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International Journal of Systematic and Evolutionary Microbiology (2014), 64, 719–724 DOI 10.1099/ijs.0.057513-0 Octadecabacter jejudonensis sp. nov., isolated from the junction between the ocean and a freshwater spring and emended description of the genus Octadecabacter Sooyeon Park and Jung-Hoon Yoon Correspondence Jung-Hoon Yoon Department of Food Science and Biotechnology, Sungkyunkwan University, Jangan-gu, Suwon, South Korea [email protected] A Gram-stain-negative, non-spore-forming, non-flagellated, rod-shaped bacterial strain, SSK21T, was isolated from the zone where the ocean and a freshwater spring meet at Jeju island, South Korea, and subjected to a polyphasic taxonomic study. Strain SSK2-1T grew optimally at 30 6C, at pH 7.0–8.0 and in the presence of 2.0 % (w/v) NaCl. Neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences revealed that strain SSK2-1T clustered with the type strains of two Octadecabacter species, showing 96.5– 96.8 % 16S rRNA gene sequence similarity. Strain SSK2-1T and Octadecabacter arcticus DSM 13978T contained Q-10 as the predominant ubiquinone and C18 : 1v7c as the common major fatty acid. The polar lipid profile of strain SSK2-1T was similar to that of O. arcticus DSM 13978T by having phosphatidylcholine, phosphatidylglycerol and one unidentified aminolipid as the major components. The DNA G+C content of strain SSK2-1T was 60.1 mol%. Differential phenotypic properties, particularly temperature range for growth, oxidase activity and nitrate reduction, together with phylogenetic distinctiveness, revealed that strain SSK2-1T is separate from recognized species of the genus Octadecabacter. On the basis of the data presented, strain SSK2-1T is considered to represent a novel species of the genus Octadecabacter, for which the name Octadecabacter jejudonensis sp. nov. is proposed. The type strain is SSK2-1T (5KCTC 32535T5CECT 8397T). Soesokkak, located at Jeju island, South Korea, and designated a Natural Environment Preservation Zone by UNESCO, is a unique locality where the ocean and a freshwater spring meet. During screening of bacteria from this junction, many novel taxa have been isolated and characterized taxonomically. One of these isolates, designated SSK2-1T, is described in this study, as it was found to be phylogenetically related most closely to the genus Octadecabacter, a member of the Alphaproteobacteria. The genus Octadecabacter was proposed by Gosink et al. (1997, 1998) with the descriptions of two species, Octadecabacter arcticus (the type species) and Octadecabacter antarcticus, which were isolated from sea ice of the Arctic and Antarctica, respectively. These are, at the time of writing, the only recognized species of the genus. The aim of the present work was to investigate if strain SSK2-1T represents a third species of the genus Octadecabacter by using a polyphasic characterization including chemotaxonomic and other phenotypic analyses and a detailed phylogenetic investigation based on 16S rRNA gene sequences. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain SSK2-1T is KF515220. 057513 G 2014 IUMS Water was collected from the junction between the ocean and a freshwater spring, called Soesokkak, and used as a source for the isolation of bacterial strains. Strain SSK2-1T was isolated by the standard dilution plating technique at 25 uC on marine agar 2216 (MA; Becton Dickinson) and cultivated routinely on MA at 30 uC. O. arcticus DSM 13978T, which was used as a reference strain for fatty acid and polar lipid analyses, was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. Cell morphology was examined by light microscopy (BX51; Olympus) and transmission electron microscopy (JEM1010; JEOL). The latter technique was also used to assess the presence of flagella on cells from an exponentially growing MA culture. For this, the cells were negatively stained with 1 % (w/v) phosphotungstic acid and the grids were examined after being air-dried. The Gram reaction was determined by using the bioMérieux Gram stain kit according to the manufacturer’s instructions. Growth under anaerobic conditions was determined after incubation for 10 days in an anaerobic jar (MGC) with AnaeroPack (MGC) on MA and on MA with potassium nitrate (0.1 %, w/v); the jar was kept overnight at 4 uC to create anoxic conditions Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 06:37:07 Printed in Great Britain 719 S. Park and J.