Seok Choa, Yong-Ho Choe, Gyeong-Im Choe, Senyon Choi, Ah Reum Choi, Ahyoung Choi, Cheong-Up Choi, Chi Won Choi, Da Mi Choi, Dong-Goong Choi, Eun Ju Choi, Ho Gil Choi, Hong-Jae Choi, Hwa Young G002 C004 F030, F031 A019, D020 B033 B004, B006 D006, D007 E031 C018 C010 S11-1 S12-2 S9-5 S2-4 B004 F014 F023 S13-1 H019 D029 B025, B046 B022, B023, B024 F008, G003 A013, A035, B050, E020, G007 B026 C001 H023 H026 H005 S3-4 D032, D033, D034, G014 F007 S2-1, E033, E034 C001, F010, H003 B038 F027 C1-2, D005 F009, F012 S11-1 D027 B038, B039 C010 D008, D012, D013, D014 S12-2 E020 A013 D008, D012, D013, D014 B044, D035 G001 H007 C014 E005 B027 C003, F007, H003 211 www.msk.or.kr Choi, Hyo-Suk B055 Choi, Hyoung T E009 Choi, Hyoung Tae E010, E011, E012, E013, E035 Choi, Hyun-Ah D005 Choi, Jae-Yu D027, D029 Choi, Ji-Hyeon G004 Choi, Jin Hee B021 Choi, Jiyoung A012, C008, C013 Choi, Jong-Soon S10-2, E015 Choi, Jongwoon C008, C013, E017 Choi, Jung-Hoon E018 Choi, Jung-Hye A027 Choi, Jung Youn G015 Choi, Kieun A018 Choi, Kyoung-Hwa F013 Choi, Mi-Hwa H005 Choi, Myung Sik S3-4 Choi, Nack-Shick E014, E016 Choi, Sang Ho H006 Choi, Sang Ki C020 Choi, Seung-Ik B006 Choi, Sunga C008, C013, E017 Choi, Ung Kyu C021 Choi, Youngcheol A012 Choi, Yusang D015, D016, D017, D018, D019 Chokesajjawatee, Nipa S1-4 Choo, Jinho F021 Choo, Yeon-Sik S13-5 Chowdhury, Wasimul B. C024 Chu, Hyuk S3-4 Chun, Jeong-Hoon D028 Chun, Jong Woo C006 Chun, Jongsik W1, A017, B009, B035, H008 Chung, In-Sik D009, D011 Chung, Jin Woong S11-4 Chung, Jinwoo H023 Chung, Sanghoon C008, C013 Chung, Won-Yoon E006 Chung, Young-Ho S10-2, C1-1, B044, E015 Cui, Yidan D001 D Dong, Ke Donohue, Tim Dufour, Yann Dwidar, Mohammed C017 F012 F012 C009 E Efkaen, Sisi Patricia Ekpeghere, K. I. Eom, Chi-Yong Eom, Hyun-Ju 212 Author Index G012 B034 F014 F010 Eom, Jeong Seon F024, F025 F Franks, Ashley E. Fujiwara, Shinsuke B007 S10-3 G Geum, Neri Ghim, Sa-Youl Go, You-Seak Goh, Ah-Ra Gwak, Joo-Han C006 A015, A016, C011, C012 H008 C020 S9-5, F038 H Ha, Changwan Ha, Nam-Chul Ha, Sang-Jun Ha, Un-Hwan Hahm, Mi-Seon Hahn, Yoonsoo Hahn, Youn-Soo Ham, Su-Jin Han, Ah-Reum Han, Gui Hwan Han, Nam Soo D015, D016, D017, D108, D019 F005, F006 S6-1 F003 C012 B045 D021 E001, E002, E003 E022 B018 A028, C001, C003, C019, E004, F007, F010, H003, H004 Han, Song Ih B042 Han, Sun-Hee B020 Hanh, Nguyen Phan Kieu F026 Heo, Dong-Hyuk S8-2 