Abstract
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Gsoil 1411T is AB245384.
An expanded phylogenetic tree based on 16S rRNA gene sequences of strain Gsoil 1411T and the type strains of representative members of the genus Paenibacillus is available as a supplementary figure in IJSEM Online.
Footnotes
,†,The genus Paenibacillus was defined in 1993 after an extensive comparative analysis of 16S rRNA gene sequences of 51 species of the genus Bacillus (Ash et al., 1991, 1993). At that time, the genus comprised 11 recognized species, with Paenibacillus polymyxa as the type species. At the time of writing, there are 69 recognized species within the genus Paenibacillus (). Members of the genus are aerobic, or facultatively anaerobic, organisms that produce ellipsoidal endospores in swollen sporangia and whose cell walls show structures typical of Gram-positive bacteria. The DNA G+C content ranges from 39 to 54 mol% and anteiso-C15 : 0 is the major cellular fatty acid (Shida et al., 1997a).
During the course of study on 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 1411T. Phenotypic, chemotaxonomic and phylogenetic analyses establish the affiliation of this isolate to the genus Paenibacillus. The data obtained also suggest that the isolate represents a novel species of this genus.
Strain Gsoil 1411T was originally isolated from soil from a ginseng field. The soil sample was suspended in 50 mM phosphate buffer (pH 7.0) and serial ten-fold dilutions of the suspension were spread on modified R2A agar plates (per litre: 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 C3H3NaO3, 0.3 g K2HPO4, 0.05 g MgSO4, 0.05 g CaCl2, 15 g agar). The plates were incubated at 30 °C for 1 month. Single colonies on the plates were purified by transferring them onto new plates and they were incubated again on modified R2A or half-strength modified R2A. The purified colonies were tentatively identified by partial sequencing of the 16S rRNA gene (Im et al., 2005). Strain Gsoil 1411T was one of the isolates which appeared on the modified R2A agar plates under aerobic conditions. It was routinely cultured on R2A agar at 30 °C and maintained as a glycerol suspension (20 %, w/v) at 70 °C.
For phylogenetic analysis of strain Gsoil 1411T, DNA was extracted using a genomic DNA extraction kit (Core Biosystem), the 16S rRNA gene was amplified by PCR and sequencing of the purified PCR product was carried out according to Kim et al. (2005). The full 16S rRNA gene sequences 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 CLUSTAL_X (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). 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).
The 16S rRNA gene sequence of strain Gsoil 1411T was a continuous stretch of 1487 bp. Comparative 16S rRNA gene sequence analyses showed that strain Gsoil 1411T is phylogenetically affiliated to species of the genus Paenibacillus. The phylogenetic tree based on the neighbour-joining algorithm (Fig. 1) showed that strain Gsoil 1411T appeared within the genus Paenibacillus, but occupied a distinct phylogenetic position (an expanded phylogenetic tree is available as Supplementary Fig. S1 in IJSEM Online). Species found to be closely related to strain Gsoil 1411T were Paenibacillus xylanilyticus XIL14T, Paenibacillus illinoisensis JCM 9907T, Paenibacillus pabuli NCIMB 12781T and Paenibacillus amylolyticus NRRL NRS-290T with 16S rRNA gene sequence similarity levels of 95.7, 95.2, 94.8 and 94.2 %, respectively.
