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School of Earth and Environmental Sciences and Research Institute of Oceanography, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea
Correspondence
Byung C. Cho
bccho{at}snu.ac.kr
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ABSTRACT |
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7c (64.1 %) and 11-methyl 18 : 17c (10.6 %). The DNA G+C content is 57.2 mol%. According to physiological data, fatty acid composition and phylogenetic analysis of the 16S rRNA gene sequence, CL-TA03T is considered to represent a new genus in the family Rhodobacteraceae and the name Shimia marina gen. nov., sp. nov. is proposed. The type strain of Shimia marina is CL-TA03T (=KCCM 42117T=JCM 13038T).
Present address: Marine Environmental Research Department, Korea Ocean Research and Development Institute (KORDI), Ansan 426-744, Republic of Korea. 
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; Selje et al., 2004; Buchan et al., 2005). The strains show diverse physiological and morphological features (e.g. phototrophy, aerobic sulfite oxidation, organic sulfur compound degradation, methylotrophy, gas vacuoles, poly-
-hydroxybutyrate granules, rosette formation) (Arahal et al., 2005; Buchan et al., 2005). Furthermore, phylogenetic analyses of marine Roseobacter sequences have shown that for two-thirds (68 %) of the Roseobacter diversity identified so far, it is not yet possible to access relevant physiological information through studies of cultured organisms (Buchan et al., 2005). In early stages of biofilm establishment, clones affiliated with the Roseobacter clade accounted for 85 % of the total sequenced clones, suggesting that organisms belonging to the Roseobacter clade are ubiquitous and rapid colonizers of surfaces in coastal environments (Dang & Lovell, 2000). In this study, a strain affiliated with the Rhodobacteraceae, CL-TA03T, was isolated in October 2002 from a biofilm formed on an acrylic slide submerged for 1 month in surface water on a coastal fish farm in Tongyeong, Korea. The scraped biofilm was suspended in seawater that had been passed through a 0.2 µm filter and autoclaved. The suspension was spread on a marine agar 2216 (MA; Difco) plate and incubated at 25 °C for 1 week. Strain CL-TA03T was isolated and subsequently purified four times on MA at 30 °C. The strain was maintained both on MA at 4 °C and in marine broth 2216 (MB; Difco), supplemented with 30 % (v/v) glycerol, at –80 °C.
The 16S rRNA gene was amplified from a single colony by PCR with Taq DNA polymerase (Bioneer) using primers 27F and 1492R (Lane, 1991). The PCR product was purified using the AccuPrep PCR Purification kit (Bioneer) and cloned using the pCR2.1 TOPO TA Cloning kit (Invitrogen). Sequencing of the 16S rRNA gene was performed with an Applied Biosystems automatic sequencer (ABI 3730xl) at Macrogen Corp., Seoul, Korea. An almost complete 16S rRNA gene sequence of strain CL-TA03T (1382 bp) was obtained. The sequence of strain CL-TA03T was compared with 16S rRNA gene sequences available in GenBank using BLASTN searches (Altschul et al., 1990). The sequence of strain CL-TA03T was aligned manually with those of type strains of species belonging to genera phylogenetically related to CL-TA03T and with type species of other genera in the Roseobacter clade within the family Rhodobacteraceae obtained from GenBank and from the Ribosomal Database Project (Cole et al., 2003) databases using known 16S rRNA secondary structure information. Phylogenetic trees were obtained by neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) methods. An evolutionary distance matrix for the neighbour-joining method was generated according to the model of Jukes & Cantor (1969). The robustness of tree topologies was assessed by bootstrap analyses based on 1000 replications for neighbour-joining and maximum-parsimony methods. Alignment analysis was carried out using the jPHYDIT program (Jeon et al., 2005) and phylogenetic analyses were carried out using MEGA3 (Kumar et al., 2004). The 16S rRNA gene sequence of CL-TA03T showed 95.6 % sequence similarity to Thalassobius gelatinovorus IAM 12617T and Thalassobius mediterraneus XSM19T, 95.4 % to Ruegeria atlantica IAM 14463T, 95.1 % to Silicibacter lacuscaerulensis ITI-1157T and 92.4–94.8 % to other type species of the Roseobacter lineage. In the phylogenetic trees, however, strain CL-TA03T did not form a robust clade with any species in the Roseobacter lineage (Fig. 1). The DNA G+C content was determined by HPLC analysis of deoxyribonucleosides as described by Mesbah et al. (1989) after DNA purification using the method of Marmur (1961) and was found to be 57.2 mol%.
