Sphingopyxis baekryungensis sp. nov., an orange-pigmented bacterium isolated from sea water of the Yellow Sea in Korea

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 sensu stricto. Although Yabuuchi et al. (2002) proposed that there is no phenotypic and phylogenetic evidence to divide the genus Sphingomonas into the four genera, the nomenclature of Takeuchi et al. (2001) is in common use (Kämpfer et al., 2002; Busse et al., 2003; Fujii et al., 2003; Godoy et al., 2003; Ushiba et al., 2003; Sohn et al., 2004). At present the genus Sphingopyxis comprises six species: Sphingopyxis macrogoltabida (Takeuchi et al., 1993, 2001), Sphingopyxis terrae (Takeuchi et al., 1993, 2001), Sphingopyxis witflariensis (Kämpfer et al., 2002), Sphingopyxis alaskensis (Vancanneyt et al., 2001; Godoy et al., 2003), Sphingopyxis chilensis (Godoy et al., 2003) and Sphingopyxis flavimaris (Yoon & Oh, 2005). Recently, an orange-pigmented Sphingopyxis-like bacterium, SW-150^T, was isolated from sea water of the Yellow Sea, Korea. To date, all members allocated to the genus Sphingopyxis are yellow-pigmented (Takeuchi et al., 1993; Kämpfer et al., 2002; Godoy et al., 2003; Yoon & Oh, 2005). Accordingly, the aim of the present work was to determine the exact taxonomic position of strain SW-150^T by using a polyphasic taxonomic characterization.

Sea water collected from Baekryung Island in the Yellow Sea in Korea was used as a source for isolation of bacterial strains. Strain SW-150^T 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 (TEM). The presence of flagella was investigated by TEM, using cells from exponentially growing cultures. The Gram-reaction was determined by using the bioMérieux Gram Stain kit, according to the manufacturer's instructions. Growth at various NaCl concentrations was investigated in marine broth 2216 (MB; 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 (4–40 °C) was measured on MA. The pH range for growth was determined in MB that was adjusted to various pH values (pH 4·5–9·5 at intervals of 0·5 pH units). Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on MA and on MA supplemented with nitrate, both of which 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 investigated 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 (Lanyi, 1987), except that artificial sea water was used for preparation of media. The artificial sea water contained (per litre 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). Acid production from carbohydrates was determined as described by Leifson (1963). Utilization of substrates as sole carbon and energy sources was tested according to the method of Baumann & Baumann (1981), supplemented with 2 % (v/v) Hutner's mineral base (Cohen-Bazire et al., 1957) and 1 % (v/v) vitamin solution (Staley, 1968). For in vivo pigment-absorption spectrum analysis, the strain was cultivated aerobically in the dark at 30 °C in MB. The cultures were washed twice by centrifugation using MOPS buffer (MOPS/NaOH, 0·01 M; KCl, 0·1 M; MgCl₂, 0·001 M; pH 7·5), and were disrupted by sonication with a Branson Sonifier 450. After removal of cell debris by centrifugation, the absorption spectrum of the supernatant was examined on a Beckman Coulter DU800 spectrophotometer. Other physiological and biochemical tests were performed with the API 20E system (bioMérieux). The morphological, cultural, physiological and biochemical properties of strain SW-150^T are shown in Table 1⇓, or are given in the species description (see below). The sonicated cell extracts showed absorption maximum at 463–464 nm, and methanol-soluble pigment was characterized by absorption maximum at 462 nm.

The 16S rRNA gene was amplified by 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). The almost complete 16S rRNA gene sequence of strain SW-150^T determined in this study comprised 1446 nucleotides, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. Comparative 16S rRNA gene sequence analyses showed that strain SW-150^T is phylogenetically most closely related to Sphingopyxis species of the family Sphingomonadaceae, particularly forming a cluster with S. flavimaris with a relatively high bootstrap resampling value of 85·8 % (Fig. 1⇓). The sequence contained four of five signature nucleotides defined for the genus Sphingopyxis, as described by Takeuchi et al. (2001) (Table 1⇑). Similarity values between the 16S rRNA gene sequences of strain SW-150^T and the type strains of Sphingopyxis species ranged from 91·6 % (with S. terrae) to 94·2 % (with S. macrogoltabida and S. chilensis). Sequence similarities to other members used in the phylogenetic analysis were lower than 93·6 % (Fig. 1⇓).

