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Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, South Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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7c and/or iso-C15 : 0 2-OH, C17 : 1, C15 : 1 and iso-C16 : 0 as major fatty acids. The DNA G+C content was 39·3 mol%. Phylogenetic trees based on 16S rRNA gene sequence analysis showed that strain SMK-10T belonged to the genus Colwellia. 16S rRNA gene sequence similarity values (94·7–96·7 %) to the type strains of all other Colwellia species and various differential phenotypic properties were sufficient to distinguish strain SMK-10T from recognized Colwellia species. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain SMK-10T (=KCTC 12480T=DSM 17314T) is classified as the type strain of a novel Colwellia species, for which the name Colwellia aestuarii sp. nov. is proposed.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain SMK-10T is DQ055844.
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), includes the genera Colwellia and Thalassomonas. This family accommodates bacteria with cells that are Gram-negative, motile or non-motile, chemo-organotrophic, curved rods or straight rods, psychrophilic and/or halophilic, and contain ubiquinone-8 as the predominant isoprenoid quinone (Yumoto et al., 1998; Nogi et al., 2004; Yi et al., 2004; Ivanova et al., 2004). At the time of writing, the genus Colwellia comprises eight species, Colwellia psychrerythraea and C. hadaliensis (Deming et al., 1988), C. demingiae, C. hornerae, C. psychrotropica and C. rossensis (Bowman et al., 1998), C. maris (Yumoto et al., 1998) and C. piezophila (Nogi et al., 2004). The genus Thalassomonas comprises two species, Thalassomonas viridans (Macián et al., 2001) and T. ganghwensis (Yi et al., 2004). Recently, a bacterial strain, SMK-10T, cells of which were Gram-negative, greyish-yellow-pigmented, curved rods, was isolated from a tidal flat sediment in Korea. 16S rRNA gene sequence comparison indicated that strain SMK-10T is phylogenetically related to the family Colwelliaceae. Accordingly, the aim of the present study was to determine the exact taxonomic position of strain SMK-10T by using a combination of polyphasic taxonomic data.
Tidal flat sediment collected from Saemankum, Pyunsan, Korea, was used as the source for the isolation of bacterial strains. Strain SMK-10T was isolated by the dilution plating technique on marine agar 2216 (MA; Difco) at 30 °C. C. hornerae CIP 105821T and C. maris CIP 106458T, which were obtained from the Collection de l'Institut Pasteur (CIP), Paris, France, were used as reference strains for fatty acid analysis. Cell morphology and presence of flagella were examined by light microscopy (Nikon E600) and transmission electron microscopy (TEM) by using cells grown on MA. The Gram reaction was determined by using the bioMérieux Gram Stain kit according to the manufacturer's instructions. Growth at various temperatures from 4 to 40 °C was measured on MA, and tolerance to various NaCl concentrations was measured in marine broth 2216 (MB; Difco). The pH range for growth and optimal pH were determined in MB that was adjusted to various pH values (initial pH 4·5–9·0 at intervals of 0·5 pH units). Growth in the absence of NaCl was investigated in R2A agar (Difco) and trypticase soy broth prepared according to the formula for the Difco medium except that no NaCl was used. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on MA and on MA supplemented with nitrate, both of which had been prepared anaerobically using nitrogen. Catalase and oxidase activity and hydrolysis of casein and starch were determined as described by Cowan & Steel (1965). Hydrolysis of hypoxanthine, tyrosine and xanthine was investigated on MA with the substrate concentrations described by Cowan & Steel (1965). Hydrolysis of aesculin, gelatin and urea, and nitrate reduction were studied as described by Lanyi (1987) with the modification that artificial sea water (containing 23·6 g NaCl, 0·64 g KCl, 4·53 g MgCl2.6H2O, 5·94 g MgSO4.7H2O and 1·3 g CaCl2.2H2O per litre distilled water; Bruns et al., 2001) was used for the preparation of media. Hydrolysis of Tweens 20, 40, 60 and 80 was determined as described by Cowan & Steel (1965) with the modification that artificial sea water was used for the preparation of media. Acid production from carbohydrates was determined as described by Leifson (1963). Utilization of various substrates as sole carbon and energy sources was tested according to the method of Baumann & Baumann (1981), using supplementation with 2 % (v/v) Hutner's mineral base (Cohen-Bazire et al., 1957), 1 % (v/v) vitamin solution (Staley, 1968) and 0·005 % (w/v) yeast extract. The API ZYM system (bioMérieux) was used to determine enzyme activities. Antibiotic sensitivity was tested by spreading bacterial suspension on MA and applying discs impregnated with the following antibiotics (concentration per disc): ampicillin (10 µg), carbenicillin (100 µg), cephalothin (30 µg), chloramphenicol (100 µg), gentamicin (30 µg), lincomycin (15 µg), kanamycin (30 µg), neomycin (30 µg), novobiocin (5 µg), oleandomycin (15 µg), penicillin G (20 U), polymyxin B (100 U), streptomycin (50 µg) and tetracycline (30 µg).