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. Cell morphology and motility were examined by phase-contrast microscopy and transmission electron microscopy (JEOL EX2) with cells grown for 1 day at 30 °C in MB and on MA. Anaerobic growth was checked on MA using the GasPak anaerobic system (BBL). Cells were rods of 0.3–0.6 µm wide and 0.8–3.6 µm long in exponential growth phase (Fig. 2). Cells occasionally formed small chains but not star-shaped aggregates. Cells were motile by several monopolar flagella (Fig. 2). Poly--hydroxybutyrate granules were not identified by transmission electron microscopy and Nile blue A staining (Ostle & Holt, 1982). Colonies on MA were circular, entire, convex, opaque and colourless or beige. After incubation for 1 week, colonies were approximately 2 mm in diameter. Bacteriochlorophyll a production was determined in 90 % acetone extracts from cells cultured in the dark and examined using a spectrophotometer (Ultraspec 2000; Pharmacia Biotech). Bacteriochlorophyll a was not detected in CL-TA03T.
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and gelatinase, amylase, DNase, nitrate reductase activities and degradation of Tween 80 were examined as described by Hansen & Sørheim (1991). In addition, nitrate reduction, production of indole, arginine dihydrolase, urease, gelatinase, -galactosidase, acid production from glucose and hydrolysis of aesculin were tested using the API 20NE kit (bioMérieux) according to the manufacturer's instructions, except that the cell suspension was prepared using artificial seawater (NaCl, 24 g; MgCl2, 5.1 g; Na2SO4, 4 g; CaCl2, 1.1 g; KCl, 0.7 g; NaHCO3, 0.2 g; KBr, 0.1 g; H3BO3, 0.027 g; SrCl2, 0.024 g; NaF, 0.003 g; distilled water to 1 l; Lyman & Fleming, 1940) as a suspension medium. Other enzyme activities were also assayed using the API ZYM kit (bioMérieux) and artificial seawater as a suspension medium. Carbon utilization was tested on basal agar medium supplemented with yeast extract (NaCl, 23.6 g; KCl, 0.64 g, MgCl2.6H2O, 4.53 g; MgSO4.7H2O, 5.94 g; CaCl2.2H2O, 1.3 g; NaNO3, 0.2 g; NH4Cl, 0.2 g; Bacto agar, 15 g; yeast extract, 0.05 g; distilled water to 1 l; Choi et al., 2006) containing 0.2 % of the carbon source. Incubation was prolonged for 1 month and growth was scored as positive when visible colonies were observed. Growth of CL-TA03T was observed at temperatures of 15–35 °C, with optimum growth between 30 and 35 °C. Growth occurred from pH 6 to 10. Strain CL-TA03T grew at sea salt concentrations of 3–7 % and could degrade starch and gelatin and reduce nitrate to nitrite. CL-TA03T was positive for cytochrome oxidase, catalase and alkaline phosphatase, but negative for valine arylamidase, -galactosidase and 
-glucosidase (Table 1). The results of the other biochemical and physiological tests are given in Table 1 and in the species description.
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and analysed by HPLC as described by Collins (1985). The major isoprenoid quinone in CL-TA03T is UQ-10. The fatty acid methyl esters in whole cells were analysed by gas chromatography according to the instructions of the Microbial Identification System (MIDI) at the Korean Culture Center of Microorganisms in Seoul, Korea. The dominant fatty acid for CL-TA03T was 18 : 17c (64.1 %), which is a characteristic common to the Roseobacter clade, followed by 11-methyl 18 : 17c (10.6 %), 16 : 0 (4.2 %) and 18 : 0 (4.1 %) (Table 1
).
