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Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejeon, 305-701, South Korea
Correspondence
Sung-Taik Lee
e_stlee{at}kaist.ac.kr
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ABSTRACT |
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7c/9t/12t) and C15 : 0 as the major fatty acids] supported the affiliation of strain KMY02T to the genus Burkholderia. The results of DNA–DNA hybridization experiments and physiological and biochemical tests allowed genotypic and phenotypic differentiation of the strain from recognized Burkholderia species. Therefore, KMY02T (=KCTC 12388T=NBRC 100964T) represents the type strain of a novel species, for which the name Burkholderia terrae sp. nov. is proposed.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain KMY02T is AB201285.
A transmission electron micrograph of a cell of strain KMY02T and a table giving levels of DNA–DNA hybridization between this strain and the type strains of closely related Burkholderia species are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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, and at the time of writing contains 38 species. These organisms have been isolated from diverse ecological niches, ranging from contaminated soils to the human respiratory tract (Coenye & Vandamme, 2003). During the characterization of bacteria isolated from a forest soil near the Korea Advanced Institute of Science and Technology (KAIST), strain KMY02T was cultivated on R2A agar at 30 °C, and was then subjected to a taxonomic investigation. The aim of this study was to determine the taxonomic position of the strain based on phenotypic, genotypic and chemotaxonomic characteristics and 16S rRNA gene sequence analysis. The results provide evidence that strain KMY02T represents a novel species within the genus Burkholderia. Strain KMY02T was isolated from a broad-leaved forest soil collected near KAIST, Daejeon, South Korea. The forest soil was homogenized by using an Ace homogenizer (Nihonseiki Kaisha). The suspension was spread on R2A agar plates (Difco) after being serially diluted 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 on to new plates and incubating again under the same conditions. The isolate was routinely cultured on R2A agar at 30 °C and maintained as a glycerol suspension (20 %, w/v) at –70 °C.
Genomic DNA was extracted using a commercial kit (Solgent), and PCR-mediated amplification of the 16S rRNA gene and sequencing of the purified PCR product were carried out according to the methods described by Kim et al. (2005). Full sequences of the 16S rRNA gene were compiled using SeqMan software (DNASTAR). 16S rRNA gene sequences of related taxa were obtained from the GenBank database. Multiple alignments were performed by using the CLUSTAL_X program (Thompson et al., 1997), and gaps were edited in the BIOEDIT program (Hall, 1999). Evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983). Phylogenetic trees were constructed by using the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1972) methods, using the MEGA3 program (Kumar et al., 2004) and with bootstrap values based on 1000 replications (Felsenstein, 1985).
A nearly complete 16S rRNA gene sequence of strain KMY02T was obtained (1470 bp). Preliminary sequence comparison against 16S rRNA gene sequences deposited in the GenBank database indicated that the isolate belonged to the family Burkholderiaceae of the Betaproteobacteria. On the basis of 16S rRNA gene sequence similarity, the closest cultured relatives to strain KMY02T were Burkholderia hospita LMG 20598T (98·7 %), Burkholderia caribensis LMG 18531T (98·0 %) and Burkholderia phymatum LMG 21445T (97·4 %). This relationship between strain KMY02T and other members of the genus Burkholderia was also evident in the phylogenetic tree (Fig. 1). Strain KMY02T and the three type strains above formed a monophyletic clade with a high bootstrap value (95 %), and this was supported by the neighbour-joining and maximum-parsimony algorithms.
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. Cell morphology was examined by light microscopy (Nikon) and transmission electron microscopy (Carl Zeiss) after negative staining with 1 % (w/v) phosphotungstic acid. Catalase and oxidase tests were performed by using the procedures outlined by Cappuccino & Sherman (2002). Substrate utilization as sole carbon source and several other physiological characteristics were determined with the API 32GN, API 20NE and API ZYM galleries according to the instructions of the manufacturer (bioMérieux). Tests for anaerobic growth were performed in a serum bottle containing R2A broth supplemented with thioglycolate (1 g l–1) and in which the upper air layer was substituted with nitrogen gas. Nitrate and nitrite reduction tests were performed in serum bottles containing R2A broth supplemented with KNO3 (10 mM) and NaNO2 (10 mM), respectively; reduction was monitored on an ion chromatograph (model 790 personal IC; Metrohm) equipped with a conductivity detector and anion exchange column (Metrosep Anion Supp 4; Metrohm). Nitrogen-fixing ability was determined by growth in 50 ml of a nitrogen-free medium (DSMZ medium no. 3) contained in a 500 ml Erlenmeyer flask. The primer system PolF–PolR (Poly et al., 2001) was used to amplify the nifH gene according to the methods described by Im et al. (2004). Degradation of DNA [using DNA agar (Difco) supplemented with 0·01 % toluidine blue (Merck)], chitin, CM-cellulose, starch (Atlas, 1993), lipid (Kouker & Jaeger, 1987) and xylan (Ten et al., 2004) was 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. Antibiotic-sensitivity tests were done using filter-paper discs containing the following: streptomycin (5, 10 and 15 µg ml–1), tetracycline (5, 10 and 15 µg ml–1), kanamycin (1·0, 1·5 and 2·0 mg ml–1) and ampicillin (20, 30 and 50 µg ml–1) (Sigma). Discs were placed on R2A plates spread with KMY02T culture and were then incubated at 30 °C for 5 days. All the phenotypic tests described above were performed in duplicate.
