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1 LG Life Sciences Ltd/R&D Park, #104-1 Moonji-dong, Yuseong-gu, Daejeon 305-380, Republic of Korea
2 Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, #52, Oun-dong, Yuseong-gu, Daejeon 305-333, Republic of Korea
Correspondence
Dal-Soo Kim
dalskim{at}lgls.co.kr
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains SD17T and SD18 are AY090110 and AY081186, respectively.
Micrographs and an expanded phylogenetic tree are available as supplementary material in IJSEM Online.
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on the basis of polyphasic data from 16S rRNA gene sequences, and then emended by Shida et al. (1997). Members of the genus Paenibacillus are facultatively anaerobic or strictly aerobic, rod-shaped, produce ellipsoidal spores in swollen sporangia and have G+C contents ranging from 45 to 54 mol% (Ash et al., 1993). Some of these organisms excrete diverse assortments of polysaccharide-hydrolysing enzymes (Kanzawa et al., 1995; Nakamura, 1987; Priest et al., 1988) and produce antibacterial compounds such as polymyxin, octopytin and baciphelacin (Slepecky & Hemphill, 1991) and an antifungal compound (Chung et al., 2000). The taxonomic classification of two isolates, SD17T and SD18, was studied here based on phylogenetic and phenotypic evidence. We propose that the two isolates should be classified in the genus Paenibacillus as Paenibacillus elgii sp. nov.
Strains SD17T and SD18 were isolated from roots of Perilla frutescens collected from Seocheon, Korea. The roots were washed under running tap water, heat-treated at 80 °C for 30 min, macerated and plated onto 1 : 10 diluted trypticase soy agar (1/10 TSA; Difco) for isolation of single colonies similar to the method described by Kim et al. (1997). These isolates were examined to screen for inhibitory activity against growth of Rhizoctonia solani AG 4 and Pythium aphanidermatum. Of approximately 3000 selected candidates, the isolates that suppressed diseases of turfgrass (Agrostis palustris) caused by Pythium aphanidermatum (pythium blight) and R. solani AG 2-2 (brown patch) were selected for further studies. Isolates SD17T and SD18 were inhibitory to the growth of various fungi such as Botrytis cinerea, Chaetomium globosum, Cladosporium resinae, Colletotrichum gloeosporioides, Corynespora cassicola, Fusarium oxysporum f. sp. lycopersici, Magnaporthe grisea, Phytophthora infestans, Pythium aphanidermatum, R. solani AG 1-1, R. solani AG 2-2, R. solani AG 4, Saccharomyces cerevisiae, Sclerotinia homoeocarpa and Trichoderma viride and bacteria including Bacillus subtilis, Burkholderia glumae, Escherichia coli, Paenibacillus polymyxa, Pseudomonas fluorescens, Xanthomonas oryzae and Xanthomonas vesicatoria, when extensively tested for their antimicrobial activity. Strains SD17T and SD18 were routinely cultured on 1/10 TSA and maintained in 20 % glycerol at –80 °C for storage.
Cell morphology was examined using a Nikon MICROPHOT-FXA phase-contrast microscope. The cells of 24 h cultures were Gram-stained using Hucker's modification (Gerhardt et al., 1994). For electron microscopy, vegetative cells were negatively stained with 1 % (w/v) phosphotungstic acid and, after air-drying, the grids were examined under a Philips CM-20 transmission electron microscope. Spores were pre-fixed in 5 % (v/v) glutaraldehyde in phosphate buffer (Gibco) for at least 1 h at room temperature. Samples were then post-fixed in 4 % (w/v) osmium tetroxide for 1 h, dehydrated using a graded series of acetone, transferred to 100 % acetone and embedded in Epon 812 (Fluka) substitute. Thin sections were cut with a diamond knife on a Leica Ultracut VCT microtome and stained with uranyl acetate and lead citrate. Grids were examined on a Philips CM-20 transmission electron microscope at an operating voltage of 60 kV. Phenotypic characterization was carried out using the standard methods of Claus & Berkeley (1986) and McFaddin (2000), together with API 20E and API 50CHB systems (bioMérieux). Oxidation of 95 selected carbon sources was tested using Biolog GP2 microplates according to the manufacturer's instructions. Growth was examined using a cap tube containing 10 ml tryptic soy broth, 100 mM Na2HPO4/NaH2PO4 buffer, with a pH of 6·0–9·0 at 30 °C. Growth was estimated by monitoring the optical density at 650 nm.
