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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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7c (54.3 %) and 19 : 0 cyclo 8c (20.4 %). The DNA G+C content is 67.3 mol%. According to the physiological data, fatty acid composition and phylogenetic analysis of the 16S rRNA gene sequence, strain CL-SP20T represents a novel species in a novel genus of the family Rhodobacteraceae, for which the name Citreimonas salinaria gen. nov., sp. nov. is proposed. The type strain of Citreimonas salinaria is CL-SP20T (=KCCM 42116T=JCM 13036T).
Present address: Marine Environmental Research Department, Korea Ocean Research and Development Institute, Ansan 426-744, Republic of Korea. 
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-3 subgroup of the Proteobacteria (Shiba, 1991
), many strains have been isolated, mainly from various marine environments (e.g. seawater, sediment, marine algae and invertebrates, vertebrates, hypersaline microbial mats and coastal biofilms), and the Roseobacter clade is now known to be one of the most abundant groups present in marine environments (Giovannoni & Rappé, 2000; Selje et al., 2004; Buchan et al., 2005). In addition, the Roseobacter clade constitutes a numerically important heterotrophic bacterial group present in hypersaline microbial mats (Jonkers & Abed, 2003). Species isolated from hypersaline environments include Antarctobacter heliothermus, Roseisalinus antarcticus, Roseovarius tolerans, Staleya guttiformis and Sulfitobacter brevis from the hypersaline Ekho Lake (Labrenz et al., 1998, 1999, 2000, 2005), Roseivivax halodurans and Roseivivax halotolerans from a saline lake in Western Australia (Suzuki et al., 1999), Palleronia marisminoris and Salipiger mucosus from hypersaline soil (Martínez-Cánovas et al., 2004, Martínez-Checa et al., 2005) and Jannaschia seosinensis from hypersaline water from a solar saltern (Choi et al., 2006a). Strain CL-SP20T, which is affiliated with the Rhodobacteraceae, was isolated from hypersaline water (31.8 % salinity) from a solar saltern in Seosin, Korea. The water was spread on marine agar 2216 (MA; Difco) and incubated at 30 °C for 2 weeks. Strain CL-SP20T was isolated and subsequently purified four times on MA at 30 °C. The strain was maintained both on MA at 4 °C and at –80 °C in marine broth (MB; Difco) supplemented with 30 % (v/v) glycerol.
The 16S rRNA gene was amplified from a single colony by using a PCR with Taq DNA polymerase (Bioneer) and 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 (ABI3730XL) at Macrogen Corp. (Seoul, Korea). The almost-complete 16S rRNA gene sequence of strain CL-SP20T (1401 bp) was obtained and then compared, using BLASTN searches (Altschul et al., 1990), with 16S rRNA gene sequences available in GenBank. The sequence of strain CL-SP20T was manually aligned with those of species from the Roseobacter clade, obtained from the GenBank and Ribosomal Database Project (Cole et al., 2003) databases, using known 16S rRNA secondary-structure information. Phylogenetic trees were obtained by using neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) methods. An evolutionary distance matrix for the neighbour-joining method was generated according to the model of Jukes & Cantor (1969). The robustness of the tree topologies was assessed using bootstrap analyses based on 1000 replications for the neighbour-joining and maximum-parsimony methods and 100 replications for the maximum-likelihood method. 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) and PAUP* 4.0 (Swofford, 1998). Likelihood parameters were estimated by the hierarchical ratio tests in MODELTEST 3.04 (Posada & Crandall, 1998).
