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1 Division of Biology and Ocean Sciences, Inha University, Incheon 402-751, Republic of Korea
2 Department of Microbiology, Oregon State University, Corvallis, OR 97331, USA
Correspondence
Jang-Cheon Cho
chojc{at}inha.ac.kr
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7c (52.5 %), C16 : 0 2-OH (13.5 %) and C18 : 1 11-methyl 7c (12.2 %). Phylogenetic analysis of the 16S rRNA gene sequences showed that the strains represented a distinct line of descent within the genus Ruegeria, with highest sequence similarities to Ruegeria atlantica DSM 5823T (97.2 %), Ruegeria lacuscaerulensis DSM 11314T (96.5 %) and Ruegeria pomeroyi DSM 15171T (95.6 %). Several phenotypic characteristics, including facultatively requiring NaCl and oxygen for growth, together with the cellular fatty acid composition, differentiated strain HTCC2662T from other members of the genus Ruegeria. Based on phenotypic, chemotaxonomic and phylogenetic traits, it is suggested that strains HTCC2662T and HTCC2663 represent a novel species of the genus Ruegeria, for which the name Ruegeria pelagia sp. nov. is proposed. The type strain is HTCC2662T (=KCCM 42378T=NBRC 102038T).
A transmission electron micrograph of cells of strain HTCC2662T is available with the online version of this paper.
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and contains Gram-negative, aerobic, oxidase- and catalase-positive, non-phototrophic bacteria that require NaCl or sea salts for growth. At the time of writing, the genus comprises three recognized species, Ruegeria atlantica, Ruegeria lacuscaerulensis and Ruegeria pomeroyi. These three species have been isolated from saline environments. R. lacuscaerulensis and R. pomeroyi were originally classified as Silicibacter lacuscaerulensis (Petursdottir & Kristjansson, 1997) and Silicibacter pomeroyi (González et al., 2003), respectively, but Silicibacter was recently combined with the genus Ruegeria by Yi et al. (2007) based on 16S rRNA gene sequence phylogeny and chemotaxonomy. In the present study, we describe the isolation and identification of a novel species of the genus Ruegeria.
Initial liquid cultures of two strains, designated HTCC2662T and HTCC2663, were obtained by using the high-throughput culturing approaches of Cho & Giovannoni (2003) and Connon & Giovannoni (2002) according to a dilution-to-extinction methodology. The strains were subsequently purified as single colonies on marine agar 2216 (MA; Difco) after incubation for 4 days at 25 °C.
DNA extraction, PCR and sequencing of the 16S rRNA gene were performed as described by Cho & Giovannoni (2003). The resultant 16S rRNA gene sequences for strains HTCC2662T (1425 bp) and HTCC2663 (1425 bp) were aligned by using the ARB software package (Ludwig et al., 2004) and 1184 unambiguously aligned nucleotide positions were used for phylogenetic analyses in PAUP* 4.0 beta 10 (Swofford, 2002). Phylogenetic trees were generated according to the neighbour-joining (Saitou & Nei, 1987) – with Kimura two-parameter model correction (Kimura, 1980) – maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) algorithms. Bootstrap analyses were performed for the neighbour-joining and maximum-parsimony trees based on 1000 resamplings. The 16S rRNA gene sequences of strains HTCC2662T and HTCC2663 were identical, suggesting that these two strains represented the same species. Preliminary comparisons with 16S rRNA gene sequences deposited in the GenBank database indicated that the two new strains were closely related to members of the genus Ruegeria. Based on multiple alignment in the ARB database, strain HTCC2662T showed highest 16S rRNA gene sequence similarity to R. atlantica DSM 5823T (97.2 %), followed by R. lacuscaerulensis DSM 11314T (96.5 %) and R. pomeroyi DSM 15171T (95.6 %). In all the phylogenetic trees generated in this study (Fig. 1), strains HTCC2662T and HTCC2663 formed a monophyletic clade together with the three recognized species of the genus Ruegeria, with relatively high bootstrap support (97 % in the neighbour-joining tree and 71 % in the maximum-parsimony tree). The new strains formed an independent phylogenetic lineage within the genus Ruegeria, suggesting that they were not associated with any of the recognized species of the genus and thus represented a novel species in the genus Ruegeria.
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) and Smibert & Krieg (1994) with MA as the basal medium at 30 °C. Cell morphology was examined by energy-filtering transmission electron microscopy (LIBLA120; Carl Zeiss), and phase-contrast and epifluorescence microscopy (Nikon 80i). Anaerobic growth was tested on MA at 30 °C using both the MGC anaerobic system and the AnaeroPack Anaero (Mitsubishi Gas Chemical Company, Inc.). Biochemical tests, enzyme activities and oxidative carbon source utilization tests were carried out on API 20NE (bioMérieux), API ZYM (bioMérieux) and Biolog GN2 microplates, respectively, with artificial seawater (ASW; per litre: 25.0 g NaCl, 1.0 g MgCl2 . 6H2O, 5.0 g MgSO4 . 7H2O, 0.7 g KCl, 0.15 g CaCl2 . 2H2O, 0.5 g NH4Cl, 0.1 g KBr, 0.27 g KH2PO4, 0.04 g SrCl2 . 6H2O, 0.025 g H3BO3) following the manufacturer's instructions. Susceptibility to ten different antimicrobial agents (listed in the species description below) was tested by the diffusion plate method. The DNA G+C content and cellular fatty acid composition were analysed for strain HTCC2662T. The DNA G+C content was analysed by using HPLC with a Discovery C18 column (5 µm, 15 cmx4.6 mm; Supelco) (Mesbah et al., 1989). Cellular fatty acid methyl esters were prepared from a culture grown on MA at 30 °C for 3 days, and analysed according to the instructions of the Microbial Identification System (MIDI) and performed at the Korean Culture Center of Micro-organisms (KCCM).
