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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, C16 : 17c and/or iso-C15 : 0 2-OH and C17 : 16c as the major fatty acids. Sphingoglycolipid, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine were the major polar lipids. The DNA G+C content was 58 mol%. 16S rRNA gene sequence analysis showed that strain SW-151T joins the evolutionary radiation enclosed by the genus Sphingopyxis. Similarities between the 16S rRNA gene sequences of strain SW-151T and the type strains of Sphingopyxis species ranged from 92·3 to 94·3 %, which is low enough to categorize strain SW-151T as a species distinct from previously described Sphingopyxis species. On the basis of phenotypic properties and phylogenetic distinctiveness, strain SW-151T (=KCTC 12232T=DSM 16223T) should be classified as a novel Sphingopyxis species, for which the name Sphingopyxis flavimaris sp. nov. is proposed.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain SW-151T is AY554010.
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, with five species and two genomospecies. Subsequently, a large number of species have been included in the genus (e.g. Takeuchi et al., 1993, 1995; Lee et al., 2001; Busse et al., 2003). Phylogenetic analyses based on 16S rRNA gene sequences have shown that the genus Sphingomonas and three related genera belong to the 
-subclass of the Proteobacteria (Anzai et al., 2000; Takeuchi et al., 2001; Yabuuchi et al., 2002). Takeuchi et al. (2001) proposed that Sphingomonas species should be assigned to three different genera, Sphingobium, Novosphingobium and Sphingopyxis, in addition to the genus Sphingomonas. However, Yabuuchi et al. (2002) showed that there is no phenotypic or phylogenetic evidence for dividing the genus Sphingomonas into the four genera and hence the genus Sphingomonas should remain undivided. These proposals have made the taxonomy of the genus Sphingomonas confused, but at present the nomenclature of Takeuchi et al. (2001) is generally used (Kämpfer et al., 2002; Busse et al., 2003; Fujii et al., 2003; Godoy et al., 2003; Ushiba et al., 2003; Sohn et al., 2004). In this study, we report on the detailed taxonomic characterization of a slightly halophilic, yellow-pigmented, Sphingopyxis-like bacterial strain, SW-151T, which was isolated from the water of the Yellow Sea in Korea.
Strain SW-151T was isolated by the usual dilution-plating technique on marine agar 2216 (MA; Difco) at 30 °C. Cell morphology was examined by light microscopy (E600; Nikon) and transmission electron microscopy. For the latter, the cells were negatively stained with 1 % (w/v) phosphotungstic acid and, after air-drying, the grids were examined by using a model CM-20 transmission electron microscope (Philips). The flagellation type was examined by transmission electron microscopy using cells from exponentially growing cultures. Growth at various NaCl concentrations was investigated in marine broth 2216 (Difco) or in trypticase soy broth (Difco). Growth in the absence of NaCl was investigated in trypticase soy broth without NaCl. Growth at various temperatures (4–45 °C) was measured on MA. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on MA and on MA supplemented with nitrate that had been prepared anaerobically using nitrogen. Catalase and oxidase activities and hydrolysis of casein, starch and Tweens 20, 40, 60 and 80 were determined as described by Cowan & Steel (1965). Hydrolysis of hypoxanthine, tyrosine and xanthine was tested on MA with the substrate concentrations described by Cowan & Steel (1965). Hydrolysis of aesculin, gelatin and urea and nitrate reduction were studied as described previously (Lányí, 1987) but with the modification that artificial sea water was used for preparation of media. The artificial sea water contained the following (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 (Levring, 1946). H2S production was tested as described previously (Bruns et al., 2001). Acid production from carbohydrates was evaluated as described by Leifson (1963). Utilization of substrates as sole carbon and energy sources was tested according to the method of Baumann & Baumann (1981) using supplementation with 2 % (v/v) Hutner's mineral base (Cohen-Bazire et al., 1957) and 1 % (v/v) vitamin solution (Staley, 1968).
Cell biomass of strain SW-151T for respiratory lipoquinone analysis and for DNA extraction was obtained from cultivation in marine broth at 30 °C. Respiratory lipoquinones were analysed as described previously (Komagata & Suzuki, 1987), using reversed-phase HPLC. Chromosomal DNA was isolated and purified according to a method described previously (Yoon et al., 1996), with the exception that RNase T1 was used together with RNase A. For fatty acid methyl ester analysis, a loop of cell mass was harvested from agar plates after cultivation for 7 days at 30 °C on MA. The fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). Polar lipids were extracted using the procedures described by Minnikin et al. (1984) and identified by two-dimensional TLC followed by spraying with appropriate detection reagents (Komagata & Suzuki, 1987). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984), with the modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC. The 16S rRNA gene sequence was amplified by the PCR, using 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).