Morphological and physiological analyses were performed as follows. Gram-staining was performed as described by Smibert & Krieg (1994). Cell morphology and motility were examined by phase-contrast microscopy and transmission electron microscopy (EX2; JEOL) with cells grown for 2 days in MB at 30 °C. Anaerobic growth was checked on MA using the GasPak anaerobic system (BBL). Bacteriochlorophyll a production was determined in 90 % acetone extracts from cells cultured in the dark, and examined with a spectrophotometer (Ultraspec 2000; Pharmacia Biotech). The temperature range for growth was determined on the basis of colony formation on MA plates incubated at temperatures ranging from 5 to 45 °C (in increments of 5 °C). The pH range (tested at pH 5–11 in increments of 1 pH unit) for growth was determined, in MB, by changes in the OD600 over time. The final pH was adjusted using NaOH and HCl solutions. The tolerance of CL-SP20T to sea salts was determined using synthetic ZoBell broth (Bacto peptone, 5 g; yeast extract, 1 g; ferric citrate, 0.1 g; distilled water, 1 l) with various concentrations (0–10 %, in increments of 1 %, and at 15, 20 and 25 %, w/v) of sea salts (Sigma). Growth in a medium containing NaCl as the sole salt was tested in a synthetic ZoBell agar medium supplemented with 3 % NaCl. Catalase and oxidase activities were determined according to the protocols described by Smibert & Krieg (1994), and gelatinase, amylase, DNase and nitrate reductase activities and degradation of Tween 80 were examined as described by Hansen & Sørheim (1991). In addition, nitrate reduction, the production of indole, arginine dihydrolase, urease, gelatinase and 
-galactosidase activities, acid production from glucose and the hydrolysis of aesculin were tested using an 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, 1 l) (Lyman & Fleming, 1940). Other enzyme activities were assayed using the API ZYM kit (bioMérieux) and artificial seawater as the 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, 1 l) (Choi et al., 2006b) containing 0.4 % carbon source. Incubation was performed for 1 month and growth was scored as negative when it was equal to, or less than, that in the negative control (lacking any carbon source). The ability to oxidize thiosulfate was tested on solid medium, according to protocols modified from those described by González et al. (1999). Strain CL-SP20T was streaked on medium supplemented with thiosulfate (HEPES, 10 g; NaCl, 20 g; K2HPO4, 0.5 g; NH4Cl, 0.5 g; MgSO4.7H2O, 0.6 g; CaCl2.2H2O, 0.3 g; sodium acetate, 20 mM; sodium thiosulfate, 20 mM; yeast extract, 0.05 g; agar, 15 g; distilled water, 1 l); negative controls were prepared with the same medium but without sodium thiosulfate. After 2 weeks incubation, the results were read according to González et al. (1999). All phenotypic assays (except analysis of the temperature range) were carried out at 30 °C.
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 (Seoul, Korea). The DNA G+C content was determined by HPLC analysis of deoxyribonucleosides as described by Mesbah et al. (1989) after DNA purification by the method of Marmur (1961).
The 16S rRNA gene sequence of CL-SP20T showed 96.3 % sequence similarity to Citreicella thiooxidans CHLG 1T, 95.3 % to Roseivivax halodurans OCh 239T, 94.9 % to Roseivivax halotolerans OCh 210T and 91.0–94.9 % to other species of the Roseobacter lineage. In spite of a high level of sequence similarity between strain CL-SP20T and Citreicella thiooxidans, strain CL-SP20T did not form a robust clade with Citreicella thiooxidans or any other species in the Roseobacter lineage, but instead formed a distinct subline in all 16S rRNA gene sequence-based phylogenetic trees constructed using different methods (Fig. 1). Thus, strain CL-SP20T could be recognized as representing a distinct genus.
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). Bright inclusions inside the cells were not observed under phase-contrast microscopy, but poly--hydroxybutyrate granules were identified by staining with Nile blue A (Ostle & Holt, 1982). Colonies on MA were circular, entire, convex, opaque, shiny and beige in colour. After incubation for 1 week, colonies were approximately 1 mm in diameter. Bacteriochlorophyll a was not detected. Growth of CL-SP20T was observed at temperatures from 15 to 40 °C, with an optimum between 30 and 35 °C. Growth occurred at pH 6–10, with an optimum at pH 7–8. Strain CL-SP20T grew with sea salts at concentrations from 1 to 10 % (with an optimum between 5 and 6 %), but did not grow on agar plates containing 3 % NaCl as the sole source of salt. Strain CL-SP20T degraded DNA and gelatin and reduced nitrate to nitrite, but did not degrade starch. It was positive for catalase, aesculin hydrolysis, alkaline phosphatase, esterase lipase and valine arylamidase, but was negative for cytochrome oxidase, urease, acid phosphatase and -glucosidase (Table 1). Strain CL-SP20T was unable to oxidize thiosulfate. The other results of biochemical and physiological tests are given in Table 1 and the species description.
