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1 School of Earth and Environmental Sciences and Research Institute of Oceanography, Seoul National University, Seoul 151-742, Republic of Korea
2 Division of Life and Marine Sciences, Inha University, Incheon 402-751, Republic of Korea
3 Department of Environmental Sciences, University of California, Riverside, CA 92521, USA
4 Department of Microbiology, Oregon State University, Corvallis, OR 97331, USA
Correspondence
Byung C. Cho
bccho{at}snu.ac.kr
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ABSTRACT |
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-hydroxybutyrateThe GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strain CL-SP27T and B5-6T are AY906863 and DQ514326, respectively.
A table giving the major characteristics that differentiate strains CL-SP27T and B5-6T from other related members of the family Rhodobacteraceae is available as supplementary material in IJSEM Online.
These authors contributed equally to this work. 
Present address: Marine Environmental Research Department, Korea Ocean Research and Development Institute (KORDI), Ansan 426-744, Republic of Korea.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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-3 subgroup of the phylum Proteobacteria (Shiba, 1991
), many novel species in the Roseobacter clade have been isolated from various marine environments (seawater, sediment, marine algae, invertebrates, vertebrates, hypersaline microbial mats and coastal biofilms). Nowadays, the Roseobacter clade within the family Rhodobacteraceae is known to be one of the most abundant groups in marine environments (Giovannoni & Rappé, 2000; Selje et al., 2004; Buchan et al., 2005). Members of the Roseobacter clade 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). In this study, two strains, designated CL-SP27T and B5-6T, were isolated from hypersaline water of a solar saltern in Korea and surface seawater from the Sargasso Sea, Atlantic Ocean. Although the two novel strains were isolated from different geographical origins, the 16S rRNA gene sequences of the strains were 99.6 % similar. A polyphasic taxonomic approach was used to determine whether the two novel strains represent the same or separate species. Based on these data, it is proposed that the isolates represent two novel species in a new genus within the family Rhodobacteraceae.
Strain CL-SP27T was isolated from a hypersaline water sample (31.8 % salinity) from a solar saltern in Seosin, Korea, using a standard dilution plating method on marine agar 2216 (MA, Difco) plates. After incubation of the plates at 30 °C for 2 weeks, strain CL-SP27T was purified as single colonies. Strain B5-6T was isolated from a surface (5 m) seawater sample from the western Sargasso Sea on R2A seawater agar medium (Lanoil et al., 2000) and routinely maintained on MA at 30 °C.
Nearly complete 16S rRNA gene sequences for strains CL-SP27T (1385 bp) and B5-6T (1405 bp) were obtained as described previously (Cho & Giovannoni, 2003; Choi et al., 2006) and used for phylogenetic analyses. The 16S rRNA gene sequences were aligned with those of members of the family Rhodobacteraceae using the jPHYDIT program (Jeon et al., 2005). Phylogenetic trees were generated by neighbour-joining (Saitou & Nei, 1987) with the Jukes & Cantor model (1969), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) methods, using MEGA3 (Kumar et al., 2004) and PAUP 4.0 (Swofford, 1998). The robustness of the tree topologies was assessed by bootstrap analyses based on 1000 replications for the neighbour-joining and maximum-parsimony trees and 100 replications for the maximum-likelihood tree. Likelihood parameters were estimated by the hierarchical ratio tests in MODELTEST, version 3.04 (Posada & Crandall, 1998). The 16S rRNA gene sequence similarity between strains CL-SP27T and B5-6T was 99.6 %. Phylogenetic analyses based on the 16S rRNA gene sequences as well as BLASTN search (Altschul et al., 1990) results showed that strains CL-SP27T and B5-6T belong to the Roseobacter clade within the family Rhodobacteraceae. The novel strains showed 93.0–93.2 % gene sequence similarity to Jannaschia helgolandensis and Oceanicola granulosus and 90.4–92.9 % to various type species belonging to the Roseobacter clade. In the phylogenetic trees generated by the three phylogenetic reconstruction methods, the novel strains formed a distinct clade within the family Rhodobacteraceae, but were not specifically associated with any species in the family (Fig. 1). Phylogenetic analyses and the low levels of 16S rRNA gene sequence similarities between the novel strains and other members of the family Rhodobacteraceae clade indicate that strains CL-SP27T and B5-6T represent a new genus in the family.
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). In addition, there was a considerable difference in the DNA G+C contents between the two novel strains (70.0 and 66.7 mol% for strains CL-SP27T and B5-6T, respectively) as measured by HPLC according to Mesbah et al. (1989). Based on these results, the novel strains represent two separate species in a new genus within the family Rhodobacteraceae.