The cells are Gram-variable and exhibit motility by peritrichous flagella. Ellipsoidal spores formed in swollen sporangia and mature spores have surface stripes. They exhibit a star-shaped morphology in thin section when visualized by transmission electron microscopy (Elo et al., 2001; see Supplementary Figs A and B, available in IJSEM Online). Distinguishing phenotypic characteristics between the isolates and phylogenetically related species are shown in Table 1.
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. Menaquinones were analysed by reversed-phase HPLC as described by Komagata & Suzuki (1987). The diamino acid of the peptidoglycan was determined by TLC (DC-Alufoline cellulose; Merck) as described by Komagata & Suzuki (1987). G+C content of the DNA was determined from the midpoint value of the thermal denaturation profile using an Ultraspec 2000 spectrophotometer (Pharmacia Biotech) equipped with a programmable Peltier temperature control unit according to the equation of Marmur & Doty (1962) as modified by De Ley (1970). Strains SD17T and SD18 contained MK-7 as the major menaquinone and meso-diaminopimelic acid in the cell-wall peptidoglycan. These chemotaxonomic characteristics were most similar to the those characteristic of the genus Paenibacillus (Shida et al., 1997). The DNA G+C content of strain SD17T was 51·7 mol%. Whole-cell fatty acid compositions of the two isolates and related species are shown in Table 2. According to the published data, the cellular fatty acid pattern of strains SD17T and SD18 is similar to those of phylogenetically closely related species of the genus Paenibacillus. The main fatty acid is anteiso-C15 : 0, comprising 54–62 % of the total. These values are similar to Paenibacillus ehimensis, but higher than those reported for Paenibacillus koreensis KCTC 2393T (Lee et al., 2004) and Paenibacillus azoreducens DSM 13822T (Meehan et al., 2001). The second major fatty acid in strains SD17T and SD18 is iso-C15 : 0, whereas it is iso-C16 : 0 in Paenibacillus ehimensis and Paenibacillus koreensis. In Paenibacillus azoreducens, saturated fatty acid C16 : 0 was the second major component. This clearly distinguished the isolates from related species.
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. The resultant sequence of strain SD17T was manually aligned with representative sequences of Paenibacillus obtained from the GenBank database, using known 16S rRNA secondary structure information (Gutell, 1994). Phylogenetic trees were inferred using the Fitch–Margoliash (Fitch & Margoliash, 1967) and neighbour-joining methods (Saitou & Nei, 1987). Evolutionary distance matrices for the neighbour-joining and Fitch–Margoliash methods were generated according to the model of Jukes & Cantor (1969). The PHYLIP package (Felsenstein, 1993) was used for all analyses. The resultant neighbour-joining tree topology was evaluated by bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings. The almost complete 16S rRNA gene sequence (1481 bp) of SD17T was determined and its primary structure was compared with closely related reference strains. A phylogenetic tree based on the nucleotide substitution rate (Knuc values) indicates that the strains belong to the genus Paenibacillus (Fig. 1). When the phylogenetic position of isolates SD17T and SD18 was compared with closely related species of the genus Paenibacillus, the strains formed a monophyletic clade with Paenibacillus koreensis KCTC 2393T and Paenibacillus ehimensis IFO 15659T (see Supplementary Fig. C). The nucleotide sequence similarity values between SD17T and other Paenibacillus species ranged from 98·6 % (with Paenibacillus ehimensis IFO 15659T) to 91·3 % (with Paenibacillus kobensis IFO 15729T). The next most closely related species is Paenibacillus koreensis, with 97·9 % sequence similarity. Therefore, the genealogical position of strains SD17T and SD18 was further examined using DNA–DNA relatedness with Paenibacillus ehimensis IFO 15659T and Paenibacillus koreensis KCTC 2393T.