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Gram reaction testing was performed by the non-staining method as described by Buck (1982). Cell morphology was observed under a Nikon light microscope at x1000 magnification, with cells grown for 3 days at 30 °C on R2A agar. Catalase and oxidase tests were performed as outlined by Cappuccino & Sherman (2002). For single carbon source assimilation studies, we used a defined liquid medium plus basal salt medium containing (per litre): 1.8 g K2HPO4, 1.08 g KH2PO4, 0.5 g NaNO3, 0.5 g NH4Cl, 0.1 g KCl, 0.1 g MgSO4 and 0.05 g CaCl2. To this medium, a vitamin solution (Widdel & Bak, 1992), trace element solution SL-10 (Widdel et al., 1983) and selenite/tungstate solution (Tschech & Pfennig, 1984) were added and the pH of the medium was adjusted to 6.8. This liquid medium was aliquoted (0.24 ml) into 96-well trays and filter-sterilized carbon sources (0.1 ml) were added to each well (individually at 0.1 %, w/v). Growth in the 96-well plates was examined visually after incubation at 30 °C for 7 days. Negative control wells did not contain any added carbon source. A positive control culture was grown in a well containing R2A medium. Fermentative acid production and oxidative acid production from carbohydrates were tested by growth in OF basal medium with bromothymol blue (Atlas, 1993) supplemented with 1 % carbohydrate [soft-agar stabs with (fermentative) and without (oxidative) sterile mineral oil overlay]. The OF medium tubes were incubated at 30 °C for 5 days. Tests for acid production from carbohydrates and some other phenotypic characteristics, as given in the species description below, were determined with API 20E galleries according to the manufacturer's instructions (bioMérieux). Anaerobic growth was tested in serum bottles with the addition of thioglycolate (1 g l1) to R2A broth and substituting the upper air layer with nitrogen gas. A test for anaerobic nitrate reduction, as the final electron acceptor, was performed in serum bottles with the addition of thioglycolate (1 g l1) to R2A broth and substituting the upper air layer with nitrogen gas, while nitrate was added as KNO3 at concentrations of 10 mM. Aerobic nitrate reduction was later confirmed by inoculations, in each case, into three serum bottles (25 ml) containing 12 ml R2A media, while nitrate was added as KNO3 at concentrations of 10 mM. Reduction of nitrate and nitrite was monitored by ion chromatography (model 790 personal IC; Metrohm) equipped with a conductivity detector and anion exchange column (Metrosep Anion Supp 4; Metrohm). Tests for degradation of DNA (DNase agar Scharlau by flooding plates with 1 M HCl), casein, chitin, starch (Atlas, 1993), lipid (Kouker & Jaeger, 1987), xylan and cellulose (Ten et al., 2004) were performed and evaluated after 7 days. Growth at different temperatures (4, 15, 25, 30, 37, 42 and 45 °C) and various pH values (pH 4.010.0 at intervals of 0.5 pH units) was assessed after 5 days incubation. Salt tolerance was tested on R2A medium supplemented with 110 % (w/v) NaCl after 5 days incubation. Growth on nutrient agar, trypticase soy agar (TSA) and MacConkey agar was also evaluated at 30 °C.
Cells of strain Gsoil 1411T were Gram-positive, motile rods that formed oval spores that lay subterminally in swollen sporangia. Peritrichous flagella were observed (Fig. 2). After 1 day on R2A, colonies were 0.51.0 mm in diameter, convex, irregular, undulate, non-glossy and slightly yellowish. On R2A agar, the optimum temperature for growth was 37 °C. Strain Gsoil 1411T showed oxidase and catalase activity and reduced nitrate to nitrite. Physiological characteristics that differentiate strain Gsoil 1411T from its closest phylogenetic relatives, P. xylanilyticus, P. illinoisensis, P. amylolyticus and P. pabuli, are listed in Table 1.
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Table 1. Differential phenotypic characteristics of strain Gsoil 1411T and the type strains of phylogenetically related Paenibacillus species Strains: 1, Gsoil 1411T; 2, P. xylanilyticus XIL14T (data from Rivas etal., 2005); 3, P. illinoisensis NRRL NRS-1356T (Shida et al., 1997b); 4, P. pabuli NRRL NRS-924T (Shida et al., 1997b); 5, P. amylolyticus NRRL NRS-290T (Shida et al., 1997a). All strains are positive for catalase activity and acid production from L-arabinose and D-mannitol, but are negative for citrate and L-malate utilization. All produce ellipsoidal or oval spores in swollen sporangia. +, Positive; , negative; V, variable; ND, no data; W, weakly positive.
For the measurement of G+C content of the chromosomal DNA, genomic DNA was extracted and purified as described by Moore & Dowhan (1995) and enzymically degraded into nucleosides. The G+C content of DNA was then determined as described by Mesbah et al. (1989) using reverse-phase HPLC. Isoprenoid quinones were extracted with chloroform/methanol (2 : 1, v/v), evaporated under vacuum conditions and reextracted in n-hexane/water (1 : 1, v/v). The crude quinone in n-hexane was purified using Sep-Pak Vac Cartridges Silica (Waters) and subsequently analysed by HPLC, as described by Hiraishi et al. (1996). Cellular fatty acids were analysed following growth on R2A 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 were then analysed by GC (Hewlett Packard 6890) using the Microbial Identification software package (Sasser, 1990).
The DNA G+C content of strain Gsoil 1411T was 53.9 mol%, which lies within the range of values reported for members of the genus Paenibacillus. Affiliation of Gsoil 1411T to the genus Paenibacillus was also supported based on analysis of the respiratory quinone system; the majority of species in the genus have MK-7 as the major quinone (Shida et al., 1997a). The fatty acid profile of isolate Gsoil 1411T is given in Table 2 and is compared with those of the type strains of phylogenetically related Paenibacillus species. Anteiso-branched C15 : 0, the major fatty acid found in members of the genus Paenibacillus (Shida et al., 1997a), was also the major fatty acid component of strain Gsoil 1411T, comprising 44.8 % of the total. However, some qualitative and quantitative differences in fatty acid content were observed between strain Gsoil 1411T and its phylogenetically closest relatives. The second most abundant fatty acid in strain Gsoil 1411T was iso-C16 : 0, comprising 20.2 % of the total. This value is much higher than that reported for the reference species tested (Table 2). We also found slight differences in the proportions of the other fatty acids.
