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Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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7c as the major fatty acid. The DNA G+C content was 67.0 mol%. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed that strain SW-255T is phylogenetically closely related to the genera Ruegeria and Silicibacter of the Alphaproteobacteria. The levels of 16S rRNA gene sequence similarity between strain SW-255T and the type strains of Ruegeria atlantica and two Silicibacter species were in the range 95.8–96.2 %. A phylogenetic tree based on gyrB sequences showed that strain SW-255T forms a distinct evolutionary lineage within the Alphaproteobacteria. Differential phenotypic properties, polar lipid profiles and DNA G+C contents, together with the phylogenetic distinctiveness, suggest that strain SW-255T should be distinguished from the members of the genera Ruegeria and Silicibacter. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, strain SW-255T represents a novel genus and species, for which the name Pseudoruegeria aquimaris gen. nov., sp. nov. is proposed. The type strain of Pseudoruegeria aquimaris is SW-255T (=KCTC 12737T=JCM 13603T).
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). 16S rRNA gene sequence analyses have since shown that Ruegeria gelatinovorans and Ruegeria algicola have phylogenetic positions that are independent of Ruegeria atlantica, the type species of the genus (González et al., 2003; Lee et al., 2005). Ruegeria gelatinovorans and Ruegeria algicola have subsequently been reclassified as Thalassobius gelatinovorus (Arahal et al., 2005) and Marinovum algicola (i.e. in a novel genus; Martens et al., 2006). The related genus Silicibacter was proposed by Petursdottir & Kristjansson (1997) with a single species, Silicibacter lacuscaerulensis; another Silicibacter species, Silicibacter pomeroyi, has since been described (González et al., 2003). Here we describe a bacterial strain, SW-255T, which is phylogenetically closely related to Ruegeria atlantica, S. lacuscaerulensis and S. pomeroyi. The aim of the present study was to determine the exact taxonomic position of strain SW-255T by means of a polyphasic characterization that included determination of phenotypic and chemotaxonomic properties and a detailed phylogenetic analysis based on 16S rRNA gene sequences.
Strain SW-255T was isolated from seawater from Hwajinpo on the coast of the East Sea, Korea, by means of the standard dilution plating technique at 25 °C on marine agar 2216 (MA; Difco). The type strains of Ruegeria atlantica, S. lacuscaerulensis and S. pomeroyi were used as reference strains for the phenotypic and fatty acid analyses; Ruegeria atlantica KCTC 12424T and S. lacuscaerulensis KCTC 2953T were obtained from the Korean Collection for Type Cultures, Taejon, Korea. S. pomeroyi DSM 15171T was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). The morphological, physiological and biochemical characteristics of strain SW-255T were investigated using routine cultivation on MA at 37 °C. The cell morphology was examined by using light microscopy (E600; Nikon) and transmission electron microscopy. The presence of flagella was determined using transmission electron microscopy (CM-20; Philips) with cells from cultures growing exponentially. For transmission electron microscopic observation, cells were negatively stained with 1 % (w/v) phosphotungstic acid and the grids were examined after air drying. Growth under anaerobic conditions was determined after incubation in a Forma anaerobic chamber on MA and on MA supplemented with nitrate, both of which had been prepared anaerobically using a nitrogen atmosphere. Growth in the absence of NaCl was investigated using trypticase soy broth prepared according to the formula of the Difco medium except that no NaCl was used. Growth at various NaCl concentrations was investigated in marine broth 2216 (Difco) or trypticase soy broth (Difco). Growth at various temperatures (4–55 °C) was measured on MA. Catalase and oxidase activities and the hydrolysis of casein, starch and Tweens 20, 40, 60 and 80 were determined as described by Cowan & Steel (1965). The hydrolysis of hypoxanthine, tyrosine and xanthine was tested on MA using the substrate concentrations described by Cowan & Steel (1965). The hydrolysis of aesculin, gelatin and urea and nitrate reduction were investigated as described previously (Lanyi, 1987), with the modification that artificial seawater was used in the preparation of the media. The artificial seawater contained (l–1 distilled water) 23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2.6H2O, 5.94 g MgSO4.7H2O and 1.3 g CaCl2.2H2O (Bruns et al., 2001). H2S production was tested as described previously (Bruns et al., 2001). Susceptibility to antibiotics was detected on MA plates by using antibiotic discs with the following amounts: polymyxin B (100 U), streptomycin (50 µg), penicillin G (20 U), chloramphenicol (100 µg), ampicillin (10 µg), cephalothin (30 µg), gentamicin (30 µg), novobiocin (5 µg) and tetracycline (30 µg). Acid production from carbohydrates was determined as described by Leifson (1963). The utilization of various substrates for growth was determined as described by Baumann & Baumann (1981) using supplementation with 2 % (v/v) Hutner's mineral salts solution (Cohen-Bazire et al., 1957) and a 1 % (v/v) vitamin solution (Staley, 1968). Other physiological and biochemical tests were performed with the API 20E and API ZYM systems (bioMérieux).