In terms of phenotypic features, strain CL-TA03T could be differentiated from the closely related genus Thalassobius by the absence of granules inside cells, the presence of weak amylase activity and the absence of growth on acetate, D-glucose, D-ribose, D-fructose, D-mannitol, myo-inositol, sorbitol, salicin, glycerol, L-arginine, L-aspartate, L-glutamate and sucrose as the sole carbon source. Furthermore, fatty acid profiles could distinguish CL-TA03T from the two Thalassobius species (Table 1). The fatty acid profile of strain CL-TA03T was different from that of T. mediterraneus mainly by the proportions of 18 : 17c and 11-methyl 18 : 17c, and from that of T. gelatinovorus mainly by the proportions of minor fatty acids including 10 : 0, 19 : 0 cyclo, 18 : 36c (6,9,12) and 2-OH 16 : 0. Moreover, phylogenetic analysis of the 16S rRNA gene sequence clearly showed that strain CL-TA03T could not be classified as a member of any known genera in the Roseobacter clade. Therefore, phylogenetic analyses based on 16S rRNA gene sequences, fatty acid profile and phenotypic features indicated that strain CL-TA03T should be classified as a novel genus and species, for which the name Shimia marina gen. nov., sp. nov. is proposed.
Description of Shimia gen. nov.
Shimia (Shi'mi.a. N.L. fem. n. Shimia named in honour of Dr Jae H. Shim, for his contributions to marine plankton ecology in Korea).
Cells are Gram-negative and rod-shaped. Growth is heterotrophic and strictly aerobic. Catalase- and oxidase-positive. Absence of granules inside cells. The predominant isoprenoid quinone is UQ-10. The dominant fatty acid is 18 : 17c. Cells do not contain bacteriochlorophyll a. The genus is a member of the family Rhodobacteraceae. The type species is Shimia marina.
Description of Shimia marina sp. nov.
Shimia marina (ma.ri'na. L. fem. adj. marina of or belonging to the sea, marine).
Displays the following properties in addition to those given in the genus description. Cells are approximately 0.3–0.6 µm wide and 0.8–3.6 µm long. Cells are motile by several monopolar flagella. On MA medium, colonies are circular, entire, convex, opaque and colourless or beige. Grows at 15–35 °C (optimum 30–35 °C) and at pH 6–10. Growth occurs at sea-salt concentrations of 3–7 % (w/v). No growth without sea salts in the medium. Amylase, gelatinase, nitrate reductase, DNase and Tween 80 hydrolysis activities are present. According to API 20NE tests, nitrate reductase, indole production, acid production from glucose, arginine dihydrolase, aesculin hydrolysis, gelatinase and urease activities are not detected. According to API ZYM tests, alkaline phosphatase and leucine arylamidase activities are present and esterase (C4), esterase lipase (C8) and acid phosphatase activities are weakly present, whereas lipase (C14), valine arylamidase, cystine arylamidase, trypsin, -chymotrypsin, naphthol-phosphohydrolase, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase activities are absent. Major fatty acids are 18 : 17c, 11-methyl 18 : 17c, 16 : 0, 18 : 0, 18 : 36c (6,9,12) and 2-OH 16 : 0. Growth occurs on acetone, -ketobutyric acid, citrate, ethanol, glycine, glycogen, L-leucine, L-lysine, L-ornithine, pyruvate, D-raffinose, succinate, tartrate and urea. No growth occurs on acetamide, L-ascorbate, benzoate, acetate, D-cellobiose, D-galactose, D-glucose, lactose, D-mannose, D-ribose, D-xylose, D-fructose, formic acid, glycerol, inulin, 2-propanol, L-arabinose, L-aspartate, L-arginine, L-glutamate, L-asparagine, L-proline, D-mannitol, maleic acid, myo-inositol, N-acetylglucosamine, L-rhamnose, salicylate, salicin, sorbitol, sucrose, thiamine or D-trehalose. The DNA G+C content of the type strain is 57.2 mol%.
The type strain is CL-TA03T (=KCCM 42117T=JCM 13038T), isolated from a biofilm in a coastal fish farm in Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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