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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
Tae-Kwang Oh
otk{at}kribb.re.kr
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ABSTRACT |
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7c, C16 : 0, C14 : 0 2-OH and C16 : 17c and/or iso-C15 : 0 2-OH as the major fatty acids. Sphingoglycolipid, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and unidentified phospholipid were the major polar lipids. The DNA G+C content was 66.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DS-4T fell within the evolutionary radiation comprising Sphingomonas species. Levels of 16S rRNA gene sequence similarity between strain DS-4T and the type strains of Sphingomonas species ranged from 93.0 to 97.6 %. DNA–DNA relatedness data and differential phenotypic properties, together with the phylogenetic distinctiveness, demonstrated that strain DS-4T differs from the recognized Sphingomonas species. On the basis of phenotypic, phylogenetic and genetic data, this strain represents a novel species of the genus Sphingomonas, for which the name Sphingomonas dokdonensis sp. nov. is proposed, with DS-4T (=KCTC 12541T=CIP 108841T) as the type strain.
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, 2006). In this study, we report on the detailed taxonomic characterization of a yellow-pigmented Sphingomonas-like bacterial strain, DS-4T, which was considered to belong to the class Proteobacteria from the results of 16S rRNA gene sequence analysis. Soil samples collected in Dokdo (37° 14' 12'' N 131° 52' 07'' E) provided the source for isolation of bacterial strains. Strain DS-4T was isolated by the standard dilution plating technique at 25 °C on nutrient agar (Difco). The type strains of six Sphingomonas species were used as reference strains for DNA–DNA hybridization. Sphingomonas aquatilis KCTC 2881T, Sphingomonas asaccharolytica KCTC 2825T, Sphingomonas koreensis KCTC 2882T, Sphingomonas mali KCTC 2826T and Sphingomonas pruni KCTC 2824T were obtained from the Korean Collection for Type Cultures (KCTC), Taejon, Korea. Sphingomonas melonis DSM 14444T was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany.
Morphological, physiological and biochemical characteristics of strain DS-4T were investigated using routine cultivation at 25 °C on trypticase soy agar (TSA; Difco) with the pH adjusted to 6.5. Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy. The presence of flagella was determined by transmission electron microscopy using cells from exponentially growing cultures. For transmission electron microscopic observation, cells were negatively stained with 1 % (w/v) phosphotungstic acid and after air-drying the grids were examined with a Philips CM-20 transmission electron microscope. 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 TSA (pH 6.5). Growth in the absence of NaCl and growth at various NaCl concentrations [0.5 % (w/v) and 1.0–10.0 % (w/v) at intervals of 1.0 %] were investigated in trypticase soy broth prepared according to the formula of the Difco medium except that no NaCl was used. The pH range for growth was determined in nutrient broth (Difco) adjusted to various pH values (pH 4.5–10.5 at intervals of 0.5). The pH was adjusted to various levels by the addition of HCl or Na2CO3 prior to sterilization. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on TSA (pH 6.5) and on TSA (pH 6.5) supplemented with nitrate, both of which had been prepared anaerobically using nitrogen. Catalase and oxidase activities and hydrolysis of casein, gelatin, hypoxanthine, starch, Tween 20, Tween 40, Tween 60 and Tween 80, tyrosine, urea and xanthine were determined as described by Cowan & Steel (1965). Hydrolysis of aesculin and nitrate reduction were studied as described by Lányi (1987). Utilization of substrates as sole carbon and energy sources was tested according to the method of Yurkov et al. (1994) and by using the API 20NE system (bioMérieux). Sensitivity to antibiotics was tested on TSA (pH 6.5) plates using antibiotic discs containing the following: 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; tetracycline, 30 µg; kanamycin, 30 µg; lincomycin, 15 µg; oleandomycin, 15 µg; neomycin, 30 µg; carbenicillin, 100 µg. Enzyme activity and other physiological properties were tested using the API ZYM and API 20E systems (bioMérieux).
Cell biomass for DNA extraction and for isoprenoid quinone and polar lipid analyses was obtained from cultures grown in trypticase soy broth (Difco) 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 with RNA. 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 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, the cell mass of strain DS-4T was harvested from TSA (pH 6.5) plates after incubation for 4 days at 25 °C. 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 with 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.
