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Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Republic of Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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7c, C14 : 0 2-OH and C16 : 0 as the major fatty acids. Major polar lipids were sphingoglycolipid, phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content was 67.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DS-28T belonged to the genus Sphingomonas. Levels of 16S rRNA gene sequence similarity between strain DS-28T and the type strains of Sphingomonas species were in the range 93.3–98.3 %. DNA–DNA relatedness data and differential phenotypic properties, together with the phylogenetic distinctiveness, demonstrated that strain DS-28T is distinguishable from recognized Sphingomonas species. On the basis of phenotypic, phylogenetic and genetic data, strain DS-28T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas insulae sp. nov. is proposed. The type strain is DS-28T (=KCTC 12872T=JCM 14603T).
A two-dimensional thin-layer chromatogram of polar lipids of Sphingomonas insulae DS-28T is available with the online version of this paper.
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with five sphingosine-containing species and two genomospecies, and subsequently the number of Sphingomonas species has been increased considerably by reclassifications of some species belonging to other genera and descriptions of novel species (Takeuchi et al., 1993, 1995; Kämpfer et al., 1997; Denner et al., 1999; Yabuuchi et al., 1999; Lee et al., 2001; Busse et al., 2003, 2005; others). Meanwhile, Takeuchi et al. (2001) proposed that Sphingomonas species should be assigned to three new genera, Sphingobium, Novosphingobium and Sphingopyxis, in addition to the genus Sphingomonas sensu stricto. At the time of writing, the genus Sphingomonas comprises at least 36 species with validly published names, including the recently described species Sphingomonas molluscorum (Romanenko et al., 2007), Sphingomonas desiccabilis and Sphingomonas mucosissima (Reddy & Garcia-Piche, 2007), Sphingomonas pseudosanguinis (Kämpfer et al., 2007), Sphingomonas jaspsi (Asker et al., 2007), Sphingomonas kaistensis (Kim et al., 2007) and Sphingomonas fennica and Sphingomonas haloaromaticamans (Wittich et al., 2007). Here we report on the taxonomic characterization of a Sphingomonas-like bacterial strain, DS-28T, which was isolated from a soil sample from Dokdo in Korea.
Strain DS-28T was isolated by means of the standard dilution plating technique at 25 °C on 10x diluted nutrient agar (NA; Difco). The type strains of six Sphingomonas species used for DNA–DNA hybridization were Sphingomonas aquatilis KCTC 2881T, Sphingomonas asaccharolytica KCTC 2825T, Sphingomonas mali KCTC 2826T, Sphingomonas pruni KCTC 2824T, Sphingomonas melonis DSM 14444T and Sphingomonas oligophenolica JCM 12082T. Morphological, physiological and biochemical characteristics of strain DS-28T were investigated using routine cultivation at 25 °C on NA. Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy. The Gram reaction was determined using the bioMérieux Gram stain kit according to the manufacturer's instructions. Growth at various temperatures (4–40 °C) was measured on NA. Growth in the absence of NaCl and growth at various NaCl concentrations (0.5 %, w/v, and 1.0–5.0 %, w/v, at intervals of 1.0 %) were investigated in trypticase soy broth prepared according to the formula of the Difco medium, except that NaCl was excluded from the medium formula. The pH range for growth was determined in nutrient broth (NB; Difco) that was adjusted to various pH values (pH 4.5–10.5 at intervals of 0.5 pH units). The pH was adjusted prior to sterilization to various levels by the addition of HCl or Na2CO3. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on NA and on NA supplemented with nitrate, both of which had been prepared anaerobically using nitrogen. Catalase and oxidase activities and hydrolysis of casein, gelatin, hypoxanthine, starch, Tweens 20, 40, 60 and 80, tyrosine, urea and xanthine were determined as described by Cowan & Steel (1965). Hydrolysis of aesculin and nitrate reduction were studied as described by Lanyi (1987) and susceptibility to antibiotics was investigated on NA plates. Utilization of various substrates, enzyme activities and other physiological and biochemical properties were tested by using the API 20E, API 20NE, API 50CH and API ZYM systems (bioMérieux); utilization of various substrates was determined by inoculating the API 50CH strip with cells suspended in AUX medium (bioMérieux).