Table 2. Cellular fatty acid profile (%) of strain Gsoil1411T and the type strains of phylogenetically related species of the genus Paenibacillus Strains: 1, Gsoil 1411T; 2, P. xylanilyticus XIL14T (data from Rivas et al., 2005); 3, P. illinoisensis NRRL NRS-1356T (Shida et al., 1997b); 3, P. pabuli NRRL NRS-924T (Shida et al., 1997a); 4, P. amylolyticus NRRL NRS-290T (Shida et al., 1997b). ND, Not detected; ECL, equivalent chain-length.
All characteristics determined for strain Gsoil 1411T are in accordance with those of the genus Paenibacillus. On the basis of its phylogenetic distance from recognized Paenibacillus species, as indicated by relatively low 16S rRNA gene sequence similarities (<96 %), and specific combination of phenotypic characteristics, it is clear that Gsoil 1411T is not affiliated to any recognized species of this genus. Therefore, on the basis of the data presented, strain Gsoil 1411T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus panacisoli sp. nov. is proposed.
Description of Paenibacillus panacisoli sp. nov.
Paenibacillus panacisoli (pa.na.ci.so'li. N.L. n. Panax -acis scientific name of ginseng; L. n. solum soil; N.L. gen. n. panacisoli of soil of a ginseng field).
Cells are Gram-positive, facultatively anaerobic, motile, spore-forming and rod-shaped with a length of approximately 2.05.0 µm and a width of 0.40.6 µm. Spores are oval, subterminal and occur in swollen sporangia. After 1 day on R2A, colonies are 0.51.0 mm in diameter, convex, irregular, undulate, non-glossy and slightly yellowish. Oxidase and catalase reactions are positive. Nitrate is reduced to nitrite. The optimum temperature for growth is 37 °C, the maximum growth temperature is between 42 and 45 °C, and the minimum growth temperature is between 15 and 20 °C. The minimum pH for growth is between 4.5 and 5.0, the optimum pH is 6.5 and the maximum pH is between 8.5 and 9.0. Tolerates 5 % (w/v) NaCl, but not 7 %. No growth occurs on TSA, MacConkey or nutrient agar. Hydrolyses xylan and casein (weakly), but not chitin, starch, cellulose, DNA or aesculin. Utilizes D-mannose, D-ribose, D-xylose, L-xylose, D-lactose, D-raffinose, gluconate (weakly), glycerol and inulin for growth, but not D-glucose, D-fructose, D-galactose, D-mannose, D-fucose, sucrose, D-trehalose, ethanol, L-rhamnose, L-sorbose, D-arabinose, L-arabinose, D-lyxose, formate, propionate, caprate, maleate, phenylacetate, benzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, malonate, pyruvate, acetate, 3-hydroxybutyrate, valerate, fumarate, salicin, citrate, lactate, malate, succinate, tartrate, glutarate, itaconate, adipate, suberate, oxalate, D-cellobiose, D-maltose, D-melibiose, D-raffinose, D-adonitol, dulcitol, D-sorbitol, D-mannitol, xylitol, inositol, amygdalin, methanol, glycogen, dextran, N-acetyl-D-glucosamine, L-cysteine, glycine, L-iso-leucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-tryptophan, L-tyrosine, L-valine, L-alanine, L-arginine, L-asparagine, L-aspartate, L-glutamate, L-glutamine, L-histidine, L-proline or L-threonine. In API 20E tests, positive for gelatin hydrolysis and tryptophan deaminase. Negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, β-galactosidase, urease, indole and hydrogen sulfide production, citrate utilization and the VogesProskauer reaction. Acid is produced from D-mannitol and weakly from L-arabinose, D-melibiose and amygdalin, but not from inositol, D-sorbitol, L-rhamnose, sucrose or D-glucose. MK-7 is the predominant menaquinone. The major fatty acids are anteiso-C15 : 0, iso-C16 : 0 and C16 : 0. The G+C content of the genomic DNA is 53.9 mol%.
The type strain, Gsoil 1411T (=KCTC 13020T=LMG 23405T), was isolated from soil of a ginseng field of Pocheon Province, South Korea.
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