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Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea
Correspondence
Sung-Taik Lee
e_stlee{at}kaist.ac.kr
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ABSTRACT |
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The fatty acid profile of Stenotrophomonas koreensis compared with other Stenotrophomonas species is available as a supplementary table in IJSEM online.
Present address: School of Civil, Urban and Geosystem Engineering, Seoul National University, Shillim-dong, Gwanak-gu, Seoul, Korea. 
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). One of these isolates (strain TR6-01T) was member of genus Stenotrophomonas in the class Gammaproteobacteria and was the subject of this taxonomic investigation.
The genus Stenotrophomonas was first described by Palleroni & Bradbury (1993) and, at present, the genus comprises the five species Stenotrophomonas acidaminiphila Assih et al. 2002, S. africana Drancourt et al. 1997, S. maltophilia (Hugh 1981) Palleroni and Bradbury 1993, S. nitritireducens Finkmann et al. 2000 and S. rhizophila Wolf et al. 2002.
In the present study, we conducted a phylogenetic analysis on the basis of 16S rRNA gene sequences, DNA–DNA hybridization tests and some important phenotypic characteristics to determine the precise taxonomic position of this strain. On the basis of the results obtained in this study, we propose that strain TR6-01T should be placed in the genus Stenotrophomonas as the type strain of a novel species.
Strain TR6-01T was originally isolated from a compost which was composed of cow dung and rice straw, near Daejeon city in South Korea. This compost sample was suspended with 50 mM phosphate buffer (pH 7·0) and the suspension was spread on R2A agar plates (Difco) after serial dilution with 50 mM phosphate buffer (pH 7·0). The plates were incubated at 30 °C for 2 weeks. Single colonies on the plates were purified by transferring them onto new plates and were incubated once again under the same conditions. The purified colonies were tentatively identified by partial sequences of the 16S rRNA gene (Im et al., 2003). TR6-01T was one of the dominant isolates on the R2A agar plates under aerobic conditions. This organism was deposited in the Korean Collection for Type Cultures as KCTC 12211T (=JCM 13256T). Other type strains of species of the genus Stenotrophomonas were obtained from the DSMZ or KCTC.
The Gram reaction was performed by the non-staining method as described by Buck (1982). Cell morphology was observed under a Nikon light microscope at x1000, with cells grown for 2 days at 30 °C on R2A agar. Catalase and oxidase tests were performed by the procedures outlined by Cappuccino & Sherman (2002). Substrate utilization as sole carbon source and some physiological characteristics were determined with API 32GN and API 20NE galleries according to the instructions of the manufacturer (bioMérieux). Anaerobic growth was performed in serum bottles adding thioglycolate (1 g l–1) to R2A broth and substituting the upper air layer with nitrogen gas. Nitrate and nitrite reduction were later confirmed by inoculating, in each case, into three serum bottles (25 ml) containing 13 ml R2A media, while nitrate and nitrite were added as KNO3 and NaNO2 at concentrations of 10 mM. The reduction of nitrate and nitrite was monitored by ion chromatograph (model 790 personal IC; Metrohm) equipped with a conductivity detector and anion exchange column (Metrosep Anion Supp 4; Metrohm). Degradation of DNA [using DNA agar (Difco) supplemented with 0·01 % toluidine blue (Merck)], degradation of casein, chitin, cellulose and starch (Atlas, 1993), degradation of lipids (Kouker & Jaeger, 1987) and degradation of xylan (Ten et al., 2004) were also investigated; reactions were read after 5 days. Growth at different temperatures and pH was assessed after 5 days incubation. Salt tolerance was tested on R2A medium supplemented with 1–10 % (w/v) NaCl after 5 days incubation. Duplicate antibiotic-sensitivity tests were done using filter-paper discs containing the following: streptomycin (5, 10 and 15 µg), tetracycline (5, 10 and 15 µg), kanamycin (1·0, 1·5 and 2·0 mg) and ampicillin (20, 30 and 50 µg) (Sigma). Discs were placed on R2A plates spread with TR6-01T culture and were then incubated at 30 °C for 5 days.
Extraction of genomic DNA, PCR-mediated amplification of the 16S rRNA gene and sequencing of purified PCR product were carried out according to Im et al. (2004). The 16S rRNA gene sequences of related taxa were obtained from GenBank. The multiple alignments were performed by CLUSTAL_X program (Thompson et al., 1997). Gaps were edited using the BIOEDIT program (Hall, 1999). The evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983). The phylogenetic tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987) using the MEGA2 program (Kumar et al., 2001) with bootstrap values based on 1000 replications (Felsenstein, 1985) (Fig. 1).