Cell biomass for DNA extraction and for the analysis of isoprenoid quinones and polar lipids was obtained from cultures grown for 3 days in NB at 25 °C. Chromosomal DNA was extracted 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 by RNA. The 16S rRNA gene was amplified and sequenced, and phylogenetic analysis was performed as described previously (Yoon et al., 1998, 2003). 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. For fatty acid methyl ester analysis, cells were harvested from NA plates after incubation for 7 days at 25 °C. The 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). Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and analysed using reversed-phase HPLC and a YMC ODS-A (250x4.6 mm) column. Polar lipids were extracted according to the procedures described by Minnikin et al. (1984) and identified by two-dimensional TLC followed by spraying with appropriate detection reagents (Minnikin et al., 1984; Komagata & Suzuki, 1987). The presence of phosphatidylcholine was identified by spraying Dragendorff's reagent. DNA–DNA hybridization was performed fluorometrically by the method of Ezaki et al. (1989) using photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed with five replications for each sample. The highest and lowest values obtained in each sample were excluded, and the means of the remaining three values were quoted as DNA–DNA relatedness values.
Morphological, physiological and biochemical characteristics of strain DS-28T are given in the species description or shown in Table 1. The almost complete 16S rRNA gene sequence of strain DS-28T, comprising 1447 nt (approx. 96 % of the Escherichia coli 16S rRNA sequence), was determined in this study. Comparative 16S rRNA gene sequence analyses showed that strain DS-28T was phylogenetically most closely related to the genus Sphingomonas (Fig. 1). In the phylogenetic tree based on the neighbour-joining algorithm, strain DS-28T fell within the radiation of the cluster comprising Sphingomonas species (Fig. 1). The phylogenetic affiliation of strain DS-28T to the genus Sphingomonas was also recovered in the trees based on the maximum-likelihood and maximum-parsimony algorithms (data not shown). Strain DS-28T exhibited 16S rRNA gene sequence similarity values of 93.3 % (S. wittichi) to 98.3 % (S. melonis and S. aquatilis) to the type strains of Sphingomonas species.
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; Takeuchi et al., 1995, 2001; Lee et al., 2001; Buonaurio et al., 2002; Busse et al., 2005; Ohta et al., 2004; Yoon et al., 2006). The fatty acid profile of strain DS-28T was composed of the following (each constituting >0.5 % of total fatty acids): unsaturated fatty acids C18 : 17c (44.2 %), C17 : 16c (8.4 %), C18 : 15c (2.5 %), C16 : 15c (2.4 %) and C17 : 18c (1.4 %); straight-chain fatty acids C16 : 0 (12.0 %), C15 : 0 (1.0 %), C17 : 0 (0.8 %) and C14 : 0 (0.7 %); hydroxy fatty acids C14 : 0 2-OH (12.7 %), C18 : 1 2-OH (1.9 %) and C15 : 0 2-OH (1.2 %); summed feature 3 comprising C16 : 17c and/or iso-C15 : 0 2-OH (6.4 %); and 11-methyl C18 : 17c (4.0 %). This fatty acid profile was similar to those of Sphingomonas species (Yabuuchi et al., 1990; Takeuchi et al., 1995, 2001; Lee et al., 2001; Buonaurio et al., 2002; Busse et al., 2005; Ohta et al., 2004; Yoon et al., 2006). Major polar lipids found in strain DS-28T were sphingoglycolipid, phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine; minor amounts of diphosphatidylglycerol, an unidentified phospholipid and an unidentified lipid were also found (see Supplementary Fig. S1, available with the online version of this paper). The polar lipid profile of strain DS-28T is distinguishable from those of Sphingomonas asaccharolytica, Sphingomonas mali and Sphingomonas pruni, in that major amounts of phosphatidylcholine are present and phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine are absent (Kämpfer et al., 1997). The DNA G+C content of strain DS-28T was 67.0 mol%.
