Download Journal of Biotechnology VI-2 Genomics, proteomics and

Journal of Biotechnology 136S (2008) S541–S547
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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec
Abstracts
VI-2 Genomics, proteomics and metabolomics in marine biotechnology
VI2-O-002
References
Effect of osmotic downshock treatment on the yield of ectione
synthesized by Halomonas sp. EG6
Csonka, L., 1989. Physiological and genetic responses of bacteria to osmotic stress.
Microbiol. Rev. 53, 121–147.
Galinski, E.A., Trüper, H., 1994. Microbial behaviour in salt-stressed ecosystems.
FEMS Microbiol. Rev. 15, 95–108.
Leifson, E., 1963. Determination of carbohydrate metabolism of marine bacteria. J.
Bacteriol. 85, 1183–1184.
Nagata, S., Wang, Y., Oshima, A., Zhang, L., Miyake, H., Sasaki, H., Ishida, A., 2008. Efficient cyclic system to yield ectoine using Brevibacterium sp. JCM 6894 subjected
to osmotic downshock. Biotechnol. Bioeng. 99, 941–948.
Omneya Osman ∗ , Shoko Tanabe-Hosoi, Schinichi Nagata
Environmental Biochemistry Group, Research Center for Inland Seas,
Kobe University, -1-1 Fukae, Higashinada-ku, Kobe 658-0022, Japan
E-mail address: [email protected] (O. Osman).
Halophilic bacterium strain EG6 was isolated from Burg el Arab
solar saltern lake in Egypt. Strain EG6 can grow in a wide range of
NaCl concentrations up to 4 M. Phylogenetic position was established by 16S rRNA gene sequencing as a member of the genus
Halomonas. Morphological, physiological and biochemical tests
were done to characterize strain EG6 Leifson (1963). Strain EG6
produced ectoine as the main compatible solute for osmotic stress
adaptation (Csonka, 1989; Galinski and Trüper, 1994). Our aim was
to establish the best condition of osmotic downshock required for
high yield of ectoine production using strain EG6 and compare it
with one of the type strains H. elongata IFO15536 (Nagata et al.,
2008). We subjected strain EG6 to osmotic downshock treatment
with different NaCl concentration from 2 M to 0–0.7 M to determine
how much concentration of NaCl was optimum for high ectoine
yield. HPLC and NMR analysis were used to quantify and confirm
ectoine purity, respectively. It was interesting to notice that the
cells of strain EG6 had a great flexibility to withstand the sudden
change in NaCl concentration, especially when cells were subjected
to osmotic downshock treatment from 2 M to 0.3 M NaCl. That
advantage was due to cellular ability to compensate the loss of
ectoine with high efficiency. This strain showed significant yield of
ectoine, about 3.70 g/L was totally released after 7 days of osmotic
downshock from 2 M to 0.3 M NaCl which is considered as the highest yield of ectoine in comparison with other NaCl concentrations
(0–0.7 M) that were used for osmotic downshock. On the other hand
H. elongata IFO15536 had a very low yield of total ectoine released,
2.89 g/L, than that of strain EG6 under same conditions (Table 1). In
addition, growth of strain EG6 was highly increased during downshock treatment in comparison to H. elongata IFO15536. Thus, we
concluded that 0.3 M NaCl was the optimum concentration for getting the maximum yield of ectoine from strain EG6 with high purity
which can be used for large scale ectoine production. Accordingly,
strain EG6 can be classified as a new species, Halomonas sp. EG6
which is of interest in the future studies.
0168-1656/$ – see front matter
doi:10.1016/j.jbiotec.2008.07.1271
VI2-O-004
An expressed sequence tag approach for cloning of phytochelatin synthase cdna clone from Eucheuma denticulatum
(Rhodophyta)
Roohaida Othman 1,2,∗ , Diana Mohd Nor 1 , Hasbullah Daud 1 ,
Adura Mohd Adnan 2
1 Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600
Bangi, Selangor, Malaysia
2 School of Biosciences and Biotechnology, Faculty of Science and
Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor,
Malaysia
E-mail address: [email protected] (R. Othman).