Strain SMK-10T was cultivated for 3 days in MB at 30 °C to obtain the cell mass required for isoprenoid quinone analysis and DNA extraction. Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and analysed using reversed-phase HPLC and a YMC ODS-A (250x4·6 mm) column. For fatty acid methyl ester analysis, cell mass of strain SMK-10T was harvested from agar plates after cultivation for 3 days at 30 °C on MA; cell mass of C. maris CIP 106458T was obtained from 5 days cultivation at 15 °C on PYSE agar (Yumoto et al., 1998) and cell mass of C. hornerae CIP 105821T was obtained from 7 days cultivation at 10 °C on MA. The fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). Chromosomal DNA was extracted and purified by the procedure described by Yoon et al. (1996). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984) with a modification that DNA was hydrolysed and the resulting nucleotides were analysed by reversed-phase HPLC. Amplification of the 16S rRNA gene was performed according to the method described by Yoon et al. (1998) using two universal primers. Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003).
Morphological, cultural, physiological and biochemical characteristics of strain SMK-10T are shown in Table 1 or are given in the species description below. The predominant ubiquinone detected in strain SMK-10T was Q-8 at a peak area ratio of approximately 98 %. The cellular fatty acid profile of strain SMK-10T is given in Table 2, together with those of some other Colwellia species. The profile of strain SMK-10T was characterized by large amounts of unsaturated, straight-chain, branched and hydroxy fatty acids; the major components (>10 % of the total) were C16 : 17c and/or iso-C15 : 0 2-OH, C17 : 1, C15 : 1 and iso-C16 : 0. The DNA G+C content of strain SMK-10T was 39·3 mol%.
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). Strain SMK-10T exhibited 16S rRNA gene sequence similarity values of 94·7–96·7 % to the type strains of seven Colwellia species and of 94·1–95·7 % to the type strains of the two Thalassomonas species. Sequence similarities to the other species included in the phylogenetic analysis were below 90·0 % (Fig. 1).
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). In the neighbour-joining phylogenetic tree, the relationship between this clade and the cluster comprising the two Thalassomonas species was supported by a bootstrap confidence value of 100 % (Fig. 1). Phylogenetic discrimination, supported by high bootstrap resampling values, between the genera Colwellia and Thalassomonas was also shown in previous studies (Yi et al., 2004; Ivanova et al., 2004). These results suggest two possibilities for the classification of strain SMK-10T: that it is a member of the genus Colwellia or a member of a new genus. There were no distinct phenotypic, particularly chemotaxonomic, properties to differentiate strain SMK-10T from the genus Colwellia (Bowman et al., 1998; Yumoto et al., 1998; Nogi et al., 2004). The predominant respiratory lipoquinone (Q-8) of strain SMK-10T was the same as that of the genus Colwellia (Yumoto et al., 1998; Nogi et al., 2004). The fatty acid profile of strain SMK-10T was similar to those of Colwellia species, although there were differences in the proportion of each fatty acid, possibly as a result of the different analytical conditions used, e.g. cultivation and apparatus for the analysis. In particular, two fatty acids, C16 : 17c and iso-C15 : 0 2-OH, could not be separated by GLC with the MIDI system in the present study. When the type strains of two Colwellia species, C. maris CIP 106458T and C. hornerae CIP 105821T, were analysed in this study, their fatty acid profiles were similar to that of strain SMK-10T. Based on our combined phylogenetic and chemotaxonomic analyses, it seems appropriate that strain SMK-10T be considered as a member of the genus Colwellia. Strain SMK-10T was distinguishable from recognized Colwellia species by several phenotypic characteristics (Table 1). Despite the fact that we did not undertake any DNA–DNA hybridization experiments, the phylogenetic distinctiveness of strain SMK-10T is sufficient to separate it from other recognized Colwellia species (Stackebrandt & Goebel, 1994). Therefore, on the basis of the data presented, strain SMK-10T should be placed in the genus Colwellia as a member of a novel species, for which the name Colwellia aestuarii sp. nov. is proposed.
Description of Colwellia aestuarii sp. nov.
Colwellia aestuarii (aes.tu.a'ri.i. L. gen. n. aestuarii of a tidal flat, from where the type strain was isolated).
Cells are Gram-negative, curved rods, 0·4–0·5x1·8–3·1 µm. Motile by means of a single polar flagellum. Growth occurs under anaerobic conditions on MA and on MA supplemented with nitrate. Growth occurs at 4 and 32 °C with an optimum temperature range of 25–30 °C. Optimal pH range for growth is 7·0–8·0; growth occurs weakly at pH 5·0 but not at 4·5. Optimal growth occurs in the presence of 2–3 % (w/v) NaCl; growth occurs in the presence of 6 % (w/v) NaCl but not in the presence of >7 % NaCl. Tweens 20, 40 and 60 are hydrolysed, but hypoxanthine, xanthine and L-tyrosine are not. In assays with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI phosphohydrolase and 
-glucosidase are present, but lipase (C14), valine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are absent. D-Xylose and salicin are utilized as sole carbon and energy sources. Benzoate, formate, L-glutamate, D-mannose, pyruvate, succinate and sucrose are not utilized. Acid is produced from D-cellobiose, D-galactose, D-glucose, D-ribose, L-rhamnose, D-xylose and maltose. Acid is not produced from D-melezitose or D-raffinose. Susceptible to carbenicillin, cephalothin, chloramphenicol, gentamicin, kanamycin, neomycin, novobiocin, oleandomycin, penicillin G, polymyxin B and streptomycin, but not to ampicillin, lincomycin or tetracycline. The major cellular fatty acids are C16 : 17c and/or iso-C15 : 0 2-OH, C17 : 1, C15 : 1 and iso-C16 : 0. The predominant ubiquinone is Q-8. The DNA G+C content is 39·3 mol%.
The type strain, SMK-10T (=KCTC 12480T=DSM 17314T), was isolated from a tidal flat sediment from Saemankum, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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