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1 Division of Biology and Ocean Sciences, Inha University, Yonghyun-Dong, Incheon 402-751, Republic of Korea
2 Polar BioCenter, Korea Polar Research Institute, KOPRI, Songdo Techno Park, Incheon 406-840, Republic of Korea
Correspondence
Jang-Cheon Cho
chojc{at}inha.ac.kr
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ABSTRACT |
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Transmission electron micrographs of cells of strain IMCC3113T are available as a supplementary figure with the online version of this paper.
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) in the family Hahellaceae of the Gammaproteobacteria currently contains two species, Hahella chejuensis (Lee et al., 2001) and Hahella ganghwensis (Baik et al., 2005), which were isolated from marine sediments. H. chejuensis has been reported as a red-pigmented bacterial species, showing antibacterial and algicidal activities associated with the presence of antibiotic prodiginines (Jeong et al., 2005; Kim et al., 2007). H. ganghwensis is a cream-coloured, chemoheterotrophic bacterium that requires sea salts for growth (Baik et al., 2005). This study focuses on the taxonomic study of a non-pigmented bacterial strain, designated IMCC3113T, isolated from Antarctic coastal seawater. On the basis of its taxonomic properties, strain IMCC3113T is considered to represent a novel species in the genus Hahella. Strain IMCC3113T was isolated from a seawater sample collected from the coast of King George Island, Weaver Peninsula, Antarctica (6 ° 14' S 5 ° 47' E). Isolation of the strain was performed using the standard dilution-plating method on marine agar 2216 (MA; Difco) at 20 °C for 1 month. After the optimum growth temperature of the strain had been determined, cultures were maintained routinely on MA or marine broth 2216 (MB; Difco) at 25 °C and preserved as a glycerol suspension (10 %, v/v) at –75 °C.
The methods used for DNA extraction, PCR and 16S rRNA gene sequencing have been described elsewhere (e.g. Cho & Giovannoni, 2003). The resultant almost-complete 16S rRNA gene sequence (1485 bp) of strain IMCC3113T was aligned with its nearest neighbours by using the ARB software package (Ludwig et al., 2004). The 16S rRNA gene sequence similarity between strain IMCC3113T and other related species was calculated using the alignment based on the secondary structure of the 16S rRNA with the ARB software. On the basis of 16S rRNA gene sequence similarities, the strain was most closely related to H. chejuensis KCTC 2396T (93.0 %), H. ganghwensis KCTC 12277T (92.1 %) and Oleiphilus messinensis DSM 13489T (90.6 %). No other bacterial species with validly published names exceeded 90 % 16S rRNA gene sequence similarity. To clarify the phylogenetic position of the strain, 1294 unambiguously aligned nucleotide positions, determined from 16S rRNA gene sequences of 28 members of the Oceanospirillales, were used for phylogenetic analyses in PAUP* 4.0 beta 10 (Swofford, 2002). Phylogenetic trees were generated using neighbour joining (Saitou & Nei, 1987) with Jukes–Cantor distance corrections (Jukes & Cantor, 1969), maximum parsimony (Fitch, 1971) and maximum likelihood (Felsenstein, 1981). The robustness of the neighbour-joining and maximum-likelihood trees was confirmed by bootstrap analyses based on 1000 and 100 resamplings of the sequences, respectively. In all of the phylogenetic trees generated in this study (Fig. 1), strain IMCC3113T, H. chejuensis KCTC 2396T and H. ganghwensis KCTC 12277T formed a monophyletic clade with strong bootstrap support. The branching-order patterns between the three strains in the clade were recovered consistently in all of the phylogenetic trees. This monophyletic clade was clustered with Cellvibrio fulvus NCIMB 8634T in the neighbour-joining and maximum-likelihood trees. However, this phylogenetic relationship was not supported by bootstrap analyses. This phylogenetic inference, coupled with 16S rRNA gene sequence similarities of <97 % (Wayne et al., 1987) between strain IMCC3113T and the two Hahella species, suggested that the strain should be assigned to the genus Hahella as a representative of a novel species.
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) and standard methods (Smibert & Krieg, 1994), using MA as the basal medium at 25 °C, unless otherwise specified. The cell morphology was examined using liquid cultures grown aerobically in MB for 2 days. The colony morphology was observed using colonies grown on MA for 5 days. Flagellar motility was investigated using wet mounts prepared from fresh cultures grown in MB at 25 °C for 2 days. The growth temperature range and optimum were tested from 3 to 42 °C. The pH range and optimum for growth were examined on MA adjusted to pH values from 4.0 to 12.0. The NaCl concentrations and optimum for growth were determined in NaCl-free artificial seawater medium (Choo et al., 2007) supplemented with 5.0 g peptone, 1.0 g yeast extract and various concentrations of NaCl (0–15 %, w/v). Biochemical tests and carbon-source oxidation tests were carried out using API 20NE and API ZYM strips (bioMérieux) and in GN2 microplates (Biolog), according to the manufacturers' instructions, using inoculation with bacterial suspensions in artificial seawater medium. Ten different antimicrobial agents (listed in the species description) were tested using the diffusion plate method (Jorgensen et al., 1999). The DNA G+C content was determined using HPLC (Mesbah et al., 1989). Cellular fatty acid methyl esters were prepared from cultures grown on MA at 25 °C for 4 days and then analysed, according to the MIDI Microbial Identification System, by the Korean Culture Center of Microorganisms (Seoul, Republic of Korea). Respiratory quinones were analysed, using reversed-phase HPLC, by the Korean Culture Center of Microorganisms (Komagata & Suzuki, 1987).
