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-glucosidase-producing bacterium isolated from soil of a ginseng field
1 Department of Oriental Medicinal Material and Processing, College of Life Science, Kyung Hee University, 1 Seocheon-dong, Giheung-gu, Yongin-si, Gyeonggi-do 446-701, South Korea
2 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, South Korea
3 Institute of Food Science and Engineering, Yantai University, Yantai 264005, People's Republic of China
Correspondence
Deok-Chun Yang
dcyang{at}khu.ac.kr
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ABSTRACT |
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-glucosidase activity was isolated from soil from a ginseng field in South Korea and was characterized in order to determine its taxonomic position. The bacterium was found to comprise Gram-negative, rod-shaped, motile cells with unipolar polytrichous flagella. On the basis of 16S rRNA gene sequence similarity, strain KMY03T was shown to belong to the family Burkholderiaceae of the Betaproteobacteria, being most closely related to Burkholderia caledonica LMG 19076T (97.8 %), Burkholderia terricola LMG 20594T (97.5 %), Burkholderia xenovorans LMG 21463T (97.4 %) and Burkholderia phytofirmans LMG 22146T (97.3 %). Chemotaxonomic data (major ubiquinone, Q-8; major fatty acids, C17 : 0 cyclo, C16 : 0, C19 : 0 cyclo 
8c and summed feature 2) supported the affiliation of the novel strain with the genus Burkholderia. The results of DNA–DNA hybridizations and physiological and biochemical tests allowed the strain to be differentiated genotypically and phenotypically from Burkholderia species with validly published names. On the basis of these data, strain KMY03T represents a novel species of the genus Burkholderia, for which the name Burkholderia ginsengisoli sp. nov. is proposed. The type strain is KMY03T (=KCTC 12389T=NBRC 100965T).
A negatively stained transmission electron micrograph of strain KMY03T is available as a supplementary figure in IJSEM Online.
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MAIN TEXT |
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-glucosidase-producing bacterial strains were isolated (An et al., 2005
). In this study, one of these isolates, designated strain KMY03T, was subjected to phenotypic, genotypic and chemotaxonomic investigations in order to determine its taxonomic position. The results provide evidence that strain KMY03T represents a novel species within the genus Burkholderia. This genus was first described by Yabuuchi et al. (1992) and currently contains 39 species, which have been isolated from diverse ecological niches ranging from contaminated soils to the respiratory tracts of humans (Coenye & Vandamme, 2003). Strain KMY03T was isolated, via direct plating onto R2A agar (Difco), from soil from a ginseng field near Daejeon in South Korea. The resulting colonies were purified by transferring them onto new plates and subjecting them to an additional incubation for 3 days at 30 °C. Purified colonies were tentatively identified from partial 16S rRNA gene sequences.
The Gram reaction was performed by using the non-staining method described by Buck (1982). Cell morphology was examined by using 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). Utilization of substrates as sole carbon sources 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. Tests for the reduction of nitrate and nitrite 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 an anion exchange column (Metrosep Anion Supp 4; Metrohm). Nitrogen-fixing ability was determined from growth in 50 ml nitrogen-free medium (DSMZ medium no. 3) contained in a 500 ml Erlenmeyer flask. Acetylene reduction was examined for all liquid cultures by injecting purified acetylene into appropriate containers closed with rubber stoppers to yield 15 % acetylene (v/v); this was followed by incubation for up to 24 h. Ethylene was measured using a Hewlett Packard 5890A gas chromatograph equipped with a flame-ionization detector and a prepacked column (HayeSep N; Supelco). The primer system PolF–PolR (Poly et al., 2001) was used to amplify a 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 values 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 performed 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 culture of strain KMY03T and were then incubated at 30 °C for 5 days. All of the phenotypic tests described above were performed in duplicate.
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). The 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 with the BioEdit program (Hall, 1999). Evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983). Phylogenetic trees were constructed by using a neighbour-joining method (Saitou & Nei, 1987) and with the maximum-parsimony method (Fitch, 1971) using MEGA 3 (Kumar et al., 2004) and with bootstrap values based on 1000 replications (Felsenstein, 1985).