Strain DS-28T exhibited mean DNA–DNA relatedness levels of 12–23 % to the type strains of six Sphingomonas species that showed 16S rRNA gene sequence similarity values of greater than 97 % to strain DS-28T, i.e. S. aquatilis KCTC 2881T (17 %), S. asaccharolytica KCTC 2825T (12 %), S. mali KCTC 2826T (12 %), S. pruni KCTC 2824T (15 %), S. melonis DSM 14444T (23 %) and S. oligophenolica JCM 12082T (17 %). Strain DS-28T differed from phylogenetically related Sphingomonas species by several phenotypic characteristics as shown in Table 1
. The phylogenetic distinctiveness, together with DNA–DNA relatedness data and differential phenotypic properties, was sufficient to allocate strain DS-28T to a species that is distinct from recognized Sphingomonas species (Wayne et al., 1987; Stackebrandt & Goebel, 1994). Therefore, on the basis of the data presented, strain DS-28T should be placed in the genus Sphingomonas as the type strain of a novel species, for which the name Sphingomonas insulae sp. nov. is proposed.
Description of Sphingomonas insulae sp. nov.
Sphingomonas insulae (in.su'lae. L. fem. gen. n. insulae of an island, referring to the source of isolation of the type strain).
Cells are Gram-negative rods (0.3–0.6 µmx1.0–5.0 µm), motile by means of peritrichous flagella. Colonies on NA are circular, convex, smooth, glistening, yellow in colour and 1.0–1.5 mm in diameter after 7 days of incubation at 25 °C. Optimal temperature for growth is 25 °C. Growth occurs at 4 and 34 °C, but not at 35 °C. Optimal pH for growth is around 6.5; growth occurs at pH 5.0 and 8.5, but not at pH 4.5 or 9.0. Growth occurs in the presence of 0–2 % (w/v) NaCl with an optimum of 0–0.5 % (w/v). Anaerobic growth does not occur on NA or NA supplemented with nitrate. Catalase-positive. Casein and Tweens 20, 40 and 60 are hydrolysed, but hypoxanthine, xanthine, starch and L-tyrosine are not. H2S is not produced. Lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. Amygdalin, arbutin, aesculin, lactose and L-fucose are utilized as sole carbon and energy sources, but erythritol, D-arabinose, D-ribose, L-xylose, adonitol, methyl-β-D-xyloside, sorbose, dulcitol, inositol, mannitol, sorbitol, methyl-
-D-mannoside, methyl--D-glucoside, inulin, melezitose, starch, glycogen, xylitol, D-lyxose, D-tagatose, D-fucose, D-arabitol, L-arabitol, 2-ketogluconate and 5-ketogluconate are not. Acid is produced from L-arabinose, but not from D-mannitol, inositol, D-sorbitol, sucrose, melibiose or amygdalin. In assays with the API ZYM system, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase and acid phosphatase are present and alkaline phosphatase is weakly present, but lipase (C14), cystine arylamidase, trypsin, -chymotrypsin, naphthol-AS-BI-phosphohydrolase, -galactosidase, β-glucuronidase, -glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, -mannosidase and -fucosidase are absent. Susceptible to cephalothin (30 µg), chloramphenicol (100 µg), gentamicin (30 µg), novobiocin (5 µg), polymyxin B (100 U), streptomycin (50 µg), tetracycline (30 µg), kanamycin (30 µg) and neomycin (30 µg), and weakly to carbenicillin (100 µg), but not to ampicillin (10 µg), lincomycin (15 µg), oleandomycin (15 µg) or penicillin G (20 U). The predominant ubiquinone is Q-10. The major fatty acids (>10 % of total fatty acids) are C18 : 17c, C14 : 0 2-OH and C16 : 0. Major polar lipids are sphingoglycolipid, phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content is 67.0 mol% (determined by HPLC). Other phenotypic characteristics are given in Table 1.
The type strain, DS-28T (=KCTC 12872T=JCM 14603T), was isolated from the soil of Dokdo, Korea.
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ACKNOWLEDGEMENTS |
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