Heo, Eun-Jung B027 Heo, Juhee D036, H013 Ho, Cuong T. B019 Hong, Hyunjin H022 Hong, Jeoung-Hyun C002 Hong, Jin-Kyung B022 Hong, Jin Pyo B056 Hong, Kee-Jong S6-3 Hong, Seok-Hyun F009 Hong, Seung-Beom S5-3 Hong, Soon Gyu A035, B026, C005, G013 Hong, Soon Ho H002 Hong, Sun-Mee G008, H007 Huh, Won-Ki E019, E021, E031, E032 Hur, Hor-Gil B003, B008, B019, B029, C010, C015, G006 Hur, Jae-Seoun A023, A024, A034, H024 Hur, Sung Bum H025 Hur, Yoon-Ki D029 Hwang, Eunha S12-2 Hwang, Gui-Hye G003 Hwang, Hee Jung D008, D012, D013, D014 Hwang, Hyun-Gook Hwang, Jae Sung Hwang, Jae Yoon Hwang, Ji-Sun Hwang, Jooyea Hwang, Jung-Sook Hwang, Soo-Myung Hwang, Sungmin Hwang, Ui Wook Hwang, Ye-Ji Hwang, Yong-Il Hwang, Young-Ok Hyun, Ryu Ki C020 G010 F026 S6-5 D008, D012, D013, D014 S13-5 D007 F013 S4-3 A016, C012 E014 G007 H021 I Im, Choon-Soo Im, Sin-Hyeog Im, Su-jin Imanaka, Tadayuki Inoue, Kengo Itoh, Takashi A030 S6-5 D015, D016, D017, D018, D019 PL1, S10-1, S10-3 B007 S1-3 J Jang, Hyoung Ryoul Jang, Il-Gyo Jang, Jeonghwan Jang, Jong-Ok Jang, Jung Im Jang, Sungil Jang, Woo-Yeong Jang, Woo-Yeoung Jang, Yu Ji Jeon, Byoung Seung Jeon, Byung-Seung Jeon, Che Ok Jeon, Dong In Jeon, Dusik Jeon, Hyun Bum Jeon, Jeong Ho Jeon, Seon-Ae Jeon, Seon Hyun Jeon, Taeck Jeon, Young Ho Jeong, Cha Yoon Jeong, Eun Kyo Jeong, Gil-Sang Jeong, Gilhwa Jeong, Hae-Seul Jeong, Hyun-Jeong Jeong, Je Yong Jeong, Jung Sun Jeong, Seong-Yoep B057 A029 B003 B021 F024, F025 D009, D011, E006 S9-5 F038 A045 A014, A018 C009 B045, B048 A019 C008, C013 A012 H006 A015, A016 G002 H001, H011, H012 S12-2 H019 G001, G002 A012 C008, C013, E017 D036, H013 A035 B053 G002 E014, E016 Jeong, Yoonhwa Jeong, Yun-Hee Jhun, Min A Ji, Chang-Jun Jiang, Cheng-Lin Jiang, Shenghua Jin, Seon-Yeong Jin, Xiao Ling Jo, Eun-Hye Jo, Gyeong-Jin Jo, Hyun-Jung Jo, Jae-Hyung Jo, Sung-Kee Joa, Jae-Ho Joh, Kiseong Jones, Kathleen R. Joo, Young Min Joshi, Yogesh Joung, Yochan Jung, Byung Hoon Jung, Da-I Jung, Dawoon Jung, Gyoo Yeol Jung, Jae Sung Jung, Jae Young Jung, Jaejoon Jung, Ji-Hee Jung, Ji Young Jung, Jia Jung, Kwang-Eun Jung, Kwang-Hwan D021 S6-1 E032 S2-2 S1-1 C010 B027 F006 A043 C002 D006 F022 G005 B049 A003, A004 D009, D011 D009, D011 A023, A024, A034 A003, A004 E027 E016 B004 S9-1 A038, A039, A040, B036 E027 B001 C014 B045, B048 B026 E007 S8-3, E020, E023, E024, E025, E026, E027, E028 Jung, Kyoung Hwa A019, D020 