Quinones were extracted from cells grown on nutrient broth (Difco) and then analysed as described by Komagata & Suzuki (1987) by using reversed-phase HPLC. 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 RNase T1 (20 U ml–1 in each case) 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 reassociation was performed fluorometrically at 50 °C according to the method of Ezaki et al. (1989), using photobiotin-labelled DNA probes and microdilution wells. The prehybridization solution and the hybridization solution contained 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), Denhardt's solution and 50 % formamide.
The cells of strain KMY03T were found to be Gram-negative, rod-shaped and motile by means of unipolar polytrichous flagella (see Supplementary Fig. S1 available in IJSEM Online). Colonies grown on R2A were circular, convex and cream-coloured. The temperature range for growth was 25–42 °C; no growth occurred at 45 °C. An internal region of the nifH gene (about 360 bp) was clearly amplified, and strain KMY03T was able to grow well in a nitrogen-free liquid medium. The physiological characteristics of strain KMY03T are summarized in the species description; Table 1 presents a comparison between selected characteristics of strain KMY03T and the most closely related type strains of the genus Burkholderia.
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). Lower levels of sequence similarity (<97.0 %) were found with respect to all other recognized species of the genus Burkholderia.
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8c (15.1 %) and summed feature 2 (C14 : 0 3-OH and/or iso-C16 : 1 I, 11.7 %). Significant differences in fatty acid profiles were observed between strain KMY03T and the other type strains of Burkholderia species, e.g. the production of a large amount of the fatty acid C17 : 0 cyclo (Table 2). The DNA G+C content of strain KMY03T was 61.6 mol%, which is quite similar to the values for members of the genus Burkholderia (Table 1
).
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On the basis of morphological, physiological and chemotaxonomic characteristics, together with data from 16S rRNA gene sequence comparisons, strain KMY03T represents a novel member of the genus Burkholderia, for which the name Burkholderia ginsengisoli sp. nov. is proposed.
Description of Burkholderia ginsengisoli sp. nov.
Burkholderia ginsengisoli (gin.sen.gi.so'li. N.L. n. ginsengum ginseng; L. n. solum soil; N.L. gen. n. ginsengisoli of soil of a ginseng field, the source of the organism).
Cells are Gram-negative rods, 1.5–2.0 µm long by 0.3–0.6 µm wide, and motile by means of unipolar polytrichous flagella. Colonies grown on R2A are circular, convex and cream-coloured. Temperature range for growth is 25–42 °C; no growth occurs at 45 °C. Optimal growth temperature is 25–30 °C. Growth occurs in the absence of NaCl and in the presence of 1.5 % (w/v) NaCl, but not at NaCl concentrations above 3.0 % (w/v). Nitrate is not reduced. Catalase, arginine dihydrolase, urease, -galactosidase and -glucosidase activities are present. Oxidase and tryptophanase activities are absent. The type strain is positive for the assimilation of adipate, L-malate, phenylacetate, salicin, L-fucose, D-sorbitol, propionate, histidine, 2-ketogluconate, L-proline, rhamnose, inositol, DL-lactate, L-alanine and L-serine and negative for the assimilation of caprate, citrate, D-melibiose, valerate, 3-hydroxybutyrate, 3-hydroxybenzoate, 4-hydroxybenzoate, D-ribose, sucrose, itaconate, suberate, acetate, 5-ketogluconate and glycogen. Alkaline phosphatase, esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and -glucuronidase activities are present; esterase (C4), lipase (C14), valine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, -galactosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are not detected. Resistant to (ml–1) ampicillin (20 µg) and tetracycline (5 µg) and sensitive to kanamycin (1 mg) and streptomycin (5 µg). Predominant ubiquinone is Q-8. The major fatty acids are C17 : 0 cyclo, C16 : 0, C19 : 0 cyclo 8c and summed feature 2. The G+C content of the genomic DNA is 61.6 mol%.
The type strain, KMY03T (=KCTC 12389T=NBRC 100965T), was isolated from soil from a ginseng field near Daejeon in South Korea.
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ACKNOWLEDGEMENTS |
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