Morphological, cultural, physiological and biochemical properties of strain SW-151T are shown in Table 1 or are given in the species description. The predominant respiratory lipoquinone detected in strain SW-151T was Q-10, at a peak area ratio of approximately 92 %. Strain SW-151T had a cellular fatty acid profile that contained large amounts of unsaturated, straight-chain and hydroxy fatty acids; the components were C18 : 17c (24·0 %), C16 : 17c and/or iso-C15 : 0 2-OH (20·3 %), C17 : 16c (17·8 %), C16 : 0 (9·1 %), C15 : 0 2-OH (6·1 %), C16 : 0 2-OH (4·9 %), 11-methyl C18 : 17c (4·5 %), C17 : 18c (3·2 %), C14 : 0 2-OH (2·8 %), C17 : 0 (2·1 %), C18 : 15c (1·7 %), C15 : 0 (1·5 %) and C15 : 16c (1·3 %). The lack of 3-hydroxy fatty acids is a characteristic also shared by Sphingopyxis and Sphingomonas species (Yabuuchi et al., 1990; Takeuchi et al., 1993, 1995; Lee et al., 2001; Busse et al., 2003). The major polar lipids found in strain SW-151T were sphingoglycolipid, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine, an unidentified polar lipid and unidentified phospholipids were also present. The DNA G+C content of strain SW-151T was 58 mol%. The 16S rRNA gene sequence of strain SW-151T determined in this study comprised 1444 nucleotides, representing approximately 96 % of the Escherichia coli 16S rRNA sequence. 16S rRNA gene sequence similarity searches with a public database (GenBank) revealed that strain SW-151T is phylogenetically related to the genus Sphingopyxis. In the neighbour-joining tree based on 16S rRNA gene sequences, strain SW-151T joined the cluster comprising Sphingopyxis species at a bootstrap resampling value of 66·9 % (Fig. 1). Strain SW-151T exhibited 16S rRNA gene sequence similarity levels of 92·3–94·3 % to all Sphingopyxis species with validly published names. Sequence similarities to other species included in the phylogenetic analysis were in the range 85·5–93·6 % (Fig. 1).
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; Kämpfer et al., 2002). The cellular fatty acid profile of strain SW-151T is similar to those of Sphingopyxis species, particularly Sphingopyxis chilensis, although there are differences in the compositions of some fatty acids, which may be caused by different cultivation conditions (Takeuchi et al., 1993; Lee et al., 2001; Vancanneyt et al., 2001; Godoy et al., 2003). There are not enough phenotypic properties to distinguish strain SW-151T from Sphingopyxis species (Takeuchi et al., 1993; Lee et al., 2001; Vancanneyt et al., 2001; Godoy et al., 2003). It seems reasonable to assign strain SW-151T to the genus Sphingopyxis. There is a widely recognized criterion for species definition in current bacteriology that suggests that strains with more than 3 % difference in 16S rRNA gene sequences be considered as representing different species (Stackebrandt & Goebel, 1994). Despite the lack of DNA–DNA hybridization experiments, the phylogenetic distinctiveness shown in this study is sufficient to separate strain SW-151T from other Sphingopyxis species with validly published names (Stackebrandt & Goebel, 1994). Therefore, on the basis of the data presented, strain SW-151T should be placed in the genus Sphingopyxis as a member of a novel species, for which the name Sphingopyxis flavimaris sp. nov. is proposed.
Description of Sphingopyxis flavimaris sp. nov.
Sphingopyxis flavimaris (fla.vi.ma'ris. L. adj. flavus yellow; L. gen. n. maris of the sea; N.L. gen. n. flavimaris of the Yellow Sea).
Cells are rods, 0·7–0·9x1·5–2·5 µm. Gram-negative. Non-spore-forming. Motile by means of a single polar flagellum. Colonies are circular, convex, yellow in colour and 0·8–1·0 mm in diameter after 7 days cultivation at 30 °C on MA. Growth occurs at 4 °C but not at 37 °C. Optimal pH for growth is 7·0–8·0; growth occurs at pH 5·0 but not at pH 4·5. Optimal growth occurs in the presence of 2–3 % (w/v) NaCl. Growth occurs in the presence of 6 % (w/v) NaCl but not without NaCl or in the presence of >8 % (w/v) NaCl. Growth does not occur under anaerobic conditions on MA or MA supplemented with nitrate. Tweens 20, 40, 60 and 80 and tyrosine are hydrolysed. Casein, hypoxanthine, starch and xanthine are not hydrolysed. H2S is not produced. D-Glucose and pyruvate are utilized as sole carbon and energy sources. D-Galactose, lactose, acetate, succinate, benzoate, formate and L-glutamate are not utilized. Acid is not produced from the following substrates: adonitol, L-arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannitol, D-mannose, D-melezitose, melibiose, myo-inositol, D-raffinose, L-rhamnose, D-ribose, D-sorbitol, sucrose, D-trehalose and D-xylose. The predominant respiratory lipoquinone is Q-10. The major fatty acids are C18 : 17c, C16 : 17c and/or iso-C15 : 0 2-OH and C17 : 16c. The major polar lipids are sphingoglycolipid, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine; minor components are phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine, an unidentified polar lipid and unidentified phospholipids. The DNA G+C content is 58 mol% (determined by HPLC).
The type strain, SW-151T (=KCTC 12232T=DSM 16223T), was isolated from sea water at Baekryung Island in the Yellow Sea in Korea.
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ACKNOWLEDGEMENTS |
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