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1 Korean Agricultural Culture Collection, Microbial Genetics Division, National Institute of Agricultural Biotechnology, National Institute of Agricultural Science and Technology, Rural Development Administration, Suwon 441-707, Korea
2 Applied Microbiology Division, National Institute of Agricultural Science and Technology, Rural Development Administration, Suwon 441-707, Korea
3 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
Correspondence
Soon-Wo Kwon
swkwon{at}rda.go.kr
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7c, summed feature 3 (C16 : 17c/C15 : 0 iso 2-OH) and C16 : 0 (together representing 71.2 % of the total). The 16S rRNA gene sequence similarities between strain GP25-8T and members of the genus Burkholderia ranged from 94.7 to 97.4 %, indicating that this novel strain was phylogenetically related to members of that genus. The novel strain showed the highest sequence similarities to Burkholderia caryophylli ATCC 25418T (97.4 %) and Burkholderia phenazinium LMG 2247T (97.2 %); the levels of DNA–DNA hybridization with these strains were 28 and 12 %, respectively. These results support the conclusion that strain GP25-8T represents a novel species within the genus Burkholderia, for which the name Burkholderia soli sp. nov. is proposed. The type strain is GP25-8T (=KACC 11589T=DSM 18235T).
A transmission electron micrograph of a cell of strain GP25-8T is available as a supplementary figure in IJSEM Online.
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) was created through the transfer of the former Pseudomonas rRNA homology group II. Burkholderia species are isolated from very diverse ecological niches (Coenye & Vandamme, 2003) and, to date, more than 30 species with validly published names have been reported, Burkholderia cepacia being the type species. In the course of a study of bacterial diversity in fields cultivated with Korean ginseng (Panax ginseng C. A. Meyer), a bacterial strain was isolated in the Eumseong region of Korea. A soil sample was serially diluted with 0.85 % NaCl (w/v) and suitable 10-fold dilutions were plated onto R2A agar (Difco). The plates were incubated at 28 °C for 4 days and strain GP25-8T was isolated.
The almost-complete 16S rRNA gene of strain GP25-8T (approx. 1400 bp) was amplified and sequenced as described by Weon et al. (2005). Alignment of 16S rRNA gene sequences was performed with the CLUSTAL W program (Thompson et al., 1994). A phylogenetic tree (Fig. 1) was constructed by using the neighbour-joining method (Saitou & Nei, 1987) with Kimura's two-parameter calculation model (Kimura, 1980). The phylogenetic tree (Fig. 1) based on 16S rRNA gene sequences clearly indicated that strain GP25-8T was related to members of the genus Burkholderia. Strain GP25-8T showed the highest levels of sequence similarity (100 and 99.1 %, respectively) with Burkholderia species A6.2 (GenBank accession number AF247491), isolated from a polycyclic aromatic hydrocarbon-contaminated soil in the USA (Friedrich et al., 2000) and Burkholderia species Ellin155 (AF408997), isolated from a pasture soil in Australia (Schoenborn et al., 2004). The sequence similarities between strain GP25-8T and the validly described Burkholderia species ranged from 94.7 to 97.4 %, showing >97 % sequence similarity only with Burkholderia caryophylli (97.4 %) and Burkholderia phenazinium (97.2 %). The phylogenetic tree indicated that strain GP25-8T clustered with B. caryophylli ATCC 25418T with a relatively high bootstrap value (90 %) and this cluster grouped with another cluster, including Burkholderia phenazinium LMG 2247T, at a bootstrap level of 88 %.
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. Probe-labelling was conducted by using the non-radioactive DIG-High prime system (Roche), and hybridized DNA was visualized using the DIG luminescence detection kit (Roche). DNA–DNA relatedness was quantified by using a densitometer (Bio-Rad). The value for DNA–DNA hybridization between strains GP25-8T and B. caryophylli LMG 2155T was 28 %, while that between strain GP25-8T and B. phenazinium DSM 10684T was 12 %.
Cellular fatty acid contents were determined for cell mass grown on trypticase soy agar at 28 °C for 48 h. Fatty acid methyl esters were extracted and prepared according to standard protocols, as described for the MIDI Microbial Identification System (Sasser, 1990), and analysed with the MIDI Microbial Identification system. The fatty acid content of strain GP25-8T was generally consistent with that of B. caryophylli LMG 2155T, although strain GP25-8T differs in that it contains a small amount of C12 : 0 and lacks C14 : 0 (Table 1). DNA G+C contents were determined by using an HPLC-based method, as described by Mesbah et al. (1989), with a reverse-phase column (Supelcosil LC-18-S; Supelco). The DNA G+C content of strain GP25-8T was 64.9 mol%.