-H. Yoon before incubation at 30 uC. Growth at 4, 10, 20, 25, 30, 35, 37 and 40 uC was measured on MA to measure the optimal temperature and temperature range for growth. The pH range for growth was determined in marine broth 2216 (MB; Becton Dickinson) adjusted to pH 4.5–9.5 (using increments of 0.5 pH units) by using sodium acetate/acetic acid and Na2CO3 buffers. pH was verified after autoclaving. Growth in the absence of NaCl and in the presence of 0.5, 1.0, 2.0 and 3.0 % (w/v) NaCl was investigated in trypticase soy broth prepared according to the formula of the BD medium except that NaCl was excluded and that 0.45 % (w/v) MgCl2 . 6H2O was added. Growth in the presence of 2.0–10.0 % NaCl (at increments of 1.0 %) was investigated in MB. Catalase and oxidase activities were determined as described by Lányı́ (1987). Hydrolysis of casein, starch, hypoxanthine, L-tyrosine and xanthine was tested on MA using the substrate concentrations described by Barrow & Feltham (1993). Hydrolysis of aesculin and Tweens 20, 40, 60 and 80 and nitrate reduction were investigated as described by Lányı́ (1987) with the modification that artificial seawater was used for the preparation of media. Hydrolysis of gelatin and urea was investigated by using nutrient gelatin and urea agar base media (Becton Dickinson), respectively, with the modification that artificial seawater was used for the preparation of media. The artificial seawater contained (per litre distilled water) 23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2 . 6H2O, 5.94 g MgSO4 . 7H2O and 1.3 g CaCl2 . 2H2O (Bruns et al., 2001). Utilization of various substrates for growth was tested according to Baumann & Baumann (1981), using supplementation with 1 % (v/v) vitamin solution (Staley, 1968) and 2 % (v/v) Hutner’s mineral salts (Cohen-Bazire et al., 1957). Acid production from carbohydrates was tested as described by Leifson (1963). Susceptibility to antibiotics was tested on MA plates using antibiotic discs (Advantec) containing the following (mg per disc unless otherwise stated): ampicillin (10), carbenicillin (100), cephalothin (30), chloramphenicol (100), gentamicin (30), kanamycin (30), lincomycin (15), neomycin (30), novobiocin (5), oleandomycin (15), penicillin G (20 U), polymyxin B (100 U), streptomycin (50) and tetracycline (30). Enzyme activities were determined, after incubation for 8 h at 30 uC, by using the API ZYM system (bioMérieux). Cell biomass of strain SSK2-1T for DNA extraction and for the analyses of isoprenoid quinones and polar lipids was obtained from cultures grown in MB for 5 days at 30 uC and cell biomass of O. arcticus DSM 13978T for polar lipid analysis was obtained from cultures grown in MB for 10 days at 10 uC. Chromosomal DNA was extracted and purified according to the method described by Yoon et al. (1996), with the modification that RNase T1 was used in combination with RNase A to minimize contamination of RNA. The 16S rRNA gene was amplified by PCR as described previously (Yoon et al., 1998) using two universal primers (59-GAGTTTGATCCTGGCTCAG-39 and 59-ACGGTTACCTTGTTACGACTT-39). Sequencing 720 of the amplified 16S rRNA gene was performed as described by Yoon et al. (2003). Alignment of sequences was carried out with CLUSTAL W software (Thompson et al., 1994). Gaps at the 59 and 39 ends of the alignment were omitted from further analysis. Phylogenetic analyses were performed as described by Yoon et al. (2003). Isoprenoid quinones were extracted and analysed as described by Komagata & Suzuki (1987), using a reversed-phase HPLC machine equipped with a YMC ODS-A (25064.6 mm) column. The isoprenoid quinones were eluted by a mixture of methanol/2-propanol (2 : 1, v/ v) using a flow rate of 1 ml min21 at room temperature and detected by UV absorbance at 275 nm. For cellular fatty acid analysis, cell mass of strain SSK2-1T and O. arcticus DSM 13978T was harvested from MA plates after cultivation for 5 days at 30 uC and for 10 days at 10 uC, respectively. The physiological age of the cell masses was standardized