Cell biomass for respiratory lipoquinone and polar lipid analyses and for DNA extraction was obtained from cultivation in MB at 30 °C. Respiratory lipoquinones were analysed as described previously (Komagata & Suzuki, 1987), by using reversed-phase HPLC. Chromosomal DNA was isolated and purified according to the method described by Yoon et al. (1996), with the exception that ribonuclease T1 was applied in combination with ribonuclease A. For fatty acid methyl ester (FAME) analysis, a loop of cell mass was harvested from MA plates after cultivation for 7 days at 30 °C. The FAMEs were extracted and 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 appropriate detection reagents (Komagata & Suzuki, 1987). 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. Strain SW-150^T contained ubiquinone-10 (Q-10) as the predominant respiratory lipoquinone, at a peak area ratio of approximately 91 %. The major components (>1 %) of the fatty acids detected in strain SW-150^T were unsaturated fatty acids C_18 : 1ω7c (33·8 %), C_17 : 1ω6c (18·8 %), 10-methyl-C_18 : 1ω7c (5·6 %) and C_17 : 1ω8c (2·4 %), straight-chain fatty acids C_16 : 0 (9·7 %), C_17 : 0 (3·9 %), C_15 : 0 (2·6 %) and C_14 : 0 (1·2 %), and hydroxy fatty acids C_14 : 0 2-OH (8·9 %), C_15 : 0 2-OH (4·2 %) and C_16 : 0 2-OH (1·1 %). This cellular fatty acid profile was similar to those of some Sphingopyxis species, particularly S. chilensis, although there are differences in the compositions of some fatty acids, which may be caused by different cultivation conditions (Takeuchi et al., 1993; Vancanneyt et al., 2001; Godoy et al., 2003; Yoon & Oh, 2005) (Table 1⇑). 3-Hydroxy fatty acids were not detected in strain SW-150^T, which was also found for other Sphingopyxis species and all species that were previously assigned to the genus Sphingomonas (Yabuuchi et al., 1990; Takeuchi et al., 1993, 1995; Lee et al., 2001; Vancanneyt et al., 2001; Busse et al., 2003; Godoy et al., 2003). The major polar lipids detected in strain SW-150^T were sphingoglycolipid, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine; minor amounts of phosphatidylmonomethylethanolamine, an unidentified glycolipid and unidentified phospholipids were also present. This polar lipid composition is similar to those of four Sphingopyxis species, the data of which have been reported by Kämpfer et al. (1997, 2002) and Yoon & Oh (2005); phosphatidylcholine, which was detected in three Sphingopyxis species with the exception of S. flavimaris, was not present (Kämpfer et al., 1997, 2002; Yoon & Oh, 2005). The DNA G+C content of strain SW-150^T was 63 mol%. The results obtained from chemotaxonomic analyses, showing the properties that are characteristic of the genus Sphingopyxis, were in agreement with the result of phylogenetic classification based on 16S rRNA gene sequences (Takeuchi et al., 1993; Kämpfer et al., 2002; Vancanneyt et al., 2001; Godoy et al., 2003).

There are not enough phenotypic, particularly chemotaxonomic, properties to distinguish strain SW-150^T from the genus Sphingopyxis (Takeuchi et al., 1993; Lee et al., 2001; Vancanneyt et al., 2001; Godoy et al., 2003). The orange pigmentation of strain SW-150^T is not observed in Sphingopyxis species. However, three orange-pigmented species, Sphingomonas aurantiaca, Sphingomonas aerolata and Sphingomonas faeni, were recently assigned to the genus Sphingomonas, which had previously comprised only yellow-pigmented species (Busse et al., 2003). Accordingly, it seems reasonable to assign strain SW-150^T within the genus Sphingopyxis. The phylogenetic distinctiveness shown in this study is low enough to exclude the possibility of assigning strain SW-150^T to a Sphingopyxis species with a validly published name, despite the lack of DNA–DNA hybridization experiments between strain SW-150^T and other Sphingopyxis species (Stackebrandt & Goebel, 1994). There are differences between strain SW-150^T and the other six Sphingopyxis species in some phenotypic properties, including pigmentation, nitrate reduction and utilization of some substrates (Table 1⇑). In particular, it is noteworthy that nitrate reduction has not been shown in other recognized Sphingopyxis species (Table 1⇑). Therefore, on the basis of the data presented, strain SW-150^T should be placed in the genus Sphingopyxis in a novel species, for which the name Sphingopyxis baekryungensis sp. nov. is proposed.