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using reversed-phase HPLC. Quinones were extracted from cells grown on nutrient broth (Difco) and analysed as described by Komagata & Suzuki (1987) using reversed-phase HPLC. Cellular fatty acids were analysed in organisms grown on a trypticase soy agar (TSA; Difco) for 2 days. The cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). The fatty acids analysed by a gas chromatograph (Hewlett Packard 6890) were identified by the Microbial ID software package (Sasser, 1990).
Cells of strain TR6-01T were aerobic, Gram-negative, non-motile rods. Colonies grown on R2A agar plates (Difco) for 2 days were smooth, circular, non-glossy, yellowish and 2–4 mm in diameter. On R2A agar, TR6-01T was able to grow at 20–37 °C, but not at 4 or 45 °C. Physiological characteristics of strain TR6-01T are summarized in the species description and comparisons of selective characteristics with related type strains are shown in Table 1.
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The DNA G+C content of strain TR6-01T was 66·0 mol%. Characterization of the respiratory quinone system supports the affiliation of TR6-01T with the Gammaproteobacteria, where the majority of species in the genus Xanthomonas and Stenotrophomonas have Q-8 as the major quinone (Finkmann et al., 2000). The fatty acid profile of strain TR6-01T (available in Supplementary Table S1 in IJSEM Online) was mainly composed of C15 : 0 iso (34·0 %), C15 : 1 iso F (18·1 %), C13 : 0 iso (8·9 %), C11 : 0 iso (6·8 %), C11 : 0 iso 3-OH (4·8 %), C14 : 0 iso (4·2 %), iso C17 : 1 
9c (3·3 %) and C13 : 0 iso 3-OH (3·1 %). A significant difference was found compared with other Stenotrophomonas species: the production of a large amount of the fatty acid C15 : 1 iso F (18·1 %). Moreover, S. acidaminiphila produced significantly larger amounts of C14 : 0 iso (17·8 %) and C16 : 0 iso (14·3 %) and S. rhizophila produced a significantly larger amount of C15 : 0 anteiso (23·8 %). Thus, fatty acid profiles can be used to differentiate the members of the genus Stenotrophomonas at the species level.
DNA–DNA hybridization experiments were performed between TR6-01T and type strains of the genus Stenotrophomonas with the method described by Ezaki et al. (1989) using photobiotin-labelled DNA probes and microdilution wells. DNA–DNA relatedness values of strain TR6-01T to the other species of genus Stenotrophomonas were 25–42 % (40 % to S. acidaminiphila DSM 13117T, 25 % to S. maltophilia KCTC 1773T, 42 % to S. nitritireducens DSM 12575T and 25 % S. rhizophila DSM 14405T), which is low enough to assign strain TR6-01T as a novel species of the genus Stenotrophomonas. On the basis of the data and observations described above, strain TR6-01T should be assigned to the genus Stenotrophomonas as the type strain of a novel species, for which the name Stenotrophomonas koreensis sp. nov. is proposed.
Description of Stenotrophomonas koreensis sp. nov.
Stenotrophomonas koreensis (ko.re.en'sis. N.L. fem. adj. koreensis pertaining to Korea, the location of the compost sample from which the type strain was isolated).
Cells are Gram-negative, strictly aerobic, non-motile, slightly curved rods, 0·2–0·4 µm in diameter and 1·5–2·0 µm in length. Colonies grown on R2A agar (Difco) for 2 days are smooth, circular, non-glossy, yellowish and convex. Grows well at pH 6·0–8·5 and at 20–37 °C, but does not grow at 4 or 45 °C. Growth occurs in the presence of 0–2·0 % (w/v) NaCl, but not 4 % (w/v) NaCl. It cannot reduce nitrate and nitrite or grow anaerobically. It cannot degrade xylan, chitin, cellulose or starch but can degrade DNA. Substrate utilization, enzyme production, acid production and other physiological characteristics are shown in Table 1
. Resistant to discs containing 15 µg tetracycline and 15 µg streptomycin, but sensitive to 20 µg ampicillin and 1·0 mg kanamycin. Q-8 is the predominant ubiquinone and C15 : 0 iso, C15 : 1 iso F, C13 : 0 iso and C11 : 0 iso are the major cellular fatty acids. The G+C content of genomic DNA is 66·0 mol% (as determined by HPLC).
The type strain, TR6-01T (=KCTC 12211T=JCM 13256T), was isolated from a compost consisting of cow dung and rice straw, near Daejeon City, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Atlas, R. M. (1993). Handbook of Microbiological Media. Edited by L. C. Parks. Boca Raton, FL: CRC Press.
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