Jung, Kyung-Ju D019 Jung, Kyung-Tae D036, H013 Jung, Mi-Ja A001, A002, A005, A006, A007, A008, A026, A027, A032 Jung, Su-Jin F011 Jung, Won Seok F035 Jung, Yong-Gyun F009, F012 Jung, You-Jung B004, B006 Jung, Young-Mi D021 K Ka, Jong-Ok Kahng, Hyung-Yeel Kamashita, Tomoyuki Kanai, Tamotsu Kandori, Hideki Kang, Beom Sik Kang, Dae-Ook Kang, Dae Woo Kang, Hyun Ah A043, B047, B049 A033, A038, A039, B036, B037 S10-3 S10-1, S10-3 S12-5 S12-1 E016 F025 F020, F032, F033 213 www.msk.or.kr Kang, Hyun Bae Kang, Hyung-Lyun Kang, Ji-Hyun Kang, Jisun Kang, Junghwa Kang, Kook Hee Kang, Seung-Gu Kang, Sung Gyun Kang, Tae-Sook Kang, Yeonho Kang, Yu Ri Kato, Chiaki Kefala, Georgia Khan, Sumera Afzahl Kigawa, Rika Kim, B.-H. Kim, Baek Joong Kim, Bo-Young Kim, Byoung-Chan Kim, Byung-Chun Kim, Byung-Yong Kim, Cha-Yeon Kim, Chang-Jin Kim, Chang Jeon Kim, Choong-Gon Kim, Chul-Joong Kim, Chun Sung Kim, Dae Hyun Kim, Do Young Kim, Dockyu Kim, Dong-Hun Kim, Donghwan Kim, Donghyun Kim, Eun Hye Kim, Eung-Soo Kim, Eungbin Kim, EunJae Kim, Hae-Seon Kim, Haemin Kim, Hak Jun Kim, Han-Suk Kim, Han Bok Kim, Hanuel Kim, Hee-Jeong Kim, Hee-Kyoung Kim, Hee Gon Kim, Hong-Man Kim, Hong-Mi Kim, Hongik Kim, Hwang Yeon Kim, Hyangmi Kim, Hye-Jin Kim, Hye Won Kim, Hye Young 214 Author Index S3-5 H026 G007 H012 D003 D021 B016 E015, G007, H006 A045 D025, D026 E010 B046 S12-2 S13-5 B032 B034 E011 H005 S1-5 A011, A031 A043 F027 S1-1, A020, A021, A022, B021 B053, B054, B055, B057 C002, F004, G008, H007 C018 D022, D023 C021 E001, E002, E003 B017, C005 B008 C007 E017 A035, B026, F032 G013 B017 G004 E007 C004 S12-3 S3-2 G010 A003, A004 D006, D007 F023 C016, H010 F005, F006 D028 C007, D021 G010 A009, A011 B024 E011, E012 E013, E035 Kim, Hyun-Jin S2-1, E030, E033, E034 Kim, Hyun-Jung D010, D028 Kim, Hyunjung B015 Kim, Ik Sang S3-4 Kim, In Seop G001, G002 Kim, Inhae S9-1 Kim, Insook F015 Kim, Jae-Jin A044 Kim, Jae-Myung D008, D012, D013, D014 Kim, Jaisoo B015, B016, B051, C017, D005, D010 Kim, Jaisoo Su C1-2 Kim, Jandi A032 Kim, Jee-Young B015 Kim, Jeong-Hee D030 Kim, Jeong-Yoon F020, F032, F033 Kim, Jeong Hwa H019 Kim, Ji Eun C003, E004 Kim, Ji Sun A028 Kim, Jihong C008, C013, E017, F017 Kim, Jihyun F. E024, E026 Kim, Jin Seok F024, F025 Kim, Jin Young G001 Kim, Jinmoon E005, E006 Kim, Jong-Guk S13-5 Kim, Jong-Shik S4-4, C002, F004, G008 Kim, Jong Soo S12-3 Kim, Jonggill A012 Kim, Joy (Hyun) S2-5 Kim, Ju-Young F036 Kim, Jun-Tae F004, G008 Kim, Jung-Hoon S2-2 Kim, Jung-Wan E018, E022 Kim, Jungok G006 Kim, Kang-Lim D007 Kim, Ki-Yeon G011 Kim, Kwang-Wook S3-5 Kim, Kwang Kyu A041 Kim, Kyoung-Ho S1-2, B002, B005, B033 Kim, Kyung-Duk B010 Kim, Kyung-Mi B011 Kim, Kyung Jin S8-1 Kim, Kyungsun B017 Kim, Laurentius Jongsoon D006 Kim, Mi-Ree B043 Kim, Mi-Seon S4-2 Kim, Mi Na A031 Kim, Min-Gyu C010 Kim, Min-Joo D006 Kim, Min-Ju S8-4 Kim, Min-Sik F009, F012 Kim, Min-Soo A001, A002, A005, A006, A007, A010, A026, A027, A032, B005, E014, E016 Kim, Min Jee F032, F034 Kim, Min Jung D024 Kim, Mincheol Kim, Minyoung Kim, Miri Kim, Moon Chang Kim, Myungkil Kim, Na-Yeon Kim, Nam-Shik Kim, Oh Cheol Kim, Ok-Sun Kim, Ok Bin Kim, Sae-Hun Kim, Sang-Dal Kim, Sang-Jin Kim, Sang-Yoon Kim, Sang Hoon Kim, Sang Jin Kim, Sang Wook Kim, Sanguk Kim, Se-Il Kim, Se Jun Kim, Sehwan Kim, Seil Kim, Seon-Won Kim, Seung Bum Kim, Seung Il Kim, Si Wouk Kim, Sin Yeon Kim, So-Jung Kim, So-Yeon Kim, So-Young Kim, Song-Gun Kim, Soo-Jin A017, B009 E005, E006 S2-1, E033, E034 E014 H005 C002 D021 F033 B009, B035, H008 E008 D002, D003 S13-5 A025, B043, B046, G007, H006 F008 A019, D020 C1-1 S3-4 S9-1 C009 E025 E026 A014, A018 F036 B056 S10-2, C1-1, B044, D035, E015 B018, C016, H010 F029 H007 B026 C001, E023 C026 S5-3, A036, A037, A042, A043, B047, B049 Kim, Soo-Ki D032, D033, D034, G014 Kim, Soo-Kyoung D015, D016, D017, D018 Kim, Su-Ok G014 Kim, Sun-Ho F016 Kim, Sung-Kee C017, D010 Kim, Sung-Tae C011 Kim, Wan-Gyu B047 Kim, Wha-Jung C011 Kim, Wonduck C016, H010 Kim, Yi-Na C020 Kim, Yi-Seul S5-3, A036, A037, A042, A043, B047, B049 Kim, Yong-Jae F003 Kim, Yong Jin F016, G015 Kim, Yoo-Taek B016 Kim, You Sun E024 Kim, Young-Chang S9-5, F037, F038 Kim, Young-Joo C014 Kim, Young-Mi B012 Kim, Young-Pil S12-2 Kim, Young Bong D027, D029 Kim, Young Min F019 Kim, Younghoon D002 Kim, Yu-Kon B055 Kim, Yu Jin C001, C003, H004 Kim, Yun-Ji A006 Kim, Yun-Jung D033, D034 Kim, Yun Jae H006 Kim, Yun Jeong C006, D030 Kim, Yunmin B009 Kishore Babu, Bandamaravuri B018 Kiyuna, Tomohiko B032 Klammt, Christian S12-2 Koh, S.