Cells of strain KMY02T were Gram-negative, slightly curved rods, and were motile by means of a single polar flagellum (see Supplementary Fig. S1 in IJSEM Online). Nitrogen fixation determined by growth in nitrogen-free medium and a nifH gene was positive. Physiological characteristics of strain KMY02T are summarized in the species description, and comparison of selective characteristics with those of the type strains of its most closely related species is given in Table 1.
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by using reversed-phase HPLC. Cellular fatty acids were analysed in organisms grown on 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 GC (Hewlett Packard 6890) were identified using the Microbial Identification software package (Sasser, 1990).
Total DNA for determination of the G+C content was extracted from cells grown on a nutrient agar plate (Difco) using the method described by Ausubel et al. (1995). RNA in the DNA solution was removed by incubation with a mixture of RNase A and T1 (each at 20 units ml–1) at 30 °C for 1 h. The G+C content of the total DNA was analysed as described by Mesbah et al. (1989) using reversed-phase HPLC. DNA–DNA hybridization was performed fluorometrically according to the method of Ezaki et al. (1989), using photobiotin-labelled DNA probes and microdilution wells.
Ubiquinone Q-8 was detected as the predominant quinone system in strain KMY02T; this is also the case in all other species of the genus Burkholderia. The cellular fatty acid profile of strain KMY02T included C16 : 0 (28·1 %), C17 : 0 cyclo (23·4 %), summed feature 7 (C18 : 17c/9t/12t, 11·8 %) and C15 : 0 (7·5 %). Significant differences were found between strain KMY02T and the other Burkholderia species investigated with regard to the fatty acid profile; the proportion of hydroxyl fatty acids is lower in strain KMY02T and it has C15 : 0 as a major component (Table 2).
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). The level of DNA–DNA relatedness between strain KMY02T and the type strains of B. hospita, B. caribensis and B. phymatum was 28, 36 and 22 %, respectively (see Supplementary Table S1 in IJSEM Online). Levels of DNA–DNA hybridization were determined to be less than 70 %, which is the threshold used to delineate a genomic species (Stackebrandt & Goebel, 1994). The results therefore support the designation of strain KMY02T as representing a separate, previously unrecognized species. On the basis of its morphological, physiological and chemotaxonomic characteristics, together with data from 16S rRNA gene sequence comparisons, strain KMY02T should be placed in the genus Burkholderia as a novel species, for which the name Burkholderia terrae sp. nov. is proposed.
Description of Burkholderia terrae sp. nov.
Burkholderia terrae (ter'rae. L. gen. n. terrae of the earth).
Cells are Gram-negative, slightly curved rods, 1·6–2·0 µm long by 0·6–0·8 µm wide, motile by means of a single polar flagellum. Colonies grown on R2A are circular, convex and cream-coloured. Temperature range for growth is 25–30 °C; no growth occurs at 42 °C. Growth occurs in the absence of NaCl and in the presence of 1·5 % (w/v) NaCl, but not above 3·0 % (w/v) NaCl. Nitrate is not reduced. Nitrogen fixation is positive. Catalase, oxidase, arginine dihydrolase, urease and 
-galactosidase activities are positive. Tryptophanase and -glucosidase activities are negative. Positive for assimilation of mannose, gluconate, caprate, phenylacetate, mannitol, D-glucose, L-fucose, D-sorbitol, L-arabinose, malate, histidine, 2-ketogluconate, 3-hydroxybutyrate, L-proline, rhamnose, N-acetylglucosamine, D-ribose, inositol, suberate, DL-lactate, L-alanine, 5-ketogluconate and L-serine. Negative for assimilation of adipate propionate, valerate, sucrose, itaconate and 3-hydroxybenzonate. Positive for alkaline phosphatase, esterase lipase C8, leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase; negative for esterase C4, lipase C14, valine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase. Resistant to 20 µg ampicillin ml–1 and 5 µg tetracycline ml–1 but susceptible to 1 mg kanamycin ml–1 and 5 µg streptomycin ml–1. Predominant ubiquinone is Q-8. The major fatty acids are C16 : 0, C17 : 0 cyclo, summed feature 7 (C18 : 17c/9t/12t) and C15 : 0. The G+C content of the genomic DNA is 62 mol%.
The type strain is KMY02T (=KCTC 12388T=NBRC 100964T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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