Cell biomass for respiratory lipoquinone analysis and for DNA extraction was obtained from cultivation in marine broth 2216 at 37 °C. Chromosomal DNA was isolated and purified according to the method described by Yoon et al. (1996), with the exception that RNase T1 was used in combination with RNase A to minimize contamination with RNA. The 16S rRNA gene was amplified by using a PCR with two universal primers, as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003). PCR amplification of the DNA gyrase B subunit gene (gyrB) was performed by using two primers, UP-1 and UP-2r, according to a method described previously (Yamamoto & Harayama, 1995). The PCR product was purified with the QIAquick PCR purification kit (Qiagen). Sequencing of the purified PCR product was performed with an Applied Biosystems automatic DNA sequencer (model 3130) using two primers, as described by Yamamoto & Harayama (1995). Alignment of the sequences was carried out with the CLUSTAL W software (Thompson et al., 1994). Gaps at the 5' and 3' ends of the alignment were omitted from further analysis. Evolutionary distance matrices were calculated by using the algorithm of Jukes & Cantor (1969) with the DNADIST program. A phylogenetic tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987) in the PHYLIP package (Felsenstein, 1993). The stability of the relationships was assessed by means of a bootstrap analysis based on 1000 resamplings of the neighbour-joining dataset, using the programs SEQBOOT, DNADIST, NEIGHBOR and CONSENSE of the PHYLIP package.
Respiratory lipoquinones were analysed as described by Komagata & Suzuki (1987) using reversed-phase HPLC. For cellular fatty acid analysis, cell mass of strain SW-255T, Ruegeria atlantica KCTC 12424T, S. lacuscaerulensis KCTC 2953T and S. pomeroyi DSM 15171T was harvested from MA plates after cultivation for 3 days at 37, 20, 45 and 30 °C, respectively. Fatty acids were extracted and fatty acid methyl esters were prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). Polar lipids were extracted according to the procedures described by Minnikin et al. (1984) and were identified by two-dimensional TLC followed by spraying with the appropriate detection reagents (Minnikin et al., 1984; Komagata & Suzuki, 1987). The presence of phosphatidylcholine was tested by spraying with Dragendorff's reagent (Sigma). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984), with the modification that the DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC.
Morphological, cultural, physiological and biochemical characteristics of strain SW-255T are given in the genus and species descriptions (see below) and in Table 1. The almost-complete 16S rRNA gene sequence of strain SW-255T determined in this study comprised 1421 nt, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain SW-255T was part of the clade comprising Ruegeria atlantica and the two Silicibacter species, with a bootstrap resampling value of 53.8 % (Fig. 1). Strain SW-255T exhibited 16S rRNA gene sequence similarity values of 95.8, 96.0 and 96.2 % with respect to the type strains of Ruegeria atlantica, S. lacuscaerulensis and S. pomeroyi, respectively. The 16S rRNA gene sequence similarity values between strain SW-255T and other species used in the phylogenetic analysis were below 95.6 %. The gyrB sequences of strain SW-255T, Ruegeria atlantica KCTC 12424T, S. lacuscaerulensis KCTC 2953T and S. pomeroyi DSM 15171T determined in this study each comprised 1146 nt. Strain SW-255T exhibited gyrB sequence similarity values of 76.2, 81.8 and 80.1 % with respect to Ruegeria atlantica KCTC 12424T, S. lacuscaerulensis KCTC 2953T and S. pomeroyi DSM 15171T, respectively. The gyrB sequence similarity between S. lacuscaerulensis KCTC 2953T and S. pomeroyi DSM 15171T was 85.4 %.
