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1 School of Biological Sciences, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Korea
2 Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, Yusung, PO Box 115, Taejon 305-600, Korea
Correspondence
Jongsik Chun
jchun{at}snu.ac.kr
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ABSTRACT |
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-Proteobacteria (94·9 % similarity). Depending on the tree-making algorithm used, the isolate either formed a monophyletic clade with T. viridans or was recovered as a sister group of a class containing the genera Thalassomonas and Colwellia. Phenotypic features of the getbol isolate were similar to those of T. viridans, but several physiological and chemotaxonomic properties, including nitrate reduction, amylase, lecithinase, Tweenase and utilization of 13 carbon sources, distinguished strain JC2041T from T. viridans. The polyphasic data presented in this study indicate that the isolate should be classified as a novel species in the genus Thalassomonas. The name Thalassomonas ganghwensis sp. nov. is therefore proposed for the getbol isolate; the type strain is JC2041T (=IMSNU 14005T=KCTC 12041T=DSM 15355T).
The GenBank accession number for the 16S rDNA sequence of strain JC2041T is AY194066.
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). Five of these isolates were members of the -Proteobacteria, including strain JC2041T and two strains of Zooshikella ganghwensis (Yi et al., 2003). In this study, the polyphasic characterization of isolate JC2041T is presented; the name Thalassomonas ganghwensis sp. nov. is proposed for this isolate.
Strain JC2041T was isolated from a tidal flat sediment sample (37°36'22·3''N; 126°22'59·4''E) using MR2A [R2A (Difco) supplemented with the artificial sea water of Lyman & Fleming (1940)] at 25 °C and routinely maintained on MA (marine agar 2216; Difco) at 30 °C. Thalassomonas viridans DSM 13754T was used as a reference strain and grown on MA at 30 °C.
Bacterial DNA preparation and PCR amplification and sequencing of 16S rDNA were carried out as described previously (Chun & Goodfellow, 1995). The resultant sequence of strain JC2041T was aligned manually against sequences obtained from GenBank. Phylogenetic trees were inferred using the Fitch–Margoliash (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1993), maximum-parsimony (Fitch, 1972) and neighbour-joining (Saitou & Nei, 1987) methods. Evolutionary distance matrices were generated according to Jukes & Cantor (1969). The resultant tree topologies were evaluated in bootstrap analyses (Felsenstein, 1985) of the neighbour-joining method based on 1000 resamplings. The alignment and phylogenetic analyses were carried out using PHYDIT (available at http://plaza.snu.ac.kr/
jchun/phydit/) and PAUP 4.0 (Swofford, 1998) as described previously (Chun et al., 2000).
A nearly complete 16S rDNA sequence of strain JC2041T was obtained (1408 bp) and used for an initial BLAST search against GenBank. The result clearly indicated that the getbol isolate was a member of the -Proteobacteria. The newly determined sequence was then aligned manually against representatives of the -Proteobacteria based on the secondary structure of bacterial 16S rRNA. Strain JC2041T showed highest 16S rDNA sequence similarity to T. viridans CECT 5083T (94·9 %); the next highest similarities were observed with the genera Colwellia (93·0–93·4 %), Idiomarina (90·9–91·1 %) and Pseudoalteromonas (88·4–90·2 %).
These relationships were also observed in phylogenetic trees, as shown in Fig. 1. The clade containing strain JC2041T, T. viridans and Colwellia species had 100 % bootstrap support and was obtained by all tree-making methods. However, the isolate either formed a monophyletic clade with T. viridans (in Fitch–Margoliash and neighbour-joining trees) or was recovered as a sister group to a clade containing T. viridans and Colwellia species (in maximum-parsimony and maximum-likelihood trees), depending on the tree-making algorithm used.
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) and SMM (Shioi's marine medium; Shiba, 1992). The test strain retained viability for about 20 days on MA at 30 °C. The isolate was strictly halophilic, requiring media containing 1–8 % (w/v) artificial sea salts (Sigma) for growth (optimum of 2–4 % sea salts). It was unable to grow on modified CSY-3 medium containing 0–15 % (w/v) NaCl alone. Growth on MA was observed in the pH range 7–11 and optimal growth occurred at pH 7–8. The temperature range for growth was 15–40 °C; optimum growth was observed at 35 °C. No growth was detected under anaerobic conditions created by a GasPack system (BBL).
The morphology of cells grown on MA at 30 °C was examined using phase-contrast microscopy, SEM and TEM. After 1 day incubation, colonies were approximately 2 mm in diameter; they reached a maximum diameter of 4–5 mm after 4 days. Colonies were circular, convex with an entire margin, glistening, translucent, viscid and yellowish. When grown on YBM [BM (basal medium; Baumann et al., 1972) containing 0·01 % yeast extract] supplemented with 0·1 % tyrosine, strain JC2041T produced diffusible brown pigments on an agar plate. Cells in late exponential phase were rod-shaped with a polar flagellum and approximately 1·5–2·3 µm long by 0·5–0·8 µm wide. Spore formation was not observed.