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. Cell morphology was observed by transmission electron microscopy (model 912AB; LEO) and phase-contrast microscopy (Axio; Zeiss) after incubation for 1 day on R2A agar. Growth under anaerobic conditions was determined in BBL anaerobic agar (Becton Dickinson). Catalase activity, oxidase activity, indole production, the Voges–Proskauer reaction and the hydrolysis of casein, DNA, gelatin, starch and Tween 80 were tested by using standard procedures (Smibert & Krieg, 1994). The hydrolysis of carboxymethylcellulose (0.1 %), chitin from crab shells (1 %, w/v), pectin (0.5 %, w/v) and tyrosine (0.5 %, w/v) was also tested. Growth at different temperatures and pH values was assessed after 14 days incubation. Salt tolerance was tested on R2A medium supplemented with 1–10 % (w/v) NaCl after 14 days incubation. Several other physiological characteristics and the utilization of various substrates as sole carbon sources were determined with the API ID 32GN, API 20NE and API ZYM galleries, according to the manufacturer's instructions (bioMérieux). The oxidation of various substrates was also determined by using the Biolog GN2 MicroPlate assay as recommended by the manufacturer. API ZYM tests were read after 4 h incubation at 37 °C; the other API tests and Biolog microplates were read after 48 h at 28 °C.
The cells of strain GP25-8T were 0.5–0.75x1.6–3.9 µm in size, Gram-negative, aerobic and non-motile (see Supplementary Fig. S1, available in IJSEM Online). The colonies were milky, convex and circular with clear margins. Growth was observed at temperatures in the range 10–40 °C and at pH 4–8. The isolate grew on R2A, nutrient agar (Difco) and trypticase soy agar (Difco), but did not grow on MacConkey agar (Difco). The differential phenotypic characteristics of strain GP25-8T and closely related Burkholderia species are shown in Table 1
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On the basis of the above results, it is proposed that strain GP25-8T represents a novel species in the genus Burkholderia, for which the name Burkholderia soli sp. nov. is proposed.
Description of Burkholderia soli sp. nov.
Burkholderia soli (so'li. L. gen. n. soli of soil, the source of the type strain).
Colonies are milky, convex and circular with clear margins. Cells are approximately 0.5–0.75 µm wide and 1.6–3.9 µm long. Gram-negative, strictly aerobic, rod-shaped and non-motile. Growth occurs at 10–40 °C (optimum, 28 °C), pH 4–8 (optimum, pH 6–7) and 0–2 % (w/v) NaCl. Catalase- and oxidase-positive. Negative for indole production and in the Voges–Proskauer test. Tween 80 and tyrosine are hydrolysed, but casein, chitin, carboxymethylcellulose, DNA, gelatin, pectin and starch are not. The following are oxidized: Tweens 40 and 80, N-acetyl-D-glucosamine, D-fructose, 
-D-glucose, D-mannitol, pyruvic acid methyl ester, itaconic acid, bromosuccinic acid, D-alanine, L-alanine, L-alanyl glycine, L-asparagine, L-glutamic acid, L-histidine, L-phenylalanine, urocanic acid, glycerol and D-glucose 6-phosphate. The following are not oxidized: -cyclodextrin, dextrin, glycogen, N-acetyl-D-galactosamine, adonitol, L-arabinose, D-arabitol, D-cellobiose, i-erythritol, L-fucose, D-galactose, gentiobiose, myo-inositol, -D-lactose, lactulose, maltose, D-mannose, D-melibiose, methyl 
-D-glucoside, D-psicose, D-raffinose, L-rhamnose, D-sorbitol, sucrose, D-trehalose, turanose, xylitol, succinic acid monomethyl ester, acetic acid, cis-aconitic acid, citric acid, formic acid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, -hydroxybutyric acid, -hydroxybutyric acid, 
-hydroxybutyric acid, p-hydroxyphenylacetic acid, -ketobutyric acid, -ketoglutaric acid, -ketovaleric acid, DL-lactic acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, sebacic acid, succinic acid, succinamic acid, glucuronamide, L-alaninamide, L-aspartic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, hydroxy-L-proline, L-leucine, L-ornithine, L-proline, L-pyroglutamic acid, D-serine, L-serine, L-threonine, DL-carnitine, -aminobutyric acid, inosine, uridine, thymidine, phenylethylamine, putrescine, 2-aminoethanol, 2,3-butanediol, DL--glycerol phosphate and -D-glucose 1-phosphate (Biolog GN microplate). Assimilates N-acetylglucosamine, D-ribose, inositol, itaconic acid, lactic acid, L-alanine, glycogen, D-mannitol, D-glucose, L-fucose, D-sorbitol, L-arabinose, propionic acid, L-histidine, 3-hydroxybutyric acid and L-proline, but does not assimilate L-rhamnose, D-sucrose, D-maltose, suberic acid, sodium malonate, sodium acetate, potassium 5-ketogluconate, 3-hydroxybenzoic acid, L-serine, salicin, D-melibiose, capric acid, valeric acid, trisodium citrate, potassium 2-ketogluconate or 4-hydroxybenzoic acid (API ID 32GN; bioMérieux). The major fatty acids are C18 : 17c, summed feature 3 and C16 : 0 (representing 71.2 % of the total). The DNA G+C content is 64.9 mol% (determined by HPLC). Other phenotypic characteristics are given in Table 1.
The type strain, GP25-8T (=KACC 11589T=DSM 18235T), was isolated in Korea from soil cultivated with Korean ginseng.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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