Eucheuma denticulatum is an economically important carrageenophyte found in the Indo-Pacific region mainly algal reef areas of
islands in Southeast Asia (Doty, 1987). Expressed sequence tags
(EST) approach was undertaken to investigate gene expression in
this red alga. Several cDNA libraries were constructed from whole
plants where a total of 10,087 ESTs were obtained. Cluster analysis
and contig assembly resulted in a total of 3649 unique transcripts.
Comparison with the database showed that only 29% exhibited significant similarity to known sequences in the Genbank database
and corresponded to a variety of genes associated with general cellular metabolism. Upon analysis of all the ESTs, a cDNA clone that
codes for phytochelatin synthase (PCS) was found; no prior isolation in algae has been reported. PCS catalyses the synthesis of
phytochelatins (PCs) which are cysteine-rich proteins with important role in heavy metal detoxification found in various types of
plants (Cobbett, 2000). PCS uses glutathione (GSH) as a substrate in
the presence of metal ions with Cd2+ as the strongest inducer. The
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Abstracts / Journal of Biotechnology 136S (2008) S541–S547
full-length clone for E. denticulatum PCS has been isolated using
PCR and rapid amplification of cDNA ends (RACE) techniques. Total
RNA was extracted from the whole plants and used as template
in first strand cDNA synthesis 5 end-RACE (Othman et al., 2006).
The full length cDNA clone with the size of 1.6 kb contains a single open reading frame which encodes a protein containing 218
amino acids. The expected amino acid sequence shared high similarity with PCS from Allium sativum (51%), Cynodon dactylon (51%)
and Arabidopsis thaliana (50%). Multiple sequence alignment of the
E. denticulatum cDNA clones with other clones showed presence
of several protein motifs and binding sites typically found in PCS,
suggesting structural similarity to other plant PCS. Phylogenetic
analyses of this algal PCS sequence have also been performed for
further characterization of its function and significance.
References
Apostolidis, A.P., Mamuris, Z., Triantaphyllidis, C., 2001. Phylogenetic relationships
among four species of Mullidae (Perciformes) inferred from DNA sequences
of mitochondrial Cytochrome b and 16S rRNA genes. Biochem. Syst. Ecol. 29,
901–909.
Kocher, T.D., Thomas, W.K., Meyer, A., Edwards, S.V., Paabo, S., Villablanca, F.X.,
Wilson, C., 1989. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Evolution 86, 6169–6200.
doi:10.1016/j.jbiotec.2008.07.1273
VI2-O-013
Expression studies of human antimicrobial peptide hepcidin
Farzana Rashid 1,∗ , Ijaz Ali, Qipeng Yuan 1
References
Cobbett, C.S., 2000. Phytochelatins and their roles in heavy metal detoxification.
Plant Physiol. 123, 825–832.
Doty, M.S., 1987. The production and use of Eucheuma. In Doty, M.A., Caddy, J.F.,
Santelices, F. (Eds.), Case Studies of Seven Commercial Seaweed Resources. FAO
Fish Tech. Paper, vol. 281. Rome, pp. 123–161.
Othman, R., Chan, P.C., Diana, M.N., Sie, L.E.K., 2006. High yield RNA extraction from
Eucheuma denticulatum and Kappaphycus alvarezii (Gigartinales, Rhodophyta). J.
Trop. Plant Physiol. 1, 81–88.
doi:10.1016/j.jbiotec.2008.07.1272
VI2-O-006
Phylogenetic relationship among mullidae species in Turkish
ichthyofauna
Emre Keskin 1,2,∗ , Hasan Huseyin Atar 2 , Alp Can 1
1
Ankara University Biotechnology Institute, Ankara, Turkey
Ankara University Agriculturl Faculty Department of Fisheries and
Aquaculture, Ankara, Turkey
2
E-mail address: [email protected] (E. Keskin).