The morphological, cultural, physiological and biochemical characteristics of strain DS-4T are given in the species description (see later) and are shown in Table 1. The almost complete 16S rRNA gene sequence (1441 nt) of strain DS-4T was determined in this study. In the phylogenetic tree based on the neighbour-joining algorithm, strain DS-4T fell within the radiation of the cluster comprising Sphingomonas species (Fig. 1). The phylogenetic affiliation of strain DS-4T to the genus Sphingomonas was also recovered in trees based on the maximum-likelihood and maximum-parsimony algorithms (data not shown). Levels of 16S rRNA gene sequence similarity between strain DS-4T and the type strains of Sphingomonas species ranged from 93.0 % (Sphingomonas taejonensis JSS54T) to 97.6 % (Sphingomonas asaccharolytica IFO 15499T).
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; Takeuchi et al., 1995, 2001; Lee et al., 2001; Busse et al., 2003; Ohta et al., 2004). The fatty acid profile of strain DS-4T comprised unsaturated fatty acids C18 : 17c (45.7 %) and C16 : 15c (1.3 %), straight-chain fatty acids C16 : 0 (19.0 %) and C14 : 0 (0.8 %), summed feature 3 comprising C16 : 17c and/or iso-C15 : 0 2-OH (14.8 %), hydroxy fatty acid C14 : 0 2-OH (12.1 %) and 11-methyl C18 : 17c (6.3 %). 3-Hydroxy fatty acids were not detected in strain DS-4T. This fatty acid profile was similar to those of Sphingomonas species (Yabuuchi et al., 1990; Takeuchi et al., 1995, 2001; Lee et al., 2001; Busse et al., 2003; Ohta et al., 2004). The major polar lipids were sphingoglycolipid, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and unidentified phospholipid (Fig. 2). Phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine were not detected in strain DS-4T (Fig. 2). The polar lipid profile of strain DS-4T was distinguishable from those of Sphingomonas assaccharolytica, Sphingomonas mali and Sphingomonas pruni in that phosphatidylcholine was present as a major polar lipid and phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine were absent (Kämpfer et al., 1997). The DNA G+C content of strain DS-4T was 66.9 mol%.
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). The phylogenetic distinctiveness, together with DNA–DNA relatedness data and differential phenotypic properties, are sufficient to categorize strain DS-4T as a member of a species that is distinct from the recognized Sphingomonas species (Wayne et al., 1987; Stackebrandt & Goebel, 1994). Therefore, on the basis of the data presented, strain DS-4T should be placed in the genus Sphingomonas as a novel species, for which the name Sphingomonas dokdonensis sp. nov. is proposed.
Description of Sphingomonas dokdonensis sp. nov.
Sphingomonas dokdonensis (dok.do.nen'sis. N.L. fem. adj. dokdonensis of Dokdo, Korea, where the organism was first isolated).
Cells are Gram-negative rods (0.4–0.6x0.8–3.5 µm). Motile by means of a single polar flagellum. Colonies on TSA (pH 6.5) are circular, convex, smooth, glistening, yellow in colour and 0.8–1.0 mm in diameter after 4 days of incubation at 25 °C. Optimal temperature for growth is 25 °C. Growth occurs at 10 and 34 °C, but not at 4 and 35 °C. Optimal pH for growth is between 6.0 and 6.5; growth occurs at pH 5.0 and 9.5, but not at pH 4.5 and 10.0. Growth occurs in the presence of 0–5 % (w/v) NaCl; optimal growth occurs without NaCl. Anaerobic growth does not occur on TSA (pH 6.5) or on TSA (pH 6.5) supplemented with nitrate. Catalase- and oxidase-positive. Casein, starch and Tween 20, Tween 40, Tween 60 and Tween 80 are hydrolysed, but hypoxanthine, xanthine and L-tyrosine are not. H2S is not produced. Lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. Pyruvate is utilized as a sole carbon and energy source, but D-mannitol, benzoate, formate, phenylacetate, succinate and L-glutamate are not. In assays with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase are present and cystine arylamidase is weakly present, but lipase (C14), trypsin, 
-chymotrypsin, -galactosidase, 
-glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are absent. Susceptible to chloramphenicol, gentamicin, tetracycline, kanamycin, neomycin and oleandomycin, but not to polymyxin B, streptomycin, penicillin G, ampicillin, cephalothin, novobiocin, carbenicillin or lincomycin. The predominant ubiquinone is Q-10. The major fatty acids (>10 % of total fatty acids) are C18 : 17c, C16 : 0, C14 : 0 2-OH and C16 : 17c and/or iso-C15 : 0 2-OH. Major polar lipids are sphingoglycolipid, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and unidentified phospholipid. The DNA G+C content is 66.9 mol% (determined by HPLC). Other phenotypic characteristics are given in Table 1.
The type strain, DS-4T (=KCTC 12541T=CIP 108841T), was isolated from soil.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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