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1 Department of Bio-Environmental Chemistry, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 305-764, Republic of Korea
2 Department of Biology and Medicinal Sciences, Pai Chai University, Daejeon 302-735, Republic of Korea
3 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea
Correspondence
Min-Ho Yoon
mhyoon{at}cnu.ac.kr
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ABSTRACT |
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An extended 16S rRNA gene sequence-based neighbour-joining tree is available as supplementary material with the online version of this paper.
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MAIN TEXT |
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, 1993). When the genus was first described, it comprised 11 species, including Paenibacillus polymyxa as the type species. Currently, the genus Paenibacillus encompasses over 70 species (Euzéby, 1997; updated January 2007). Members of the genus Paenibacillus are aerobic or facultatively anaerobic organisms with typically Gram-positive cell walls and produce ellipsoidal endospores in swollen sporangia. The DNA G+C contents range from 39 to 54 mol% and anteiso-C15 : 0 is the major cellular fatty acid (Shida et al., 1997). During the course of an investigation of the culturable aerobic and facultatively anaerobic bacterial community in soil from a ginseng field in Pocheon Province, South Korea, a large number of bacteria were isolated. In this study, we have characterized one of these isolates, strain Gsoil 139T. Phenotypic, chemotaxonomic and phylogenetic analyses have established the affiliation of this isolate to the genus Paenibacillus, and the data obtained in this work suggest that the isolate represents a novel species of this genus.
Strain Gsoil 139T was originally isolated from soil from a ginseng field in Pocheon Province. The soil sample was suspended in 50 mM phosphate buffer (l–1: 5.355 g K2HPO4, 2.62 g KH2PO4; pH 7.0) and serial decimal dilutions of the suspension were spread-plated onto modified R2A agar plates containing the following (l–1): 0.25 g tryptone, 0.25 g peptone, 0.25 g yeast extract, 0.125 g malt extract, 0.125 g beef extract, 0.25 g Casamino acids, 0.25 g soytone, 0.5 g glucose, 0.3 g soluble starch, 0.2 g xylan, 0.3 g sodium pyruvate, 0.3 g K2HPO4, 0.05 g MgSO4, 0.05 g CaCl2 and 15 g agar. The plates were then incubated at 30 °C for 1 month. On the 107-diluted plate, 40–50 colonies appeared, of which strain Gsoil 139T was one. Single colonies were purified by transferring them onto new plates (modified R2A medium) and repeated incubation. Strain Gsoil 139T was one of the isolates that appeared on the modified R2A agar plates under aerobic conditions. It was routinely cultured on R2A agar (Difco) at 30 °C and maintained as a glycerol suspension (20 %, w/v) at –70 °C.
For phylogenetic analysis of strain Gsoil 139T, DNA was extracted using a genomic DNA extraction kit (Core Biosystems); the 16S rRNA gene was amplified by a PCR and then sequencing of the purified PCR product was carried out according to Kim et al. (2005). The complete 16S rRNA gene sequence was 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). Gaps were edited in the BioEdit program (Hall, 1999). Evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983) and phylogenetic trees were constructed by using the neighbour-joining method (Saitou & Nei, 1987) and the maximum-parsimony method (Fitch, 1971) using the MEGA3 program (Kumar et al., 2004) with bootstrap values based on 1000 replications (Felsenstein, 1985).
Comparative analysis of the 16S rRNA gene sequence of strain Gsoil 139T (1469 bp) showed that this strain is phylogenetically affiliated to Paenibacillus species. The phylogenetic tree (Fig. 1) based on the neighbour-joining algorithm showed that strain Gsoil 139T fell within the radiation of the cluster comprising Paenibacillus species (see the expanded phylogenetic tree; Supplementary Fig. S1 available in IJSEM Online), joining Paenibacillus hodogayensis JCM 12520T with a bootstrap resampling value of 99.0 %. The closest phylogenetic neighbours of strain Gsoil 139T are P. hodogayensis SGT (95.6 %) and Paenibacillus koleovorans TBT (93.8 %). The 16S rRNA gene sequence similarity to type strains of all other Paenibacillus species with validly published names was below 93.8 %; according to Wayne et al. (1987), the phylogenetic definition of a species requires strains to show approximately 
70 % DNA–DNA relatedness. The available data indicate that organisms having less than 97.0 % 16S rRNA gene sequence similarity will not exhibit more than 60 % reassociation, irrespective of the hybridization method applied (Stackebrandt & Goebel, 1994; Keswani & Whitman, 2001). This phylogenetic result demonstrated that strain Gsoil 139T was not related to any previously described Paenibacillus taxa at the species level.
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. Cell morphology and motility were observed with a Nikon light microscope (x1000 magnification) using the hanging drop technique with cells allowed to grow on R2A agar for 3 days at 30 °C. Catalase and oxidase tests were performed as outlined by Cappuccino & Sherman (2002). Acid production from 49 carbon sources was tested at 30 °C with API 50 CH in combination with API 50 CHB/E medium (bioMérieux), which was used for inoculum preparation, and evaluated after 2 days. Growth at a variety of temperatures (4, 15, 18, 25, 30, 37, 42 and 45 °C) was assessed on R2A agar and at pH 5.0–10.0 (in increments of 0.5 pH units) was assessed in R2A broth. Some physiological characteristics and the utilization of various substrates as sole carbon sources were determined using API 32GN, API 20NE and API 20E galleries according to the instructions of the manufacturer (bioMérieux). Tests for the degradation of DNA [in which DNase agar (Scharlau) plates were flooded with 1 M HCl], casein, chitin, starch (Atlas, 1993), olive oil (Kouker & Jaeger, 1987), xylan and cellulose (Ten et al., 2004) were performed and evaluated after 7 days. Salt tolerance was tested on R2A medium supplemented with 1–10 % (w/v) NaCl after 5 days incubation. Growth on nutrient agar, trypticase soy agar (TSA; Difco) and MacConkey agar was also evaluated at 30 °C.