-C. B034 Koh, Young Jin A023, A024, A034, A040, H024 Koo, Chang-Duck B030 Kook, Joong-Ki D022, D023, D024 Krock, Hans J. S13-4 Ku, Hyun-Ok D013 Kum, Hyun-Woo E009 Kwak, Jun-Yong F030 Kwon, Hak Cheol C014 Kwon, Ho-Keun S6-5 Kwon, Hyeok-Do B013 Kwon, Kae Kyoung C1-1, A025, B040, B043, B044, B046 Kwon, Min Sik C023 Kwon, Ohsuk F020, F032, F033, F034, F035, F036 Kwon, Sang Oh B044, D035 Kwon, Soon-Kyeong E024, E026 Kwon, Soon-Wo S5-3, A036, A037, A042, A043, B047, B049 Kwon, Suk-Tae S10-5 Kwon, Tae-Hyung G008 L Lee, Byung Soo Lee, Chan-Gul Lee, Chang-Muk Lee, Chang-Ro Lee, Changhan Lee, Cho Hee Lee, Choong-Gu Lee, Don-Hwa Lee, Dong-Heon Lee, Dong-Hun Lee, Dong Gyu Lee, Dong Hyuck Lee, Dongsun Lee, DongYup Lee, Eun-Gyeong Lee, Eun-Joo Lee, Eun-Sook Lee, Gun-Seung Lee, Hae Ran Lee, Hak-Sung S3-5 G014 B049 S2-1, E030, E033, E034 F015, F017 G010 S6-5 S4-2 A033, A038, A039, B036, B037 B030, B031 B044 G001 S12-4 H004 F035, F036 B027 B020 B020 B042 D011 215 www.msk.or.kr Lee, Hakmi H022 Lee, Han-Ki D021 Lee, HanBoreum C021 Lee, Hee-Jung D029 Lee, Hong Kum A035, B026, C005 Lee, Hun-Goo E031 Lee, Hyang Burm B053, B054, B055, B056, B057 Lee, Hye-Jung B016 Lee, Hyi-Seung S13-3 Lee, Hyo-Jin B041, D007 Lee, Hyo Jung B045, B048 Lee, Hyoung Ro A028 Lee, Hyun-Ju C017, E001, E002, E003 Lee, Hyun-Jung C1-4 Lee, Hyun Sang C006 Lee, Hyun Sook H006 Lee, Hyune Hwan F022 Lee, In-Jung S13-5 Lee, In-Koo S13-5 Lee, Insun C018, D021 Lee, Jae-Chan A020, A021, A022, B021 Lee, Jae Ho F019 Lee, Jae Il G001, G002 Lee, Jae Seong H019 Lee, Jaehoon D025, D026 Lee, Jang-Hyun D027 Lee, Jeong Im G001, G002 Lee, Ji-Youl C1-2, D005 Lee, Jin-Mok S8-4 Lee, Jin-Uk C002 Lee, Jin-Won S2-2 Lee, Jin-Woo B040 Lee, Jin Ha B051 Lee, Jin Sung A044, G013 Lee, Jinho G012 Lee, Jong Kyu S12-3 Lee, Joon-Hee S11-3, D015, D016, D017, D018, D019 Lee, Jung-Hyun A025, B040, E015, H006 Lee, Jung-Sook S5-2, A041 Lee, Junghoon E029 Lee, Kang-Mu C024, D001, D002, D031 Lee, Kang Hyun A009, A011, A031 Lee, Kangmu D003 Lee, Kangseok F005, F006 Lee, Keon Ah E028 Lee, Keun Chul A041 Lee, Ki Young A033, E020 Lee, Kieun H022 Lee, Kihyun B009, B035 Lee, Kon-Ho H026 Lee, Kui-Jae C004 Lee, Kun-Jae C010 Lee, Kwang-Jun D036, H013 Lee, Kyenam D036, H013 216 Author Index Lee, Kyu-Ho S3-2 Lee, Kyung-Chang F011 Lee, Lan Hee E001, E003 Lee, Minho F005 Lee, Miyoung H022 Lee, Mok-Young B020 Lee, Myunju B031 Lee, Oh Hyoung A045 Lee, Sang-Hoon B023 Lee, Sang-Seob S5-1, C1-2, B015, B016, B051, C017, D005, D010, H014, H015 Lee, Sang-Yual B014 Lee, Sang Yup F035, F036 Lee, Seung-Cheol S7-4 Lee, Seung-Ju C1-2 Lee, Sun Bok A033 Lee, Sung-Jae A012 Lee, Sung-Suk E009, H005 Lee, Sung Gu E035 