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1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
2 Department of Food and Life Science, Sungkyunkwan University, Chunchun-dong 300, Jangan-gu, Suwon, Korea
3 Probionic Corporation, Bio-venture Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
Correspondence
Yong-Ha Park
yhpark{at}mail.kribb.re.kr
| ABSTRACT |
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The phylogenetic tree from which Fig. 2
was taken is available as supplementary data in IJSEM Online (http://ijs.sgmjournals.org).
| INTRODUCTION |
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The genus Paenibacillus was created with 11 Bacillus spp. by Ash et al. (1993)
. Since its creation, continuous transfers of Bacillus spp. to the genus and descriptions of novel Paenibacillus spp. have increased the number of recognized Paenibacillus spp. considerably (Heyndrickx et al., 1996
; Shida et al., 1997a
, b
; Pettersson et al., 1999
; Yoon et al., 1998b
; Tcherpakov et al., 1999
; Van der Maarel et al., 2000
; Elo et al., 2001
). At the time of writing, there were 28 validly described species belonging to the genus Paenibacillus. It has been shown that many recently described Bacillus spp. possess the general characteristics of the genus Paenibacillus (Shida et al., 1997a
). There may also be additional Bacillus spp. or strains that possess characteristics of the genus Paenibacillus, and rod-shaped endospore formers with the characteristics of the genus Paenibacillus may be relatively common in nature. Accordingly, descriptions of novel Paenibacillus spp. will contribute to the field of taxonomy and to our understanding of the biological diversity of the genus Paenibacillus. The aim of the present study was to unravel the taxonomic positions of strains AM49T and AM141T by using a polyphasic taxonomic approach. On the basis of the data presented here, it is proposed that strains AM49T and AM141T be placed into the genus Paenibacillus as two distinct novel species, Paenibacillus kribbensis and Paenibacillus terrae, respectively.
| METHODS |
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Morphological characterization.
Colony and cell morphologies were examined by using colonies and cells grown on TSA. Observation of cell micromorphology was performed using light microscopy and transmission electron microscopy (TEM). Flagellum type was examined by TEM using cells from exponentially growing cultures. For TEM observations, cells were negatively stained with 1 % (w/v) phosphotungstic acid and, after air-drying, the grids were examined by using a model CM-20 transmission electron microscope (Philips).
Physiological characterization.
Oxidase activity was determined by oxidation of 1 % p-aminodimethylaniline oxalate. Catalase activity was determined by bubble formation in a 3 % (v/v) H2O2 solution. Hydrolysis of aesculin and nitrate reduction were determined as described previously (Lanyi, 1987
). Hydrolyses of casein, gelatin, hypoxanthine, starch, Tween 80, tyrosine and xanthine, and urease activity were determined as described by Cowan & Steel (1965)
. Acid production from carbohydrates was determined by using the API 50CH system (bioMérieux). Utilization of various substrates as sole carbon and energy sources was determined as described by Shirling & Gottlieb (1966)
. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber with TSA that was prepared anaerobically. Tolerance to NaCl was measured in TSB containing 16 % (w/v) NaCl. Growth at various temperatures was measured on TSA at 450 °C.
Isolation of DNA.
Chromosomal DNA was isolated and purified as described previously (Yoon et al., 1996
), with the exception that ribonuclease T1 was used together with ribonuclease A.
Chemotaxonomic characterization.
The isomer type of the diaminopimelic acid of the peptidoglycan layer was analysed by the method of Komagata & Suzuki (1987)
. Menaquinones were analysed as described previously (Komagata & Suzuki, 1987
) using reversed-phase HPLC. For quantitative analysis of cellular fatty acid compositions, a loop of cell mass was harvested and FAMEs were prepared and identified following the instructions of the Microbial Identification System (MIDI).
DNA G+C content.
This was determined by the method of Tamaoka & Komagata (1984)
. DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC.
16S rDNA sequencing and phylogenetic analysis.
16S rDNA was amplified by PCR using two universal primers as described previously (Yoon et al., 1998a
). The PCR product was purified with a QIAquick PCR purification kit (Qiagen). Sequencing of the purified 16S rDNA was performed using an ABI PRISM BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems) as recommended by the manufacturer. The purified sequencing reaction mixtures were electrophoresed automatically using an Applied Biosystems model 377 automated DNA sequencer. Alignment of sequences was carried out using the CLUSTAL W software (Thompson et al., 1994
). Gaps at the 5' and 3' ends of the alignment were omitted from further analyses. Phylogenetic trees were inferred by using three tree-making algorithms, i.e. the neighbour-joining (Saitou & Nei, 1987
), maximum-likelihood (Felsenstein, 1981
) and maximum-parsimony (Kluge & Farris, 1969
) methods contained within the PHYLIP package (Felsenstein, 1993
). Evolutionary distance matrices for the neighbour-joining method were calculated by the algorithm of Jukes & Cantor (1969)
using DNADIST. The stability of relationships was assessed by a bootstrap analysis based on 1000 resamplings of the neighbour-joining dataset by using the programs SEQBOOT, DNADIST, NEIGHBOR and CONSENSE of the PHYLIP package.
DNADNA hybridization.
This was performed fluorometrically by the method of Ezaki et al. (1989)
using photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed with five replications for each sample. Of the values obtained, the highest and lowest values in each sample were excluded and the remaining three values were used for the calculation of similarity values. Hence, DNADNA similarity values are expressed as the mean of three values.
| RESULTS AND DISCUSSION |
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Description of Paenibacillus kribbensis sp. nov.