by observing the growth development of colonies on the agar plates followed by harvesting them from the same quadrant on the agar plates according to the standard MIDI protocol (Sherlock Microbial Identification System, version 6.1). Fatty acids were saponified, methylated and extracted using the standard protocol of the MIDI (Sherlock Microbial Identification System, version 6.1). The fatty acids were analysed by GC (Hewlett Packard 6890) and identified by using the TSBA6 database of the Microbial Identification System (Sasser, 1990). Polar lipids were extracted according to the procedures described by Minnikin et al. (1984) and separated by two-dimensional TLC using chloroform/methanol/water (65 : 25 : 3.8, by vol.) for the first dimension and chloroform/methanol/ acetic acid/water (40 : 7.5 : 6 : 1.8, by vol.) for the second dimension as described by Minnikin et al. (1977). Individual polar lipids were identified by spraying with molybdophosphoric acid, molybdenum blue, ninhydrin and a-naphthol reagents (Minnikin et al., 1984; Komagata & Suzuki, 1987) and with Dragendorff’s reagent (Sigma). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984) with the modification that DNA was hydrolysed and the resultant nucleotides were analysed with a reversed-phase HPLC machine equipped with a YMC ODS-A (25064.6 mm) column. The nucleotides were eluted by a mixture of 0.55 M NH4H2PO4 (pH 4.0) and acetonitrile (40 : 1, v/v), using flow rate of 1 ml min21 at room temperature and detected by UV absorbance at 270 nm. Morphological, cultural, physiological and biochemical characteristics of strain SSK2-1T are given in the species description and in Table 1. The almost-complete 16S rRNA gene sequence of strain SSK2-1T determined in this study comprised 1384 nt, representing approximately 95 % of the Escherichia coli 16S rRNA gene sequence. In the neighbourjoining phylogenetic tree based on 16S rRNA gene sequences, strain SSK2-1T joined the cluster comprising the type strains of O. arcticus and O. antarcticus by a bootstrap resampling value of 99.3 % (Fig. 1). The relationships among strain SSK2-1T and the type strains of O. Downloaded from www.microbiologyresearch.org by International Journal of Systematic and Evolutionary Microbiology 64 IP: 88.99.165.207 On: Thu, 15 Jun 2017 06:37:07 Octadecabacter jejudonensis sp. nov. Table 1. Differential phenotypic characteristics between strain SSK2-1T and the type strains of the two recognized species of the genus Octadecabacter Strains: 1, SSK2-1T; 2, O. arcticus 238T; 3, O. antarcticus 307T. Data of reference strains are taken from Gosink et al. (1997). +, Positive reaction; 2, negative reaction; W, weakly positive reaction. All strains are positive for catalase activity, and utilization of D-glucose and succinate (weakly positive for the two reference strains). All strains are negative for anaerobic growth, Gram-staining, motility and hydrolysis of gelatin. Characteristic Growth at: 4 and 10 uC 20 and 30 uC Oxidase Nitrate reduction Hydrolysis of starch Utilization of: D-Fructose Acetate Citrate Pyruvate L-Glutamate DNA G+C content (mol%) 1 2 3 2 + + + + + 2 2 2 2 + 2 2 2 2 + + + 2 2 60.1 2 2 2 2 2 2 + 56 W W W 57 arcticus and O. antarcticus were also maintained in the trees constructed using the maximum-likelihood and maximumparsimony algorithms (Fig. 1). Strain SSK2-1T exhibited 16S rRNA gene sequence similarity values of 96.5 and 96.8 % to O. arcticus 238T and O. antarcticus 307T, respectively, and of less than 96.3 % to the type strains of the other recognized species used in the analyses. The predominant isoprenoid quinone detected in strain SSK2-1T and O. arcticus DSM 13978T was ubiquinone-10 (Q-10), which is typical of the vast majority of the class Alphaproteobacteria; minor amounts of Q-8 (approx. 1 %) and Q-9 (approx. 