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7c (54.3 %) (a feature shared by members of the Roseobacter clade); this was followed by 19 : 0 cyclo 8c (20.4 %), 18 : 36c (6,9,12) (7.7 %), 11-methyl 18 : 17c (5.5 %), 16 : 0 (5.3 %), 18 : 0 (4.0 %) and 12 : 1 3-OH (2.9 %) (Table 1
). The DNA G+C content was 67.3 mol%.
Strain CL-SP20T is closely related to Citreicella thiooxidans on the basis of 16S rRNA gene sequence similarity. However, the phylogenetic analysis showed that CL-SP20T does not form a robust clade with Citreicella thiooxidans strains; furthermore, CL-SP20T can be differentiated from this most closely related micro-organism by its inability to oxidize thiosulfate, its ability to reduce nitrate, its gelatinase activity, the absence of oxidase, urease, acid phosphatase and -glucosidase, its carbon-utilization characteristics and its responses to temperature and sea salts (Table 1). The fatty acid profile of strain CL-SP20T is also obviously different from that of Citreicella thiooxidans, i.e. in the proportions of the major fatty acids (18 : 17c and 19 : 0 cyclo 8c) and by the presence or absence of other fatty acids (Table 1). Overall, the polyphasic taxonomic evidence based on phylogenetic analysis of 16S rRNA gene sequences, fatty acid profiles and phenotypic features indicate that strain CL-SP20T should be classified within a novel genus and species, for which the name Citreimonas salinaria gen. nov., sp. nov. is proposed.
Description of Citreimonas gen. nov.
Citreimonas (Cit.re.i.mo'nas. L. n. citreum lemon; L. fem. n. monas a unit, monad; N.L. fem. n. Citreimonas a lemon-shaped monad).
Cells are Gram-negative, lemon-shaped rods. Obligately heterotrophic and aerobic. Catalase-positive and oxidase-negative. The predominant fatty acids are 18 : 17c and 19 : 0 cyclo 8c. Cells do not contain bacteriochlorophyll a and do not oxidize thiosulfate. The genus is a member of the family Rhodobacteraceae. The type species is Citreimonas salinaria.
Description of Citreimonas salinaria sp. nov.
Citreimonas salinaria (sa.li.na'ri.a. L. fem. adj. salinaria of or belonging to a salt-works).
Shows the following properties in addition to those in the genus description. Cells are non-motile and approximately 0.5–0.8 µm wide and 1.0–1.5 µm long. On MA medium, colonies are circular with an entire margin, convex, opaque, shiny and beige in colour. Grows at temperatures in the range 15–40 °C (optimum, 30–35 °C) and at pH 6–10. Growth occurs at sea salts concentrations of 1–10 % (w/v). No growth occurs without sea salts in the medium. DNase, gelatinase, nitrate reductase, esterase, esterase lipase and leucine arylamidase activities are present and alkaline phosphatase and valine arylamidase activities are weakly present. Amylase, indole production, acid production from glucose, arginine dihydrolase, urease, -galactosidase, Tween 80 degradation, lipase, cystine arylamidase, trypsin, -chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase activities are absent. Growth occurs on L-arabinose, cellobiose, D-fructose, D-glucose, D-mannose, sucrose, D-trehalose, D-xylose, acetone, acetate, Casamino acids, pyruvate, citrate, succinate, glycerol, N-acetylglucosamine, inulin, salicin, L-arginine, L-leucine, L-lysine, L-ornithine and L-proline. No growth occurs on -ketobutyric acid, acetamide, D-galactose, D-raffinose, D-ribose, L-rhamnose, polyethylene glycol, ascorbate, benzoate, formic acid, lactose, maleic acid, salicylate, tartrate, ethanol, myo-inositol, 2-propanol, D-mannitol, sorbitol, L-aspartate, L-asparagine, DL-cysteine, L-glutamate, glycine, glycogen, thiamine or urea. The DNA G+C content is 67.3 mol%.
The type strain, CL-SP20T (=KCCM 42116T=JCM 13036T), was isolated from hypersaline water from a solar saltern in Seosin, Korea.
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ACKNOWLEDGEMENTS |
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