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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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ABSTRACT |
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9c as the major fatty acids. The DNA G+C content was 65.1 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain DS-16T joined the cluster comprising Stenotrophomonas species. The levels of 16S rRNA gene sequence similarity between strain DS-16T and the type strains of Stenotrophomonas species ranged from 95.5 to 97.5 %. DNA–DNA relatedness data and differential phenotypic properties, together with the phylogenetic distinctiveness of strain DS-16T, demonstrated that this novel strain differs from Stenotrophomonas species with validly published names. On the basis of phenotypic, phylogenetic and genetic data, strain DS-16T (=KCTC 12543T=CIP 108839T) should be classified in the genus Stenotrophomonas as a member of a novel species, for which the name Stenotrophomonas dokdonensis sp. nov. is proposed.
Phenotypic characteristics of Stenotrophomonas dokdonensis sp. nov. and the type strains of other Stenotrophomonas species are provided in a supplementary table available in IJSEM Online.
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). At the time of writing, the genus comprises four species with validly published names: Stenotrophomonas maltophila (Palleroni & Bradbury, 1993), Stenotrophomonas nitritireducens (Finkmann et al., 2000), Stenotrophomonas acidaminiphila (Assih et al., 2002) and Stenotrophomonas rhizophila (Wolf et al., 2002). In this study, we report on the taxonomic characterization of a Stenotrophomonas-like bacterial strain, DS-16T, which was isolated from a soil sample from Dokdo island (37° 14' 12'' N, 131° 52' 07'' E), Korea.
Strain DS-16T was isolated by means of the standard dilution plating technique at 25 °C on 10x diluted nutrient agar (Difco). The type strains of four Stenotrophomonas species were used as reference strains: S. maltophila KCTC 1773T, S. nitritireducens KCTC 12168T, S. acidaminiphila KCTC 12167T and S. rhizophila KCTC 12169T were obtained from the Korean Collection for Type Cultures, Taejon, Korea. The morphological, physiological and biochemical characteristics of strain DS-16T were investigated using routine cultivation on trypticase soy agar (TSA; Difco) at 30 °C. The cell morphology was examined by using light microscopy (E600; Nikon) and transmission electron microscopy. The presence of flagella was determined with a transmission electron microscope (CM-20; Philips), using cells from exponentially growing cultures. For transmission electron microscopic observation, the cells were negatively stained with 1 % (w/v) phosphotungstic acid and the grids were air-dried. The Gram reaction was determined using the bioMérieux Gram stain kit according to the manufacturer's instructions. Growth at various temperatures (4–45 °C) was measured on TSA. Growth in the absence of NaCl and at various NaCl concentrations [0.5 % (w/v) and 1.0–5.0 % (w/v) using 1.0 % increments] was investigated using trypticase soy broth prepared according to the formula of the Difco medium except that no NaCl was included. The pH range for growth was determined in nutrient broth (Difco) adjusted to various pH values (pH 4.5–10.5, using increments of 0.5 pH units) prior to sterilization by the addition of HCl or Na2CO3. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on TSA and on TSA supplemented with nitrate, both of which had been prepared anaerobically using nitrogen. Catalase and oxidase activities and the hydrolysis of casein, gelatin, hypoxanthine, starch, Tweens 20, 40, 60 and 80, tyrosine, urea and xanthine were investigated as described by Cowan & Steel (1965). Hydrolysis of aesculin and nitrate reduction were studied as described previously (Lanyi, 1987). Sensitivity to antibiotics was tested on TSA plates using antibiotic discs containing the following concentrations: 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; and carbenicillin, 100 µg. Assimilation of various substrates and enzyme activity were tested by using the API 20E, API 20NE, API 50CH and API ZYM systems (bioMérieux). The cells were suspended in AUX medium according to the manufacturer's instructions to inoculate the API 50CH system.