2 %) were also present strain SSK2-1T. The major fatty acids (.10 % of the total) found in strain SSK2-1T were C18 : 1v7c (66.6 %) and 11-methyl C18 : 1v7c (14.3 %). In Table 2, the fatty acid profile of strain SSK21T is compared with that of O. arcticus DSM 13978T analysed under identical conditions. The fatty acid profile of strain SSK2-1T was similar to that of O. arcticus DSM 13978T in that C18 : 1v7c is the predominant fatty acid and a significant amount of 11-methyl C18 : 1v7c is present, although there were differences in the proportions of some fatty acids (Table 2). The major polar lipids found in strain SSK2-1T were phosphatidylcholine, phosphatidylglycerol and one unidentified aminolipid (Fig. 2). In Fig. 2, the polar lipid profile of strain SSK2-1T is compared with that of O. arcticus DSM 13978T. The polar lipid profile of strain SSK2-1T was similar to that of O. arcticus DSM 13978T in that phosphatidylcholine, phosphatidylglycerol and one unidentified aminolipid are major http://ijs.sgmjournals.org polar lipids, but was distinguishable from that of O. arcticus DSM 13978T in that one additional major polar lipid (L4) is absent. The DNA G+C content of strain SSK2-1T was 60.1 mol%, a value slightly higher than those reported for species of the genus Octadecabacter (Table 1). From the results obtained from the chemotaxonomic analysis and the phylogenetic analysis based on 16S rRNA gene sequences, it is reasonable to classify strain SSK2-1T as a member of the genus Octadecabacter (Figs 1 and 2; Table 1). Strain SSK2-1T was distinguishable from the type strains of O. arcticus and O. antarcticus by differences in some phenotypic characteristics, including temperature range for growth, oxidase activity, nitrate reduction, starch hydrolysis and utilization of several substrates (Table 1). These differences, in combination with the phylogenetic distinctiveness of strain SSK2-1T, suggest that the novel strain is separate from the two recognized species of the genus Octadecabacter (Stackebrandt & Goebel, 1994). On the basis of the data presented, therefore, strain SSK2-1T is considered to represent a novel species of the genus Octadecabacter, for which the name Octadecabacter jejudonensis sp. nov. is proposed. Emended description of the genus Octadecabacter Gosink et al. 1997 The description of the genus Octadecabacter is as given by Gosink et al. (1997) with the following amendments. Oxidase activity is variable. Nitrate reduction is variable. The predominant ubiquinone is Q-10. The major polar lipids are phosphatidylcholine, phosphatidylglycerol and one unidentified aminolipid. The major fatty acid is C18 : 1v7c. The G+C content of the chromosomal DNA is 56.0–60.1 mol%. Description of Octadecabacter jejudonensis sp. nov. Octadecabacter jejudonensis (je.ju.do.nen9sis. N.L. masc. adj. jejudonensis pertaining to Jeju island of South Korea, from where the type strain was isolated). Cells are Gram-stain-negative, non-spore-forming, nonflagellated and rod-shaped, approximately 0.2–0.5 mm in diameter and 0.8–5.0 mm in length. Gas vesicles are produced. Colonies on MA are circular, slightly convex, smooth, glistening, reddish orange and 0.5–1.0 mm in diameter after incubation for 5 days at 30 uC. Optimal growth occurs at 30 uC; growth occurs at 15 and 35 uC, but not at 10 or 37 uC. Optimal pH for growth is between 7.0 and 8.0; growth occurs at pH 6.0, but not at pH 5.5. Growth occurs in the presence of 1.0–6.0 % (w/v) NaCl with an optimum of approximately 2.0 % (w/v) NaCl. Mg2+ ions are required for growth. Anaerobic growth does not occur on MA or on MA supplemented with nitrate. Catalase- and oxidase-positive. Nitrate is reduced to nitrite. Aesculin, casein, hypoxanthine, starch, Tweens 20, 40, 60 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 06:37:07 721 S. Park and J.-H. Yoon Loktanella litorea DPG-5T (JN885197) 77.9 75.8 0.01 Loktanella koreensis GA2-M3T (DQ344498) Loktanella salsilacus LMG 21507T (AJ440997) Jannaschia helgolandensis Hel 10T (AJ438157) 65.6 Jannaschia pohangensis H1-M8T (DQ643999) 100 87.6 94.7 Jannaschia seosinensis CL-SP26T (AY906862) 99.8 Litoreibacter janthinus KMM 3842T (AB518880) Litoreibacter meonggei MA1-1T (JN021667) 99.9 Litoreibacter arenae GA2-M15T (EU342372) 100 84.3 Octadecabacter arcticus 238T (U73725) Octadecabacter antarcticus 307T (U14583) 99.3 Octadecabacter jejudonensis SSK2-1T (KF515220) 99.6 97.2 Litoreibacter albidus KMM 3851T (AB518881) Sulfitobacter pontiacus CHLG 10T (Y13155) Sulfitobacter marinus SW-265T (DQ683726) Roseobacter litoralis ATCC 49566T (X78312) 63.3 83.9 60.3 100 Nereida ignava 2SM4T (AJ748748) Marivita cryptomonadis CL-SK44T (EU512919) Marivita litorea CL-JM1T (EU512918) Marivita geojedonensis DPG-138T (JN885198) 63.0 Marivita hallyeonensis DPG-28T (JF260872) Marivita byunsanensis SMK-114T (FJ467624) 53.2 Thalassobius maritimus GSW-M6T (HM748766) 99.6 Donghicola eburneus SW-277T (DQ667965) Donghicola xiamenensis Y-2T (DQ120728) Primorskyibacter sedentarius KMM 9018T (AB550558) Thalassobius gelatinovorus IAM 12617T (D88523) Thalassobius mediterraneus CECT 5383T (AJ878874) Thalassobius aestuarii JC2049T (AY442178) Shimia marina CL-TA03T (AY962292) 99.7 Shimia isoporae SW6T (FJ976449) Stappia stellulata IAM 12621T (D88525) Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of strain SSK2-1T, the type strains of the two recognized species of the genus Octadecabacter and representatives of some other members of the Alphaproteobacteria. Bootstrap values (expressed as percentages of 1000 replications) of .50 % are shown at branch points. Filled circles indicate that the corresponding nodes were also recovered in the trees generated with the maximum-likelihood and maximum-parsimony algorithms. Stappia stellulata IAM 12621T was used as an outgroup. Bar, 0.01 substitutions per nucleotide position. and 80, urea and L-tyrosine are hydrolysed, but gelatin and xanthine are not. Cellobiose, D-fructose, D-galactose, Dglucose, D-xylose, acetate, citrate, L-malate and succinate are utilized, but L-arabinose, maltose, D-mannose, sucrose, trehalose, benzoate, formate, pyruvate, L-glutamate and salicin are not. Acid is produced from L-arabinose, D-galactose, D-glucose, maltose, D-ribose and D-xylose, but not from cellobiose, D-fructose, lactose, D-mannose, melezitose, melibiose, raffinose, L-rhamnose, sucrose, 722 trehalose, myo-inositol, D-mannitol or D-sorbitol. In assays with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase and acid phosphatase activities are present, but lipase (C14), valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, naphthol-AS-BI-phosphohydrolase, a-galactosidase, bgalactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and afucosidase activities are absent. Susceptible to ampicillin, Downloaded from www.microbiologyresearch.org by International Journal of Systematic and Evolutionary Microbiology 64 IP: 88.99.165.207 On: Thu, 15 Jun 2017 06:37:07 Octadecabacter jejudonensis sp. nov. Table 2. Cellular fatty acid compositions (%) of strain SSK21T and the type strain of Octadecabacter arcticus All data were obtained from this study. Fatty acids that represented ,0.5 % in both strains were omitted. TR, Trace (,0.5 %); 2, not detected. SSK2-1T Fatty acid Acknowledgements This work was supported by a grant from the National Institute of Biological Resources (NIBR) funded by the Ministry of Environment (MOE) and the Program for Collection, Management and Utilization of Biological Resources and BK 21 program from the Ministry of Science, ICT & Future Planning (MSIP) of the Republic of Korea. O. arcticus DSM 13978T References Straight-chain C16 : 0 C18 : 0 Unsaturated C18 : 1v7c Hydroxy C10 : 0 3-OH C12 : 1 3-OH 11-methyl C18 : 1v7c Summed feature 3* 6.5 4.4 4.4 2 66.6 74.9 2 4.9 14.3 2.9 9.1 1.1 7.6 Barrow, G. I. & Feltham, R. K. A. (editors) (1993). Cowan and Steel’s Manual for the Identification of Medical Bacteria, 3rd edn. Cambridge: Cambridge University Press. Baumann, P. & Baumann, L. (1981). The marine Gram-negative eubacteria: genera Photobacterium, Beneckea, Alteromonas, Pseudomonas, and Alcaligenes. In The Prokaryotes, pp. 1302–1331. Edited by M. P. Starr, H. Stolp, H. G. Trüper, A. Balows & H. G. Schlegel. Berlin: Springer. TR Bruns, A., Rohde, M. & Berthe-Corti, L. (2001). Muricauda ruestringensis gen. nov., sp. nov., a facultatively anaerobic, appendaged bacterium from German North Sea intertidal sediment. Int J Syst Evol Microbiol 51, 1997–2006. *Summed feature 3 contained C16 : 1v7c and/or C16 : 1v6c. Cohen-Bazire, G., Sistrom, W. R. & Stanier, R. Y. (1957). Kinetic studies of pigment synthesis by nonsulfur purple bacteria. J Cell Comp Physiol 49, 25–68. carbenicillin, cephalothin, chloramphenicol, gentamicin, neomycin, novobiocin, oleandomycin, penicillin G, polymyxin B and streptomycin, but not to kanamycin, lincomycin or tetracycline. The predominant ubiquinone is Q-10. 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