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1 Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, P O Box 115, Yusong, Daejeon 305-600, Korea
2 Department of Biotechnology, Yonsei University, Seoul 120-749, Korea
Correspondence
Jung-Sook Lee
jslee{at}kribb.re.kr
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ABSTRACT |
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Additional data relating to whole-cell fatty acid compositions are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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, based on phylogenetic data from 16S rDNA sequences, and then emended by Shida et al. (1997). Members of the genus Paenibacillus produce ellipsoidal spores in swollen sporangia, are facultatively anaerobic or strictly aerobic, rod-shaped, and have G+C contents ranging from 45 to 54 mol% (Ash et al., 1993).
Bacillus ehimensis and Bacillus chitinolyticus, both of which exhibit chitinolytic activity, were first described by Kuroshima et al. (1996). At that time, these authors studied the morphological and biochemical characteristics of B. ehimensis and B. chitinolyticus, but did not describe their phylogenetic relationship with related taxa. However, the current authors believe that B. ehimensis and B. chitinolyticus are closer to the genus Paenibacillus than the genus Bacillus, based on a phylogenetic analysis of their 16S rDNA sequences. Therefore, to clarify the taxonomic position of B. ehimensis and B. chitinolyticus, the current study examined their phenotypic and chemotaxonomic characteristics, 16S rDNA sequences, and phylogenetic relationship.
The following strains were used in this study: B. chitinolyticus KCTC 3791T; B. ehimenis KCTC 3748T, KCTC 3747 and KCTC 3757; Bacillus sphaericus KCTC 3346T; Bacillus subtilis KCTC 3135T; Brevibacillus brevis KCTC 3743T; Halobacillus halophilus KCTC 3685T; Jeotgalibacillus alimentarius KCTC 13914T; Paenibacillus chinjuensis WN9T; Paenibacillus koreensis YC300T; Paenibacillus polymyxa KCTC 3627T; Paenibacillus validus KCTC 3401T; and Virgibacillus pantothenticus KCTC 3539T. All strains, except for H. halophilus and J. alimentarius, were cultured on trypticase soy agar (Difco). H. halophilus KCTC 3685T and J. alimentarius KCTC 13914T were cultivated on marine agar 2216 (Difco). All strains were incubated for 48 h at 30 °C.
For cellular fatty acid analysis, B. ehimensis KCTC 3748T, KCTC 3747 and KCTC 3757; B. chitinolyticus KCTC 3791T; P. koreensis YC300T and P. chinjuensis WN9T were cultured on trypticase soy agar for 48 h at 30 °C. The fatty acids were then analysed as previously described (Lee et al., 1996; Yang et al., 1993).
The whole-cell fatty acid compositions of the strains are shown in Table 1. The dominant fatty acids for the studied strains were C15 : 0 anteiso, C16 : 0 iso, C17 : 0 anteiso, C16 : 0, and C15 : 0 iso. Detailed fatty acid compositions of the strains are available as Supplementary Table A in IJSEM Online.
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, 27F and 1492R, were used for the PCR amplification of the 16S rDNA. The amplified PCR product was then purified using a QIAquick PCR purification kit (Qiagen). Finally, the purified 16S rDNA was sequenced using an ABI Prism BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems) and automatic DNA sequencer (Model 377; Applied Biosystems).
The 16S rDNA sequences were aligned with representative sequences from the genera Bacillus and Paenibacillus and related taxa using CLUSTAL W software (Thompson et al., 1994). Gaps at the 5' and 3' ends of the alignment were omitted from further analysis. Evolutionary distance matrices were calculated using the algorithm of Jukes & Cantor (1969) along with the DNADIST program included in the PHYLIP software package (Felsenstein, 1993). A phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987), as implemented in the NEIGHBOR program from the same software package. The stability of the groupings was assessed by a bootstrap analysis of 1000 datasets using the programs SEQBOOT, DNADIST, NEIGHBOR, and CONSENSE from the PHYLIP software package. The GenBank accession numbers for the 16S rDNA sequences used for the phylogenetic analysis are shown in Fig. 1.
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) indicated that B. chitinolyticus KCTC 3791T and B. ehimensis strains KCTC 3748T, KCTC 3747 and KCTC 3757 were all clustered with the genus Paenibacillus. B. ehimensis strains KCTC 3748T, 3747 and 3757 showed identical 16S rDNA sequences, suggesting that they are members of the same species, and were most closely related to P. koreensis YC300T (98·3 % sequence similarity). At the same time, B. chitinolyticus KCTC 3791T was found to be phylogenetically closely related to P. chinjuensis WN9T (95·2 % sequence similarity).