DNA sequence comparisons of three mitochondrial DNA genes
(Kocher et al., 1989) were used to reveal phylogenetic relationships among four species and a sub-species of mullidae family.
To our knowledge, this is the first report using mitochondrial DNA
sequence data to infer phylogenetic relationships between Mullus
barbatus and its subspecies; Mullus barbatus ponticus. Cytochrome
b, 12S ribosomal RNA, and cytochrome oxidase II regions of 242
individuals belonging to species Mullus barbatus, Mullus surmuletus,
Upeneus moluccensis, Upeneus pori and sub-species Mullus barbatus
ponticus were sequenced and phylogenetic trees were constructed
using four different methods: neighbor joining (NJ), minimum
evolution (ME), maximum parsimony (MP) and unweighted pairgroup method of arithmetic average (UPGMA) (Apostolidis et al.,
2001). The phylogenetic trees constructed, support the existing taxonomic data of two mullid genera (Mullus, Upeneus). Molecular data
shows no significant difference between same species of different
geographical populations. The results suggest that the molecular
difference is not large enough between Mullus barbatus and Mullus
barbatus ponticus to consider them as subspecies.
Acknowledgements
The authors wish to thank Dr. Hilal Ozdag for helpful advise and
comments. This work was supported by Ankara University Biotechnology Institute.
1
Beijing University of Chemical Technology, Beijing 100029, China
E-mail address: fari [email protected] (F. Rashid).
Hepcidin, a small cysteine rich cationic peptide synthesized in hepatocytes and initially isolated from human plasma and urine is
a key regulator of intestinal iron absorption (Krause et al., 2000;
Park et al., 2001). We performed the cloning and expression of a
low molecular weight human antimicrobial peptide hepcidin (hepc
20) in recombinant form. Full length synthesized gene of the hepcidin (20 amino acids) protein was cloned into different expression
vectors and transformed into E. coli and Pichia P. pastoris strain
GS115. The influence of different factors on biomass and hepcidin
protein production during induction phase was subsequently studied. The expression level and activation were detected by Tricine
SDS-PAGE and immuno blotting respectively. Studies revealed that
active recombinant hepcidin peptide can be successfully expressed
in E. coli and methylotrophic yeast P. pastoris. The BMMY medium
was found to be optimal for recombinant hepcidin protein expression and growth of the recombinant yeast strains. The Dissolved
Oxygen (DO) and the optimal concentration of methanol required
for the growth of recombinant organisms were also recorded. The
expressed hepcidin protein concentration was less than 10 mg/L.
The recombinant protein had a high specificity against antibody.
It also exhibited antibacterial activity against S. aureus and Bacillus subtilis. The study provides a basis for further researches on the
early diagnosis of different peptides and the mechanism of their
action.
References
Krause, A., Neitz, S., Magert, H.J., Schulz, A., Forssmann, W.G., Schulz-Knappe, P.,
Adermann, K., 2000. LEAP-1, a novel highly disulfide bonded human peptide,
exhibits antimicrobial activity. FEBS Lett. 480, 147–150.
Park, C.H., Valore, E.V., Waring, A.J., Ganz, T., 2001. Hepcidin, a urinary antimicrobial
peptide synthesized in the liver. J. Biol. Chem. 276, 7806–7810.
doi:10.1016/j.jbiotec.2008.07.1274
VI2-O-001
Environmental adaptation: Genomic analysis of the piezotolerant and psychrotolerant deep-sea iron reducing bacterium
Shewanella piezotolerans WP3
Fengping Wang 1 , Jianbin Wang, Huahua Jian 1 , Bing Zhang, Feng
Wang 1 , Jun Yu, Songnian Hu, Xiang Xiao 1
1
Key Laboratory of Marine Biogenetic Resources, State Oceanic Administration, Xiamen 361005, PR China
Members of the genus Shewanella inhabit various environments,
and are well known for their versatile respiratory capabilities, cou-