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. At that time, they studied the morphological and biochemical characteristics of Bacillus ehimensis and Bacillus chitinolyticus, yet did not describe their phylogenetic relationship with related taxa. Recently, the taxonomic status of Bacillus ehimensis and Bacillus chitinolyticus was examined based on 16S rRNA gene sequences, DNA–DNA hybridization and other taxonomic characteristics (Lee et al., 2004). The study reported that the two species belonged to the genus Paenibacillus and proposed that Bacillus ehimensis and Bacillus chitinolyticus be transferred to the genus Paenibacillus as Paenibacillus ehimensis comb. nov. and Paenibacillus chitinolyticus comb. nov. DNA–DNA hybridization was determined based on a membrane filter technique using a DIG High Prime DNA labelling and detection starter kit II (Roche Molecular Biochemicals). Genomic DNA (200 ng) was denatured by the alkaline method and immobilized on a nylon membrane (Hybond-N+; Amersham) by applying a low vacuum and the DNA preparations (1 µg) were labelled using the DIG High Prime DNA labelling and detection starter kit II according to the manufacturer's protocol. The membranes were then pre-hybridized in a hybridization solution at 52 °C for 30 min. The actual pre-hybridization was carried out in a hybridization solution containing labelled DNA (25 ng ml–1) at 52 °C for 16 h. After hybridization, the membranes were washed twice in a primary washing solution (2x SSC and 0·1 % SDS) and then subsequently washed twice in a secondary solution (0·5x SSC and 0·1 % SDS) at 68 °C. Detection reagents were added to the membranes for 5 min at room temperature, the membranes were then exposed to autoradiography film (Hyperfilm-ECL; Amersham) for 10 min and the signal intensities were determined using the TINA 2.0 program. The signal produced by self-hybridization of the probe with homologous target DNA was taken as 100 %, and percentage relatedness values were calculated for the duplicate samples.
DNA–DNA hybridization results confirmed that isolates SD17T and SD18 belonged to the same species. Relatedness between the two strains was 89·5 %. Strain SD17T showed 19·8 and 17·4 % DNA–DNA relatedness with Paenibacillus ehimensis IFO 15659T and Paenibacillus koreensis KCTC 2393T, respectively. The latter two strains showed 26 % mutual DNA–DNA relatedness (Lee et al., 2004). DNA–DNA relatedness of these strains with strain SD18 was also very low (17·2 % with Paenibacillus ehimensis IFO 15659T and 15·0 % with Paenibacillus koreensis KCTC 2393T).
The phylogenetic definition of a species of Wayne et al. (1987) includes strains with approximately 70 % or more DNA–DNA relatedness. Organisms that have less than 97 % 16S rRNA gene sequence similarity will not reassociate to more than 60 %, no matter which hybridization method is applied (Stackebrandt & Goebel, 1994). We did not determine the DNA–DNA hybridization value with Paenibacillus azoreducens because of low 16S rRNA gene sequence similarity (94 %). This result, together with phylogenetic analysis, demonstrates that isolates SD17T and SD18 are distinct from all previously described Paenibacillus taxa at the species level.
On the basis of morphological, physiological, chemotaxonomic characteristics, 16S rRNA gene sequence analysis and DNA–DNA relatedness, strains SD17T and SD18 represent a novel species of the genus Paenibacillus for which the name Paenibacillus elgii sp. nov. is proposed.
Description of Paenibacillus elgii sp. nov.
Paenibacillus elgii (el'gi.i. N.L. gen. n. elgii arbitrary name formed from the company name LG where taxonomic studies on this species were performed).
Cells are facultatively anaerobic, Gram-variable rods (0·8–1·0 µm wide, 3·0–5·0 µm long), motile with peritrichous flagella. Ellipsoidal spores are formed in swollen sporangia and mature spores have stripes on the surface. Colonies on nutrient agar are circular, flat, smooth, opaque and white. Temperature range for growth is 20–45 °C; growth occurs at pH 6·0–8·5 (optimum 7·0). Isolates are able to grow in the presence of 2 % NaCl. Catalase-positive, oxidase-negative. Reduction of nitrate is positive. H2S is not produced but indole is produced. Casein, aesculin and starch are hydrolysed. Acid is produced from glucose, maltose, mannitol, mannose and trehalose. Glucose, ribose, N-acetylglutamate and Tween 40 are assimilated. The G+C content of the DNA is 51·7 mol%. The major isoprenoid quinone is menaquinone MK-7. The major cellular fatty acid is anteiso-C15 : 0. Cell-wall peptidoglycan contains meso-diaminopimelic acid.
The type strain is SD17T (=KCTC 10016BPT=NBRC 100335T) and SD18 (=KCTC 3756) is a reference strain. Isolated from roots of Perilla frutescens in Seocheon County, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Chun, J. & Goodfellow, M. (1995). A phylogenetic analysis of the genus Nocardia with 16S rRNA gene sequences. Int J Syst Bacteriol 45, 240–245.[CrossRef][Medline]
Chung, Y. R., Kim, C. H., Hwang, I. & Chun, J. (2000). Paenibacillus koreensis sp. nov., a new species that produces an iturin-like antifungal compound. Int J Syst Evol Microbiol 50, 1495–1500.[Abstract]
Claus, D. & Berkeley, R. C. W. (1986). Genus Bacillus Cohn 1872. In Bergey's Manual of Systematic Bacteriology, vol. 2, pp. 1105–1140. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.