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7c (72.9 %) (Table 2). This fatty acid profile was similar to those of Ruegeria atlantica and the two Silicibacter species, although there were differences in the proportions of some fatty acids, perhaps because of differences in cultivation conditions (González et al., 2003; Table 2). The major polar lipids detected in strain SW-255T were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid and an unidentified glycolipid (Fig. 2). The major polar lipids detected in Ruegeria atlantica KCTC 12424T, S. lacuscaerulensis KCTC 2953T and S. pomeroyi DSM 15171T are shown in Fig. 2 and Table 1
. The DNA G+C content of strain SW-255T was 67.0 mol%.
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, but it is clearly distinguishable on the basis of the difference in DNA G+C content. It can also be differentiated from Ruegeria atlantica on the basis of the phylogenetic relationships among strain SW-255T, Ruegeria atlantica and the two Silicibacter species (Fig. 1). Strain SW-255T is distinguishable from the two Silicibacter species by phenotypic differences (Table 1). The neighbour-joining tree based on gyrB sequences showed that strain SW-255T forms a phylogenetic lineage that is independent of those of Ruegeria atlantica, S. lacuscaerulensis and S. pomeroyi (Fig. 3). Strain SW-255T is also distinguishable from the type strains of Ruegeria atlantica, S. lacuscaerulensis and S. pomeroyi in terms of the polar lipid patterns (Fig. 2, Table 1). In particular, strain SW-255T differs from Ruegeria atlantica in that phosphatidylcholine is absent, and it differs from S. pomeroyi in that diphosphatidylglycerol is present as a major polar lipid and phosphatidylcholine is absent (Fig. 2). Strain SW-255T is also distinguishable from S. lacuscaerulensis by the presence of an unidentified phospholipid and an unidentified glycolipid (Fig. 2). Accordingly, strain SW-255T should be classified within a novel genus and species distinct from the genera Ruegeria and Silicibacter. On the basis of the data presented, strain SW-255T represents a novel genus and species within the class Alphaproteobacteria, for which the name Pseudoruegeria aquimaris gen. nov., sp. nov. is proposed.
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Cells are Gram-negative, aerobic rods (0.3–0.8x1.0–8.0 µm). The predominant ubiquinone is Q-10. The major fatty acid is C18 : 17c. The DNA G+C content is 67.0 mol% (HPLC). The type species is Pseudoruegeria aquimaris.
Description of Pseudoruegeria aquimaris sp. nov.
Pseudoruegeria aquimaris (a.qui.ma'ris. L. n. aqua water; L. gen. n. maris of the sea; N.L. gen. n. aquimaris of the water of the sea).
Cells are Gram-negative rods (0.3–0.8x1.0–8.0 µm). Colonies on MA are circular to slightly irregular, raised, smooth, greyish yellow in colour and 2.0–3.0 mm in diameter after 3 days incubation at 37 °C. Growth occurs at 15 and 49 °C, but not at 10 or 50 °C. Optimal pH for growth is between 7.0 and 8.0; growth occurs at pH 5.5, but not at pH 5.0. Growth occurs in the presence of 8 % (w/v) NaCl, but not in the absence of NaCl or in the presence of more than 9 % (w/v) NaCl. Anaerobic growth does not occur on MA or on MA supplemented with nitrate. Aesculin and hypoxanthine are hydrolysed, but xanthine, L-tyrosine and Tweens 40 and 60 are not. D-fructose and L-malate are utilized as carbon and energy sources, but benzoate and formate are not. Acid is produced from D-cellobiose, D-galactose, D-mannose, melibiose, L-rhamnose, D-trehalose and myo-inositol, but not from D-melezitose, D-raffinose, D-ribose or D-sorbitol. Susceptible to ampicillin, carbenicillin, cephalothin, gentamicin, kanamycin, neomycin, novobiocin, oleandomycin, polymyxin B, streptomycin and tetracycline, but not to lincomycin. The predominant ubiquinone is Q-10. The major fatty acid (>10 % of total fatty acids) is C18 : 17c. The major polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid and an unidentified glycolipid. The DNA G+C content of the type strain is 67.0 mol% (determined by HPLC). Other phenotypic characteristics are given in Table 1.
The type strain, SW-255T (=KCTC 12737T=JCM 13603T), was isolated from seawater from the beach at Hwajinpo, East Sea, Korea.
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ACKNOWLEDGEMENTS |
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