Morphological and physiological analyses were also performed. Morphology and motility of cells grown on MA and marine broth 2216 (MB) were examined by phase-contrast microscopy. Accumulation of poly -hydroxybutyrate (PHB) granules was determined by Nile blue A staining (Ostle & Holt, 1982). Transmission electron microscopic investigations were performed after negative staining of cells with 2 % uranyl acetate. Catalase and oxidase activities were determined according to the protocols described by Smibert & Krieg (1994). Gelatinase, amylase, DNase, nitrate reductase activities and degradation of Tween 80 were examined as described by Hansen & Sørheim (1991). Anaerobic growth was assessed on MA using the GasPak anaerobic system (BBL). Bacteriochlorophyll a production was determined spectrophotometrically in 90 % acetone extracts from cells cultured in the dark. 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). The kit was used according to the manufacturer's instructions, except that the cell suspension was prepared using artificial seawater [ASW (24 g NaCl, 5.1 g MgCl2, 4 g Na2SO4, 1.1 g CaCl2, 0.7 g KCl, 0.2 g NaHCO3, 0.1 g KBr, 0.027 g H3BO3, 0.024 g SrCl2, 0.003 g NaF, 1 l distilled water); Lyman & Fleming, 1940] as a suspension medium. Other enzyme activities were also assayed using the API ZYM kit (bioMérieux) with ASW as the suspension medium. The temperature range for growth was determined on the basis of colony formation on MA plates that were incubated at 5–45 °C with increments of 5 °C. The pH range (pH 4–12 at intervals of 1 pH unit) for growth was determined by changes in OD600 with time in MB. Tolerance of sea salts was determined using synthetic ZoBell broth (distilled water l–1, 5 g Bacto peptone, 1 g yeast extract, 0.1 g ferric citrate) at various concentrations [0 to 10 % at intervals of 1 % and 15, 20 and 25 % (w/v)] of sea salts (Sigma). Ionic requirements were determined using synthetic ZoBell agar with the following combinations of salts (all w/v): (i) 3 % NaCl; (ii) 3 % NaCl, 0.6 % MgCl2.6H2O and 0.3 % MgSO4.7H2O; (iii) 3 % NaCl, 0.6 % MgCl2.6H2O, 0.3 % MgSO4.7H2O and 0.06 % KCl, and (iv) 3 % NaCl, 0.6 % MgCl2.6H2O, 0.3 % MgSO4.7H2O, 0.06 % KCl and 0.2 % CaCl2.2H2O. Carbon utilization was tested on basal agar medium supplemented with yeast extract (23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2.6H2O, 5.94 g MgSO4.7H2O, 1.3 g CaCl2.2H2O, 0.2 g NaNO3, 0.2 g NH4Cl, 15 g Bacto agar, 0.05 g yeast extract, 1 l distilled water; 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. No visible colonies were observed on control plates with no carbon source. Susceptibility to antibiotics was determined by the diffusion plate method. The following antibiotics were tested (µg per disc): streptomycin (10), gentamicin (10), cephalexin (30), vancomycin (30), mitomycin (1), kanamycin (30), penicillin (10), erythromycin (15), tetracycline (30), nalidixic acid (30), chloramphenicol (30), ciprofloxacin (5) and ampicillin (10).
Cells of strains CL-SP27T and B5-6T were Gram-negative, non-motile rods approximately 0.7–1.4x1.0–4.5 µm and 0.4–0.8x1.3–2.8 µm, respectively. Irregular rod-forms were frequently found in both fresh and old cultures of strain CL-SP27T. Elongated cells of up to 30 µm were observed in old cultures. PHB granules were identified in cells of both novel strains by transmission electron microscopy (Fig. 2) and Nile blue A staining. Detailed morphological, physiological and biochemical characteristics of the strains are listed in the genus and species descriptions. The novel strains were not able to grow in medium with NaCl as a sole salt, but were able to grow in medium containing Na+ and Mg2+. Most biochemical characteristics tested were similar between the two novel strains except glucose fermentation and esterase activity. Carbon source utilization patterns, however, were quite different between the two novel species (Table 1). Biochemical characteristics, carbon source utilization patterns and antibiotic susceptibilities can be used to differentiate the two novel strains.
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and analysed by HPLC as described by Collins (1985). The major isoprenoid quinone in strains CL-SP27T and B5-6T is UQ-10. Fatty acid methyl esters (FAMEs) in whole cells were analysed by GC according to the instructions of the Microbial Identification System (MIDI). Dominant fatty acids for the strains were 18 : 1
7c (54.2–65.3 %), followed by cyclo 19 : 08c (8.2–21.0 %) (Table 2). Strains CL-SP27T and B5-6T could be differentiated by the proportions of the major fatty acids (18 : 17c and cyclo 19 : 08c) and by the presence or absence of several fatty acids, including 16 : 0, 17 : 0, 18 : 36c and 11-methyl 18 : 17c (Table 2).
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Description of Maribius gen. nov.