Lee, Sung Hyuk A025 Lee, Sungkyoung D025, D026 Lee, Tae Soo H019 Lee, U Youn H019 Lee, Won-Ho F038 Lee, Woo-Kon H026 Lee, Woong S4-2 Lee, Yeol Gyun E015 Lee, Yeon Ju S13-3, E022 Lee, Yeonhee H022 Lee, Yin-Won F023 Lee, Yong-Hwan H016 Lee, Yong Heon F029 Lee, Yong Hoon C004 Lee, Yong Joo S8-1 Lee, Yoo Kyoung A035 Lee, Young-Eun C002 Lee, Young-Hee D034 Lee, Young-Hyuk B055 Lee, Young-Ok B027 Lee, Young Sun A038, A039, A040, B036 Lee, Yun-Jung D032, D033, D034 Lee, Yung Mi B026, C005 Li, Wen-Jun S1-1 Lim, Gwang-Hui B027 Lim, Gyu-Bum E019 Lim, Hyang Soon E011 Lim, Jae-Hong C010 Lim, Jeesun C024, D001, D002, D003, D004 Lim, Jeongheui C018, D021 Lim, Young Woon A044 Liu, Fang C010 Lory, Stephen PL2 Lou, Kai S1-1 Lovley, Derek R. B007 Luo, Dan Luo, Yuan Rong G009 C1-1 M Maeng, Pil Jae Margesin, Rosa Maslennikov, Innokentiy V. Matsui, Toshiro Merrell, D. Scott Min, Kyung-Bae Min, Mun-Kyoung Misawa, Norihiko Moon, Chuloo Moon, DongGeRaMi Moon, Eun Young Moon, Hye-Yun Moon, Jin Seok Mun, Hye Yeon Mun, Mi Ran Murtas, Giovanni Myung, Heejoon Myung, Nosang V. S8-1, F016 B026 S12-2 S7-2 S3-3, D009, D011 S9-5, F037 F034 S13-2 B010 F028 A035 F020 C001 B053, B054, B055, B057 C018 S9-4 H018 C010, G006 N Nakanosho, Masahiro Nam, Doo Hyun Nam, Gi-Baeg Nam, Hyeran Nam, Ji-Hyun Nam, Jong Hee Nam, Seong-Won Nam, Young-Do Nedashkovskaya, Olga I. Nguyen, Anh Dung Nguyen, Huan H. Nguyen, Thi Thuy Nguyen, Thuy Vu An Noh, Hyun-Ju Noh, Hyung-Jun S10-3 F026 F012 H022 B030, B031 S6-5 S11-1, D001, D004 A001, A002, A005, A008, B002, B005, B033 A025 A024 S6-2 A024 H002 G013 B049 O Oh, Byung-Taek Oh, Doo-Byoung Oh, Hee-Bok Oh, Hyun-Myung Oh, Hyun-Woo Oh, Jae Sung Oh, Jeong-Il Oh, Kye-Heon Oh, Kyong-Hee C004 F032, F033, F034, F035, F036 D028 A013, B050, E020 A009, A011 A039 S8-4, E007 B037, B038, B039 B031 Oh, Kyoung Hee Oh, Min-Kyu Oh, Yu-Kyung Ohkuma, Moriya Olsen, Cara H. Otgonbayar, Gan-Erdene A044 A018 D029 B032 D009, D011 H003 P Paik, Hyun-Dong Paik, Soon-Young Pak, Jae-Hong Park, Ah Reum Park, Chae Haeng Park, Chan-Hee Park, Chan-Sun Park, Chan-Won Park, Chankyu Park, Dong-Jin Park, Doo-Sang Park, Eun-Jin Park, Ha-Young Park, Ha Ju Park, Hae Youn Park, Hee-Moon Park, Ho-Yong Park, Hyo-Jin Park, In-Cheol Park, Jae-Won Park, Jae-Yeon Park, Jae-Yoon Park, Jang Min Park, Jeong Mi Park, Ji Hyun Park, Jihye Park, Jin-Soo Park, Jin-Sook Park, Jong-Tae Park, Jong Myong