DNA–DNA hybridization was carried out by 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. DNA preparations (1 µg) were labelled using the DIG High Prime DNA labelling and detection starter kit II, according to the manufacturer's protocol. Membranes were prehybridized in hybridization solution at 52 °C for 30min. Hybridization was carried out in a hybridization solution containing labelled DNA (25 ng ml–1) at 52 °C for 16 h. After hybridization, membranes were washed twice in primary washing solution (2x SSC and 0·1 % SDS), and then washed twice in secondary washing solution (0·5x SSC and 0·1 % SDS) at 68 °C. Detection reagents were added to the membranes for 5 min at room temperature, and then excess liquid was squeezed out. Membranes were exposed to autoradiography film (Hyperfilm-ECL; Amersham) for 10 min, and signal intensities were determined using the TINA 2.0 program. The signal produced by self-hybridization was taken as 100 %, and percentage hybridization values were calculated from duplicate samples.
The DNA–DNA hybridization results confirmed that B. ehimensis strains KCTC 3748T, 3747 and 3757 were members of the same species (Kuroshima et al., 1996). They exhibited high levels of relatedness (93–105 %) with each other. The DNA–DNA hybridization values between B. ehimensis KCTC 3748T and P. koreensis YC300T were less than 26 %. The phylogenetic definition of a species generally includes ‘strains with approximately 70 % or greater DNA–DNA relatedness' (Wayne et al., 1987). According to the available compiled data, organisms that have less than 97·0 % sequence similarity will not reassociate to more than 60 %, regardless of the hybridization method employed (Stackebrandt & Goebel, 1994). We did not determine the DNA–DNA hybridization value between B. chitinolyticus KCTC 3791T and P. chinjuensis WN9T because the sequence similarity between the two strains was 95·2 %. The phylogenetic and DNA–DNA hybridization results demonstrate that B. ehimensis and B. chitinolyticus are phylogenetically closer to the genus Paenibacillus than to the genus Bacillus and are not related to any of the previously described Paenibacillus taxa at the species level.
The specific forward primer PAEN515F (5'-GCTCGGAGAGTGACGGTACCTGAGA-3') and universal reverse primer 1377R were used in PCR amplifications for presumptive identification of strains belonging to the genus Paenibacillus. The PCR conditions followed the method of Shida et al. (1997). The universal primers 27F and 1492R were used for the PCR amplification of the 16S rDNA.
The results of the 16S rDNA fragment amplification for the three B. ehimensis strains, one B. chitinolyticus strain, four Paenibacillus strains, two Bacillus strains, one Brevibacillus strain, one Halobacillus strain, one Virgibacillus strain and one Jeotgalibacillus strain are shown in Fig. 2. The control reaction resulted in the predicted band of 1·5 kb from all tested strains (Fig. 2a). However, the PCR amplifications with primers PAEN515F and 1377R produced a 0·8 kb PCR fragment from the Paenibacillus strains, the three B. ehimensis strains and the B. chitinolyticus strain (Fig. 2b), suggesting that B. ehimensis and B. chitinolyticus are indeed members of the genus Paenibacillus.
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; Danne et al., 2002; Heyndrickx et al., 1995, 1996; Kuroshima et al., 1996; Nakamura, 1984, 1987; Yoon et al., 2001) and are given in Table 2.
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Description of Paenibacillus ehimensis (Kuroshima et al. 1996) comb. nov.
Paenibacillus ehimensis (e.hi.men'sis. N. L. masc. adj. ehimensis referring to Ehime Prefecture, Japan, the source of the soil samples from which the organisms were isolated).
Basonym: Bacillus ehimensis Kuroshima et al. 1996.
The description is the same as that given by Kuroshima et al. (1996). The type strain is KCTC 3748T (=IFO 15659T).
Emended description of Paenibacillus chitinolyticus (Kuroshima et al. 1996) comb. nov.
Paenibacillus chitinolyticus (chi.ti.no.ly'ti.cus. N.L. n. chitinum chitin; M.L. adj. lyticus dissolving; N.L. adj. chitinolyticus decomposing chitin).
Basonym: Bacillus chitinolyticus Kuroshima et al. 1996.
The description is the same as that given by Kuroshima et al. (1996). The type strain is KCTC 3791T (=IFO 15660T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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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]
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) end of the carbon chain. Trace, <0·5 %.