De Ley, J. (1970). Re-examination of the association between melting point, buoyant density, and chemical base composition of deoxyribonucleic acid. J Bacteriol 101, 738–754.
Elo, S., Suominen, I., Kämpfer, P., Juhanoja, J., Salkinoja-Salonen, M. & Haahtela, K. (2001). Paenibacillus borealis sp. nov., a nitrogen-fixing species isolated from spruce forest humus in Finland. Int J Syst Evol Microbiol 51, 535–545.[Abstract]
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
Felsenstein, J. (1993). PHYLIP (phylogeny inference package), version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle, USA.
Fitch, W. M. & Margoliash, E. (1967). Construction of phylogenetic trees. Science 155, 279–284.
Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors) (1994). Methods for General and Molecular Bacteriology. Washington, DC: American Society for Microbiology.
Gutell, R. R. (1994). Collection of small subunit (16S- and 16S-like) ribosomal RNA structures. Nucleic Acids Res 22, 3502–3507.
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
Kanzawa, Y., Harada, A., Takeuchi, M., Yokota, A. & Harada, T. (1995). Bacillus curdlanolyticus sp. nov. and Bacillus kobensis sp. nov., which hydrolyze resistant curdlan. Int J Syst Bacteriol 45, 515–521.[CrossRef][Medline]
Kim, D. S., Weller, D. M. & Cook, R. J. (1997). Bacillus sp. L324-92 for biological control of three root diseases of wheat grown with reduced tillage. Phytopathology 87, 551–558.
Komagata, K. & Suzuki, K. (1987). Lipids and cell-wall analysis in bacterial systematics. Methods Microbiol 19, 161–203.
Kuroshima, K., Sakane, T., Takata, R. & Yokota, A. (1996). Bacillus ehimensis sp. nov. and Bacillus chitinolyticus sp. nov., new chitinolytic members of the genus Bacillus. Int J Syst Bacteriol 46, 76–80.
Lee, J.-S., Jung, M.-C., Kim, W.-S. & 10 other authors (1996). Identification of lactic acid bacteria from kimchi by cellular FAMEs analysis. Korean J Appl Microbiol Biotechnol 24, 234–241.
Lee, J.-S., Pyun, Y.-R. & Bae, K. S. (2004). Transfer of Bacillus ehimensis and Bacillus chitinolyticus to the genus Paenibacillus with emended descriptions of Paenibacillus ehimensis comb. nov. and Paenibacillus chitinolyticus comb. nov. Int J Syst Evol Microbiol 54, 929–933.
Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5, 109–118.[Medline]
McFaddin, J. F. (2000). Biochemical Tests for Identification of Medical Bacteria, 3rd edn. Philadelphia: Lippincott Williams & Wilkins.
Meehan, C., Bjourson, A. J. & McMullan, G. (2001). Paenibacillus azoreducens sp. nov., a synthetic azo dye decolorizing bacterium from industrial wastewater. Int J Syst Evol Microbiol 51, 1681–1685.[Abstract]
Nakamura, L. K. (1987). Bacillus alginolyticus sp. nov. and Bacillus chondroitinus sp. nov., two alginate-degrading species. Int J Syst Bacteriol 37, 284–286.[CrossRef]
Priest, F. G., Goodfellow, M. & Todd, C. (1988). A numerical classification of the genus Bacillus. J Gen Microbiol 134, 1847–1882.[Medline]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Shida, O., Takagi, H., Kadowaki, K., Nakamura, L. K. & Komagata, K. (1997). Transfer of Bacillus alginolyticus, Bacillus chondroitinus, Bacillus curdlanolyticus, Bacillus glucanolyticus, Bacillus kobensis, and Bacillus thiaminolyticus to the genus Paenibacillus and emended description of the genus Paenibacillus. Int J Syst Bacteriol 47, 289–298.[CrossRef][Medline]
Slepecky, R. A. & Hemphill, H. E. (1991). The genus Bacillus – nonmedical. In The Prokaryotes, pp. 1663–1696. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K.-H. Schleifer. New York: Springer.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.[CrossRef]
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.[CrossRef]
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