Carbon source utilization was tested on BM supplemented with 0·1 % yeast extract. Other physiological properties were tested using standard procedures, as described previously (Yi et al., 2003). These reactions, as well as some other properties of strain JC2041T, are indicated in the species description. The test strain could be differentiated from T. viridans and phylogenetically related genera by a number of characteristics (Table 1).
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7c (11·3 %), were the most abundant, together with a mixture of 16 : 17c and i-15 : 0 2-OH (20·6 %; the MIDI system could not differentiate between these two fatty acids). Smaller, albeit substantial, amounts of 10 : 0 (4·9 %), i-16 : 0 (7·1 %), 16 : 19c (4·7 %) and 17 : 18c (4·4 %) were also detected. This fatty acid composition was most similar to that observed in the genus Thalassomonas and clearly differentiated the isolate from other phylogenetically related genera (Table 1
). Only minor quantitative differences in amounts of 15 : 0, 16 : 0, 17 : 18c and 18 : 17c were found between our isolate and T. viridans.
Isoprenoid quinones of the test strain and T. viridans DSM 13754T were isolated from freeze-dried biomass according to Minnikin et al. (1984), purified on preparative TLC (silica gel F254; Merck) and analysed by HPLC (Waters) equipped with a reverse-phase column (Spherisorb ODS-2 80; Waters) as described by Collins (1985). Ubiquinone-8 (Q-8) was the predominant isoprenoid quinone in both strains. The genus Colwellia is also reported to contain Q-8 as its major quinone (Yumoto et al., 1998).
The DNA G+C content was determined by HPLC of deoxyribonucleosides as described by Mesbah et al. (1989) using a reverse-phase column (Supelcosil LC-18-S; Supelco). The G+C content of strain JC2041T was 42 mol%.
Although the phylogenetic position of strain JC2041T is somewhat different depending on the tree building method used for analysis, the close relationship of our isolate to T. viridans was obvious in sequence similarity comparisons and phylogenetic trees based on 16S rDNA. Moreover, our isolate showed phenotypic traits that were similar to those of T. viridans, rather than to Colwellia species. However, the low sequence similarity, 94·9 %, and many physiological and chemotaxonomic properties, i.e. nitrate reduction, amylase, lecithinase, Tweenase and utilization of 13 carbon sources, distinguished our isolate from T. viridans (Table 1). Based on the polyphasic evidence presented in this study, it is fair to conclude that strain JC2041T merits novel species status in the genus Thalassomonas. The name Thalassomonas ganghwensis sp. nov. is proposed for strain JC2041T.
Description of Thalassomonas ganghwensis sp. nov.
Thalassomonas ganghwensis (gang.hwen'sis. N.L. fem. adj. ganghwensis named after Ganghwa Island in Korea, the geographical origin of the type strain).
Gram-negative. Oxidase- and catalase-positive. Strictly aerobic, chemoheterotrophic and halophilic. Cells are rod-shaped (1·5–2·3x0·5–0·8 µm) and motile with a polar flagellum. Colonies are circular, convex, glistening, translucent, viscid, yellowish and have an entire margin on MA. Does not grow without sea water or the addition of artificial sea salts (1–8 %) to the medium. Grows at 15–40 °C and pH 7–11. Optimal growth is observed at 35 °C, pH 7–8 and 2–4 % artificial sea salts. Abundant growth is observed on MA, CSY-3 and SMM media. Spores are not formed. Reduces nitrate to nitrite. Negative for fermentation of glucose. Decomposes casein, DNA, aesculin, gelatin and Tween 80, but not agar, alginate, cellulose, chitin, lecithin or starch. Produces 
-galactosidase, but not arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urease, acetoin, fluorescein, H2S, indole or polyhydroxybutyrate. Produces alkaline phosphatase, esterase (C4), leucine arylamidase, valine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but not esterase lipase (C8), lipase (C14), cystine arylamidase, trypsin, 
-chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase or -fucosidase. Utilizes acetate, D-galactose, D-glucose and L-tyrosine as sole carbon sources. Glycine, L-asparagine, L-lysine, succinate and tartrate are weakly utilized. Does not utilize acetamide, benzoate, citrate, D-cellobiose, D-fructose, D-mannitol, D-mannose, D-raffinose, D-ribose, D-salicin, D-sorbitol, D-trehalose, D-xylose, ethanol, inositol, inulin, 2-propanol, lactose, L-arginine, L-ascorbate, L-ornithine, L-rhamnose, polyethylene glycol, salicylate or thiamin. The major isoprenoid quinone is ubiquinone-8. The major fatty acids are 16 : 0, 18 : 17c and a mixture of 16 : 17c and i-15 : 0 2-OH.
The type strain, JC2041T (=IMSNU 14005T=KCTC 12041T=DSM 15355T), was isolated from sediment of getbol, the Korean tidal flat. The DNA G+C content of the type strain is 42 mol%.
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ACKNOWLEDGEMENTS |
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Yi, H., Chang, Y.-H., Oh, H. W., Bae, K. S. & Chun, J. (2003). Zooshikella ganghwensis gen. nov., sp. nov., isolated from tidal flat sediments. Int J Syst Evol Microbiol 53, 1013–1018.
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