Cell biomass for DNA extraction and for isoprenoid quinone analysis was obtained from cultures grown in trypticase soy broth at 30 °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). 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. For fatty acid methyl ester analysis, cell mass of strain DS-16T was harvested from TSA plates after incubation for 3 days at 30 °C. The fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). The DNA G+C content was determined by using the method of Tamaoka & Komagata (1984) with the modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC. 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, cultural, physiological and biochemical characteristics of strain DS-16T are given in the species description (see later) or are shown in Table 1 and Supplementary Table S1 in IJSEM Online. The almost complete 16S rRNA gene sequence of strain DS-16T determined in this study comprised 1500 nt, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. In the phylogenetic tree based on the neighbour-joining algorithm, strain DS-16T joined the clade comprising Stenotrophomonas species at a bootstrap confidence value of 77.2 % (Fig. 1). The relationships between strain DS-16T and Stenotrophomonas species were also recovered in the trees based on the maximum-likelihood and maximum-parsimony algorithms (Fig. 1). Strain DS-16T exhibited a 16S rRNA gene sequence similarity value of 97.5 % with respect to S. rhizophila e-p10T and similarities of 95.5–95.9 % to the type strains of the other Stenotrophomonas species. Sequence similarities to all other species included in the phylogenetic analysis were below 97.0 % (Fig. 1). The fatty acid profile of strain DS-16T comprised the following: branched fatty acids iso-C16 : 0 (27.6 %), iso-C15 : 0 (19.6 %), iso-C17 : 19c (11.9 %), iso-C11 : 0 (5.6 %), anteiso-C15 : 0 (3.4 %), iso-C17 : 0 (3.4 %), iso-C14 : 0 (2.1 %) and anteiso-C17 : 0 (1.5 %); hydroxy fatty acids iso-C11 : 0 3-OH (4.1 %) and iso-C12 : 0 3-OH (2.8 %); unsaturated fatty acids C18 : 17c (1.5 %) and C16 : 17c alcohol (1.0 %); straight-chain fatty acid C16 : 0 (1.8 %); and summed feature 3, comprising C16 : 17c and/or iso-C15 : 0 2-OH (7.8 %). This fatty acid profile was similar to those of Stenotrophomonas species (Finkmann et al., 2000; Assih et al., 2002; Wolf et al., 2002). The predominant respiratory lipoquinone detected in strain DS-16T was Q-8, at a peak area ratio of approximately 98 %. The DNA G+C content of strain DS-16T was 65.1 mol%. The mean DNA–DNA relatedness levels between strain DS-16T and the type strains of the four Stenotrophomonas species were in the range 9–21 %, indicating that these strains represent different genomic species (Wayne et al., 1987). Strain DS-16T differed from the recognized Stenotrophomonas species in several phenotypic characteristics (Table 1). The phylogenetic distinctiveness and differential phenotypic properties were sufficient to categorize strain DS-16T as a member of a species that is distinct from previously recognized Stenotrophomonas species (Stackebrandt & Goebel, 1994). Therefore, on the basis of the data presented, strain DS-16T should be placed in the genus Stenotrophomonas as a member of a novel species, for which the name Stenotrophomonas dokdonensis sp. nov. is proposed.
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Cells are Gram-negative, non-motile rods (0.2–0.4x0.7–2.6 µm). Colonies on TSA are circular, convex, sticky, glistening, cream–yellow in colour and 1.5–2.5 mm in diameter after 3 days incubation at 30 °C. Optimal temperature for growth is 30 °C. Growth occurs at 10 and 40 °C, but not at 4 or 41 °C. Optimal pH for growth is between 6.0 and 7.0; growth occurs at pH 5.5 and 8.5, but not at pH 5.0 or 9.0. Growth occurs in the presence of 0–6 % (w/v) NaCl; optimal growth occurs in the presence of 0.5 % (w/v) NaCl. Anaerobic growth does not occur on TSA or on TSA supplemented with nitrate. Catalase-positive. Urease-negative. Tweens 20, 40 and 60 are hydrolysed. H2S and indole are not produced. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. Susceptible to polymyxin B, chloramphenicol, cephalothin, carbenicillin and oleandomycin, but not to lincomycin. The predominant ubiquinone is Q-8. The major fatty acids (>10 % of total fatty acids) are iso-C16 : 0, iso-C15 : 0 and iso-C17 : 19c. The DNA G+C content is 65.1 mol% (determined by HPLC). Other phenotypic characteristics are given in Table 1
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The type strain, DS-16T (=KCTC 12543T=CIP 108839T), was isolated from soil.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of Medical Bacteria. London: Cambridge University Press.
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