Park, Joo-Hong Park, Joon-Seok Park, Jungsoon Park, Kwang-Jin Park, Kyung Sun Park, Nokyoung Park, Nyun-Ho Park, Sae Woong Park, Seong-Kyu Park, So Young Park, Su-Jin Park, Sun Jue Park, Sun Wook Park, Sung-Jin D027 H017 S4-2 G015 G013 S6-1 E014, E016 B049 E029, F015, F017 A020, A021, A022, B021 A009, A011 S1-2, A001, A005, A006, A008, A026, A032, B048 D015, D016, D017, D018, D019 C005 B017 A009 E001, E002, E003 S6-1 S5-3, A037 D032, G014 C009 D022, D023, D024 B018 F007 D020 H022 C014 A029, A030, B025 E022 A015 F012 S6-1 B028 S8-4 S12-3 G009 C002, F004, G008, H007 F019 A001 A012 D015 E013 E035 C011 217 www.msk.or.kr Park, Sungsu S11-1, C024, D001, D002, D003, D004 Park, Taesung E032, H008 Park, Woojun S11-2, B001, B028, F001, F002 Park, Yong-Ha C018, D021 Park, Yong Keun F024, F025, F029 Park, Yong Sin A045 Park, Yongjin C024, D031 Park, YongKeun G004 Park, Young-Hyun B036 Park, Yu Ri C016 Park, Yun-Seon F028 Peck, Kyong-Ran C1-2, D005 Peterkofsky, Alan S2-1, E034 Piao, Shunfu F005, F006 Pinhassi, Jarone G007 R Rathnayake, Ajith Indrawansha H019 Rejish Kumar, V. J C018, D021 Rhee, Joon Haeng S6-5 Rhee, Kwang-Ho H026 Rhee, Kwang-Hyun B050 Rhie, Gi-Eun D028, D030 Rho, Hyun Soo S4-1 Roe, Jung-Hye F009, F011, F012 Roh, Changhyun G005 Roh, Seong Woon A001, A002, A005, A006, A007, A008, A010, A026, A027, A032, B002, B033 Roh, Young Hoon G009 Rung, Doo Il E035 Ryu, Choong-Min C012 Ryu, Jae-Ha S7-3 Ryu, Ji-Young B029 Ryu, Sun-Hwa E009, H005 S Sadowsky, Michael J. Sahoo, Anupama Sang, Byoung-In Sano, Chie Sato, Takaaki Sato, Takako Schinner, Franz Seo, Byeong Joo Seo, Eun-Young Seo, Hyun-Seok Seo, Jeong-Sun Seo, Sang Woo Seok, Soon-Ja Seok, Yeong-Jae Shahid, Sudipto Shik, Chun-Shik 218 Author Index B029, C010 S6-5 A014, A018, B010, C009, G011 B032 S10-1 B046 B026 C018, D021 B006, C003, C019, H009 A025 H008 S9-1 S5-3 S2-1, E030, E033, E034 A019, D020 E031 Shin, Eunju H022 Shin, Hee-Sung F003 Shin, Hee Jae S13-3 Shin, Ji-Hye B031 Shin, Kee-Sun A005, A008, A011, A027, A031 Shin, Mun-Sik S9-5, F037, F038 Shin, Na-Ri A002, D030 Shin, Sung Jae S3-5 Shin, Sunme B017 Shon, Min jeong F020 Shon, San Ho A038, A039 Sim, Sangjun C008, C013 Sim, Se-Hoon F005, F006 Sin, Yesl C007 So, Byung-Jae D008, D012, D013, D014 So, Jae-Seon S6-5 Sohn, Hosung S3-5 Sommer, Peter S6-4 Son, Ji Hyun C006 Son, Jung-A A043, B047, B049 Son, Kwang-Hee E001, E002, E003 Son, Su-Wan