The results of morphological, physiological and biochemical tests are given in the species description and in Table 1. Strains HTCC2662T and HTCC2663 showed identical phenotypic and physiological traits. The two strains were therefore regarded as representing the same species based on 16S rRNA gene sequence similarities (100 %) and phenotypic characterizations. The two strains could clearly be differentiated from recognized Ruegeria species, mainly by NaCl requirement for growth, oxygen requirement for growth and acid production from glucose (Table 1). The DNA G+C content of strain HTCC2662T was 58.4 mol%, which is within the range of values (55–68 mol%) reported for the genus Ruegeria. The major cellular fatty acids of strain HTCC2662T were generally consistent with those of the three recognized Ruegeria species (Table 1); however, strain HTCC2662T could be differentiated based on the proportions of several fatty acids, including C18 : 17c, C16 : 0 2-OH, C18 : 1 11-methyl 7c, C16 : 0 and C12 : 0 3-OH.
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Description of Ruegeria pelagia sp. nov.
Ruegeria pelagia (pe.la'gi.a. L. fem. adj. pelagia of the sea).
Cells are Gram-negative and non-motile short rods that are 0.8–2.1 µm in length and 0.7-1.2 µm in width (see Supplementary Fig. S1 available in IJSEM Online). Colonies on MA are circular, smooth, convex, butyrous or (sometimes) viscous with entire margins and dark brown-coloured. Dark-brown precipitates are observed around the colonies. Growth occurs at 16–42 °C (optimally at 30–37 °C), pH 4–12 (optimally at pH 5–6) and 0–10 % NaCl (optimally at 3.5 %). NaCl is not essential for growth. Chemoheterotrophic and facultatively aerobic. No bacteriochlorophyll a is present. Does not produce poly-
-hydroxyalkanoate granules. Oxidase- and catalase-positive. In API 20NE tests, positive for -galactosidase, aesculin hydrolysis and acid production from glucose, but negative for nitrate reduction, indole production, urease, arginine dihydrolase and gelatin liquefaction. In API ZYM tests, positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, 
-glucosidase and N-acetyl--glucosaminidase activity, but negative for lipase (C14), cystine arylamidase, trypsin, -chymotrypsin, -galatosidase, -galactosidase, -glucuronidase, -glucosidase, -mannosidase and -fucosidase activity. In tests with Biolog GN2 microplates, the following carbon substrates produce positive results: -cyclodextrin, dextrin, glycogen, Tweens 40 and 80, N-acetyl-D-glucosamine, adonitol, L-arabinose, D-arabitol, D-cellobiose, D-fructose, D-galactose, -D-glucose, myo-inositol, -D-lactose, maltose, D-mannitol, D-mannose, D-psicose, D-sorbitol, sucrose, trehalose, turanose, xylitol, pyruvic acid methyl ester, succinic acid monomethyl ester, acetic acid, cis-aconitic acid, citric acid, formic acid, D-galacturonic acid, D-galactonic acid lactone, -hydroxybutyric acid, -hydroxybutyric acid, 
-hydroxybutyric acid, -ketobutyric acid, -ketoglutaric acid, -ketovaleric acid, DL-lactic acid, propionic acid, D-saccharic acid, succinic acid, bromosuccinic acid, succinamic acid, L-alaninamide, D-alanine, L-alanine, L-alanyl glycine, asparagine, L-aspartic acid, L-glutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-histidine, hydroxy-L-proline, L-leucine, L-ornithine, L-phenylalanine, L-proline, L-pyroglutamic acid, D-serine, L-serine, L-threonine, DL-carnitine, -aminobutyric acid, urocanic acid, inosine, uridine, thymidine, putrescine, 2-aminoethanol, 2,3-butanediol, glycerol and DL--glycerol phosphate. The following carbon substrates produce negative results in Biolog GN2 plates: N-acetyl-D-galactosamine, i-erythritol, L-fucose, gentiobiose, lactulose, D-melibiose, methyl -D-glucoside, D-raffinose, L-rhamnose, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, p-hydroxyphenlyacetic acid, itaconic acid, malonic acid, quinic acid, sebacic acid, glucuronamide, phenylethylamine, -D-glucose 1-phosphate and D-glucose 6-phosphate. Susceptible to chloramphenicol (25 µg), erythromycin (15 µg), rifampicin (50 µg), streptomycin (10 µg) and tetracycline (30 µg), but resistant to ampicillin (10 µg), gentamicin (10 µg), kanamycin (30 µg), penicillin G (10 µg) and vancomycin (30 µg). The cellular fatty acid profile comprises C18 : 17c (52.5 %), C16 : 0 2-OH (13.5 %), C18 : 1 11-methyl 7c (12.2 %), C18 : 1 2-OH (6.8 %), C10 : 0 3-OH (2.9 %), C16 : 0 (2.0 %), C12 : 0 3-OH (1.0 %), C18 : 0 (0.9 %), C18 : 0 2-OH (0.5 %), C16 : 1 2-OH (0.4 %), C17 : 0 2-OH (0.3 %), C12 : 0 (0.3 %) and C20 : 17c (0.3 %). The DNA G+C content is 58.4 mol% (by HPLC).
The type strain, HTCC2662T (=KCCM 42378T=NBRC 102038T), was isolated from Bermuda Atlantic Time Series Station in the western Sargasso Sea, Atlantic Ocean.
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ACKNOWLEDGEMENTS |
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