Cells of strain Gsoil 139T were Gram-positive, non-motile rods, 0.3–0.5 µm in diameter and 1.5–3.0 µm long after 2 days culture on R2A agar. They formed oval spores positioned centrally in swollen sporangia. Colonies grown on R2A agar plates for 2 days were 0.5–1.5 mm in diameter, circular, convex, non-glossy and white. On R2A agar, the optimum temperature for growth was 37 °C. Strain Gsoil 139T showed oxidase and catalase activities and hydrolysed DNA weakly. Physiological characteristics that can be used to differentiate strain Gsoil 139T from its closest phylogenetic relatives (P. hodogayensis and P. koleovorans) are listed in Table 1.
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and degraded enzymically into nucleosides; the DNA G+C content was then determined as described by Mesbah et al. (1989) using reversed-phase HPLC. Isoprenoid quinones were extracted with chloroform/methanol (2 : 1, v/v), evaporated under vacuum conditions and re-extracted in n-hexane/water (1 : 1, v/v). The crude quinone in n-hexane was purified using silica Sep-Pak Vac cartridges (Waters) and subsequently analysed by HPLC, as described previously (Hiraishi et al., 1996). Cellular fatty acid profiles were determined for strains grown on TSA for 3 days at 30 °C. The cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). The fatty acids were then analysed by gas chromatography (6890; Hewlett Packard) using the Microbial Identification software package (Sasser, 1990). Duplicate experiments were performed.
The DNA G+C content of strain Gsoil 139T was 48.1 mol%, which lies within the range observed for members of the genus Paenibacillus. The respiratory quinone system present in Gsoil 139T supports its affiliation with the genus Paenibacillus, in which most species have MK-7 as the major quinone (Shida et al., 1997). The fatty acids found in isolate Gsoil 139T are shown in Table 2 and are compared with values available for phylogenetically related Paenibacillus strains. Anteiso-branched C15 : 0, the major fatty acid present in members of the genus Paenibacillus (Shida et al., 1997), was also the major fatty acid component of strain Gsoil 139T, comprising 27.9 % of the total. However, some qualitative and quantitative differences in fatty acid content could be observed between strain Gsoil 139T and the phylogenetically closest relatives. The next most predominant fatty acid found in strain Gsoil 139T was iso-C16 : 0, comprising 26.8 %. This value is much higher than those reported for the reference species (Table 2).
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Description of Paenibacillus ginsengarvi sp. nov.
Paenibacillus ginsengarvi (gin.seng.ar'vi. N.L. n. ginsengum ginseng; L. n. arvum a field; N.L. gen. n. ginsengarvi of a ginseng field, the source of the type strain).
Cells are Gram-positive, non-motile rods that form oval spores positioned centrally in swollen sporangia. Oxidase and catalase reactions are positive. Nitrate is not reduced to nitrite. Anaerobic growth does not occur. Grows between 18 and 45 °C (optimum 37 °C) and at pH 5.0–8.5 (optimum pH 6.5–7.0). Tolerates NaCl at 2 % (w/v) but not 3 %. Growth occurs on TSA and nutrient agar but not on MacConkey agar. Hydrolyses DNA weakly, but does not hydrolyse chitin, starch, cellulose, xylan, lipids or casein. Positive for 
-glucosidase and -galactosidase. The following substrates are utilized for growth: D-glucose, maltose, D-melibiose, D-ribose, sucrose, salicin and gluconate. The following substrates are not utilized for growth: D-mannose, L-fucose, L-rhamnose, L-arabinose, propionate, caprate, phenylacetate, 3-hydroxybenzoate, 4-hydroxybenzoate, malonate, acetate, 3-hydroxybutylate, valerate, citrate, lactate, malate, 5-ketogluconate, 2-ketoglutarate, itaconate, adipate, suberate, D-sorbitol, D-mannitol, inositol, glycogen, N-acetyl-D-glucosamine, gelatin, urea, nitrate, L-serine, L-tryptophan, L-alanine, L-arginine, L-histidine and L-proline. Positive for -galactosidase and the Voges–Proskauer test in API 20E tests. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, urease, gelatin hydrolysis, indole and hydrogen sulphide production are negative. Acids are produced from glycerol, D-arabinose, L-arabinose, D-ribose, D-xylose, methyl -D-xyloside, D-glucose, methyl 
-D-glucoside, amygdalin, arbutin, D-cellobiose, maltose, D-melibiose, sucrose, trehalose, D-raffinose and D-turanose. Acids are not produced from erythritol, L-xylose, adonitol, D-galactose, D-fructose, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl -D-mannoside, N-acetyl-D-glucosamine, salicin, D-lactose, inulin, D-melezitose, starch, glycogen, xylitol, -gentiobiose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. MK-7 is the predominant menaquinone. The major fatty acids are anteiso-C15 : 0, iso-C16 : 0 and iso-C15 : 0. The G+C content of the genomic DNA of the type strain is 48.1 mol%.
The type strain, Gsoil 139T (=KCTC 13059T =DSM 18677T), was isolated from soil from a ginseng field in Pocheon Province, South Korea.
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ACKNOWLEDGEMENTS |
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