C1-2 Song, Hong-Gyu B011, B012, B013, B014 Song, Hye Jung G010 Song, Jae-Young H026 Song, Jae Jun F034 song, Jung-A E017 Song, Ki-Joon H020 Song, Kiwon S2-3 Song, Kyung C010 Song, Saemee F006 Song, Yong Bhum E032 Song, Yu-Jin B038 Song, Yun-Jin B039 Sugiyama, Junta B032 Suh, Yaesul B003 Sumayo, Marilyn C012 Sung, Chang-Keun B021 Sung, Hye-Ri A015, A016 Surh Young-Joon S7-1 Suwannarangsee, Surisa F033 T Takedomi, Shogo Takemasa, Ryo Tang, Shu-Kun Thawng, Cung Nawl Tomita, Junko Tran, Hoa S10-3 S10-1 S1-1 C015 B032 B007 U Ueki, Toshiyuki B007 Um, Youngsoon Unden, Gottfried Unno, Tatsuya A014, A018, B010, C009, G011 E008 B003 V Van, Nguyen Dinh D027 W Wang, Chinling Wang, Tianshi Wang, Yun Weon, Hang-Yeon Whang, Kyung-Sook Whitmire, Jeannette M. Whon, Tae Woong Won, Choul-Jae Won, Mi-Young Woo, Eun Ju Woo, Jung-Hee Woo, Sung-Il C1-3 E018 S1-1 S5-3, A036, A037, A042, A043, B047, B049 B041, B042 D011 A008 S3-5 G014 A044 F004, G007, G008, H007 D021 X Xu, Li-Hua Xu, Yongbin S1-1 F005, F006 Y Yang, Eun-Bin Yang, Hee-Jong Yang, Hyun Ok Yang, Jae-Seong Yang, Ji Yang, Ki-Hoon Yang, Seung-Jo Yang, Seung-Joon Yang, Sujeong Yang, Sung-Hyun Yeo, Kwonjoo Yeom, Gyoseon Yeom, Jinki Yi, Dae-Gwan Yi, Hana Yim, Joung Han Yokooji, Yuusuke E021 E014, E016 C014 S9-1 H005 E007 B050 C001 B015 A025 S12-2 C022, C023 S11-2, F001, F002 E021 B007 C005 S10-1 Yoo, Cheon-Kwon Yoo, In-Hwa Yoo, Ji-Sun Yoo, Ki-Seon Yoo, Seung-Hee Yoo, Yun-Jung Yoon, Byoung-Dae Yoon, Byung-Dae Yoon, Gun-Mook Yoon, Jang W. Yoon, Jeong-Jun Yoon, Jin Ho Yoon, Jung-Hoon Yoon, Juyoung Yoon, Mi Young Yoon, Min Yoon, Sang Sun Yoon, Seo-Yeon Yoon, So-Jung Yoon, Suk-Hwan Yoon, Tae-Young Yoon, Won Suck Yoon, Yeo Kyoung You, Won-Sun You, Yung-Hyun Youn, Min Yu, Dong-Joo Yu, Jaeyon Yu, Ji-Eun Yu, Kijong Yu, Seungho Yu, Yeong Man Yun, Bong-Sik Yun, Cheol-Won Yun, Dae-Myoung Yun, Hye-Ok Yun, Ji-Hyun Yun, Seulgi Yun, Sung-Hwan Yun, Sung Ho D028, D030, D036, H013 F003 F012 C003 A042 D009, D011, E006 E016 E014 C001 D002 G015 F027, F028 A015, A016 S11-1 B052, C024, D031 D001, D004 S3-1, B052, C024, D031 B030 D036, H013 B009, H008 C022, C023 G004 H016 D036 S13-5 D002 G002 D025, D026 S9-5, F037 C007 B031 F016 C004 S8-2, F018 C022, C023 B055 A005 F035 F023 C1-1, B044, D035 Z Zhang, Li-Li Zhao, Li-Xin Zhi, Xiao-Yang Zhou, Peng Zo, Young-Gun S1-1 S1-1 S1-1 B007 S1-4, H009 219 www.msk.or.kr 124 Colloquium