The phenotypic and biochemical characteristics determined for strain IMCC3113T are given in Table 1 and the species description. The DNA G+C content of the strain was 56.4 mol% and the major cellular fatty acid constituents are given in Table 2. Overall, cells of strain IMCC3113T were Gram-negative, psychrotolerant, aerobic, chemoheterotrophic, non-motile, granule-containing and irregularly rod-shaped. Transmission electron micrographs of the cells are shown in Supplementary Fig. S1 (available in IJSEM Online). The phylogenetic analyses in this study showed that strain IMCC3113T belonged to the genus Hahella. However, strain IMCC3113T and the two Hahella species could be differentiated from each other on the basis of the levels of 16S rRNA gene sequence similarity (92.1–93.0 %) and several phenotypic properties, including cell size, the temperature range for growth, oxidase activity, nitrate reduction, several enzyme activities and the proportions of major fatty acids, as shown in Tables 1 and 2. Therefore strain IMCC3113T represents a novel species of the genus Hahella, for which the name Hahella antarctica sp. nov. is proposed.
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Gram-negative, non-motile, aerobic, psychrotolerant and chemoheterotrophic. Cells are straight or irregular rods, 0.9–5.9 µm long and 0.4–0.8 µm wide. Colonies grown on MA at 25 °C for 5 days are 0.5–1.0 mm in diameter, circular, pulvinate with entire margins, dry, hard and cream-coloured. Growth occurs at 3–25 °C (optimum, 25 °C), pH 5–10 (optimum, pH 7) and with 0.5–5.0 % NaCl (optimum, 2.0 %). Other phenotypic and physiological characteristics are given in Table 1
. Oxidizes the following carbon substrates (Biolog GN2 microplates): melibiose, acetic acid, cis-aconitic acid, citric acid, D-galacturonic acid, D-gluconic acid, D-glucuronic acid, β-hydroxybutyric acid, propionic acid, bromosuccinic acid, glucuronamide, L-aspartic acid, L-histidine, hydroxy-L-proline, L-leucine, L-serine and inosine. Does not oxidize the following carbon substrates: 
-cyclodextrin, dextrin, Tween 40, Tween 80, -D-glucose, maltose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, adonitol, L-arabinose, D-arabitol, cellobiose, i-erythritol, D-fructose, L-fucose, D-galactose, gentiobiose, myo-inositol, -D-lactose, lactulose, D-mannitol, D-mannose, methyl β-D-glucoside, D-psicose, raffinose, L-rhamnose, D-sorbitol, sucrose, trehalose, turanose, xylitol, pyruvic acid methyl ester, succinic acid monomethyl ester, -ketobutyric acid, -ketovaleric acid, formic acid, D-galactonic acid lactone, D-glucosaminic acid, -hydroxybutyric acid, 
-hydroxybutyric acid, p-hydroxyphenylacetic acid, -ketoglutaric acid, DL-lactic acid, malonic acid, quinic acid, D-saccharic acid, sebacic acid, succinic acid, succinamic acid, L-ornithine, L-phenylalanine, L-pyroglutamic acid, D-serine, DL-carnitine, -aminobutyric acid, urocanic acid, uridine, thymidine, phenylethylamine, putrescine, 2-aminoethanol, 2,3-butanediol, glycerol, DL--glycerol phosphate, -D-glucose 1-phosphate, D-glucose 6-phosphate, L-alaninamide, D- and L-alanine, L-alanyl glycine, L-asparagine, L-glutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-proline and L-threonine. Susceptible to gentamicin (10 µg), kanamycin (30 µg), penicillin G (10 µg), rifampicin (50 µg), streptomycin (10 µg) and tetracycline (30 µg), but resistant to ampicillin (10 µg), chloramphenicol (25 µg), erythromycin (15 µg) and vancomycin (30 µg). Cellular fatty acid profile is given in Table 2. Major respiratory quinone is Q-9. DNA G+C content is 56.4 mol%.
The type strain, IMCC3113T (=KCCM 42675T=NBRC 102683T), was isolated from a surface seawater sample from Maxwell Bay, King George Island, western Antarctica.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Cho, J.-C. & Giovannoni, S. J. (2003). Parvularcula bermudensis gen. nov., sp. nov., a marine bacterium that forms a deep branch in the -Proteobacteria. Int J Syst Evol Microbiol 53, 1031–1036.
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