Paenibacillus kribbensis (krib.ben'sis. N.L. adj. kribbensis arbitrary name formed from the acronym of the Korea Research Institute of Bioscience and Biotechnology, KRIBB, where taxonomic studies on this species were performed).
Cells are facultatively anaerobic rods with dimensions of 1·31·8x4·07·0 µm on TSA. Gram-variable. Ellipsoidal spores are formed in swollen sporangia. Motile by means of peritrichous flagella. Colonies are cream-coloured, circular to slightly irregular in shape, flat to low convex and translucent on TSA. Optimal growth temperature is between 30 and 37 °C; growth occurs at 10 and 44 °C, but not at 4 or 45 °C. Optimal pH for growth is between pH 6·5 and 8·0; growth is inhibited below pH 4·0. Grows optimally in the presence of 02 % (w/v) NaCl; growth occurs in the presence of 4 % (w/v) NaCl, but not in the presence of 5 % (w/v) NaCl. Catalase-positive. Oxidase- and urease-negative. Aesculin, casein, gelatin, starch and Tween 80 are hydrolysed. Hypoxanthine, tyrosine and xanthine are not hydrolysed. Nitrate is reduced to nitrite. L-Arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannose, melibiose, D-raffinose, L-rhamnose, D-ribose, stachyose, sucrose, D-trehalose, D-xylose, myo-inositol, D-mannitol and sodium gluconate are utilized; disodium succinate and trisodium citrate are weakly utilized as sole carbon and energy sources. D-Melezitose, adonitol, D-sorbitol, sodium acetate and sodium benzoate are not utilized as sole carbon and energy sources. In the API 50CH system, when API CHB suspension medium is used, acid is produced from L-arabinose, ribose, D-xylose, methyl
-D-xyloside, galactose, glucose, fructose, mannose, inositol, mannitol, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, inulin, raffinose, starch, glycogen and gentiobiose; acid is weakly produced from glycerol, methyl
-D-mannoside, methyl
-D-glucoside, N-acetylglucosamine and gluconate. Acid is not produced from erythritol, D-arabinose, L-xylose, adonitol, sorbose, rhamnose, dulcitol, sorbitol, melezitose, xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, 2-keto-D-gluconate or 5-keto-D-gluconate. Cell-wall peptidoglycan contains meso-diaminopimelic acid. Predominant menaquinone is MK-7. Major fatty acid is anteiso-C15 : 0. DNA G+C content is 48 mol% (as determined by HPLC). Isolated from a soil sample from Taejon City, Korea. The type strain is strain AM49T, which has been deposited in the Korean Collection for Type Cultures as KCTC 0766BPT and the Japan Collection of Microorganisms as JCM 11465T.
Description of Paenibacillus terrae sp. nov.
Paenibacillus terrae (ter'rae. L. gen. n. terrae of the earth).
Cells are facultatively anaerobic rods with dimensions of 1·31·8x4·07·0 µm on TSA. Gram-variable. Ellipsoidal spores are formed in swollen sporangia. Motile by means of peritrichous flagella. Colonies are cream-coloured, irregular in shape, thin and translucent on TSA. Optimal growth temperature is 30 °C; growth occurs at 10 and 40 °C, but not at 4 or 41 °C. Optimal pH for growth is between pH 6·5 and 8·0; growth is inhibited below pH 4·0. Grows optimally in the presence of 02 % (w/v) NaCl; growth occurs in the presence of 3 % (w/v) NaCl, but not in the presence of 4 % (w/v) NaCl. Catalase-positive. Oxidase- and urease-negative. Aesculin, casein, gelatin and starch are hydrolysed; Tween 80 is weakly hydrolysed. Hypoxanthine, tyrosine and xanthine are not hydrolysed. Nitrate is reduced to nitrite. D-Cellobiose, D-galactose, D-glucose, lactose, maltose, D-mannose, melibiose, D-raffinose, L-rhamnose, stachyose, sucrose, D-trehalose, myo-inositol, D-mannitol and sodium gluconate are utilized; disodium succinate and trisodium citrate are weakly utilized as sole carbon and energy sources. L-Arabinose, D-fructose, D-melezitose, D-ribose, D-xylose, adonitol, D-sorbitol, sodium acetate and sodium benzoate are not utilized as sole carbon and energy sources. In the API 50CH system, when API CHB suspension medium is used, acid is produced from glycerol, L-arabinose, ribose, D-xylose, galactose, glucose, fructose, mannose, inositol, mannitol, methyl
-D-mannoside, methyl
-D-glucoside, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, inulin, raffinose, starch, glycogen, gentiobiose and D-turanose; acid is weakly produced from methyl
-D-xyloside and 5-keto-D-gluconate. Acid is not produced from erythritol, D-arabinose, L-xylose, adonitol, sorbose, rhamnose, dulcitol, sorbitol, melezitose, xylitol, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate or 2-keto-D-gluconate. Cell-wall peptidoglycan contains meso-diaminopimelic acid. Predominant menaquinone is MK-7. Major fatty acid is anteiso-C15 : 0. DNA G+C content is 47 mol% (as determined by HPLC). Isolated from a soil sample from Taejon City, Korea. The type strain is strain AM141T, which has been deposited in the Korean Culture Center of Microorganisms as KCCM 41557T and the Japan Collection of Microorganisms as JCM 11466T.
| ACKNOWLEDGEMENTS |
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