Maribius (Ma.ri.bi'us. L. neut. n. mare the sea; N.L. masc. n. bius from Gr. n. bios life; N.L. masc. n. Maribius sea life).
Cells are Gram-negative, non-motile rods. Growth is obligately heterotrophic and strictly aerobic. On MA medium, colonies are circular, entire, convex, opaque and beige in colour. Catalase- and oxidase-positive. Accumulate PHB granules. Require Mg2+ and Na+ for growth. The predominant isoprenoid quinone is UQ-10. Dominant fatty acids are 18 : 17c and cyclo 19 : 08c. Cells do not contain bacteriochlorophyll a. The G+C content of the genomic DNA is 66.7–70.0 mol%. Phylogenetically, the genus is a member of the family Rhodobacteraceae. The type species is Maribius salinus.
Description of Maribius salinus sp. nov.
Maribius salinus (sa.li'nus. N.L. masc. adj. salinus salted, salty).
Displays the following properties in addition to those given in the genus description. Cells are 0.7–1.4 µm wide and 1.0–4.5 µm long. Growth occurs within the temperature range of 10–35 °C (optimum 30–35 °C), at pH values of between 7 and 8 and at sea salt concentrations of 1–10 % (w/v). Gives a positive result in tests for amylase, urease, esterase (C4), esterase lipase (C8), leucine arylamidase and -galactosidase activities and Tween 80 and aesculin hydrolysis. Negative results in tests for DNase, gelatinase, nitrate reductase, alkaline and acid phosphatase, lipase (C14), valine arylamidase, cystine arylamidase, trypsin, naphthol-AS-BI-phosphohydrolase, -chymotrypsin, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase, arginine dihydrolase and -fucosidase activities, indole production and glucose acidification. Several fatty acids, including 16 : 0, 17 : 0, 18 : 0, 18 : 36c, 3-OH 10 : 0, ECL 11.799, 11-methyl 18 : 17c, are present at >1 %. Growth occurs on citrate, D-cellobiose, D-fructose, D-mannitol, D-raffinose, D-sorbitol, D-xylose, ethanol, formic acid, L-arginine, L-leucine, L-lysine, L-ornithine, myo-inositol, N-acetylglucosamine, pyruvic acid and succinate. No growth occurs on D-galactose, D-glucose, D-mannose, D-ribose, D-trehalose, glycerol, glycine, L-glutamate, L-proline, L-rhamnose or sucrose. The type strain is susceptible to gentamicin, cephalexin, vancomycin, mitomycin, kanamycin, penicillin, erythromycin, tetracycline, chloramphenicol, ciprofloxacin and ampicillin, but resistant to nalidixic acid and streptomycin. The DNA G+C content is 70.0 mol%.
The type strain, CL-SP27T (=KCCM 42113T=JCM 13037T), was isolated from hypersaline water from a solar saltern in Korea.
Description of Maribius pelagius sp. nov.
Maribius pelagius (pe.la'gi.us. L. masc. adj. pelagius of or belonging to the sea).
Displays the following properties in addition to those given in the genus description. Cells are 0.4–0.8 µm wide and 1.3–2.8 µm long. Growth occurs within the temperature range of 10–40 °C (optimum 30–35 °C), at pH values of between 6 and 9 and at sea salt concentrations of 2–15 % (w/v). Gives a positive result in tests for amylase, urease, esterase (C4), esterase lipase (C8), leucine arylamidase, -galactosidase activities, hydrolysis of Tween 80 and aesculin hydrolysis and glucose acidification. Negative results in tests for DNase, gelatinase, nitrate reductase, alkaline and acid phosphatases, lipase (C14), valine arylamidase, cystine arylamidase, trypsin, -chymotrypsin, naphthol-AS-BI-phosphohydrolase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase, -fucosidase and arginine dihydrolase activities and for indole production. Several fatty acids, including 18 : 0, 3-OH 10 : 0 and ECL 11.799 are present at >1 %. Growth occurs on citrate, D-fructose, D-glucose, D-ribose, D-xylose, ethanol, glycerol, L-arginine, N-acetylglucosamine and pyruvic acid. No growth occurs on D-cellobiose, D-galactose, D-mannitol, D-mannose, D-raffinose, D-sorbitol, D-trehalose, formic acid, glycine, L-glutamate, L-leucine, L-lysine, L-ornithine, L-proline, L-rhamnose, myo-inositol, sucrose or succinate. The type strain is susceptible to gentamicin, cephalexin, vancomycin, mitomycin, kanamycin, penicillin, erythromycin, tetracycline, chloramphenicol, ciprofloxacin, ampicillin and streptomycin, but resistant to nalidixic acid. The DNA G+C content is 66.7 mol%.
The type strain, B5-6T (=KCCM 42336T=JCM 14009T), was isolated from surface water of the Sargasso Sea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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