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1 Bioresource Collection and Research Center, Food Industry Research and Development Institute, PO Box 246, Hsinchu 30099, Taiwan
2 Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-Ku, Tokyo 113-0032, Japan
Correspondence
Fwu-Ling Lee
fll{at}firdi.org.tw
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ABSTRACT |
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Maximum-likelihood and maximum-parsimony trees based on 16S rRNA and gyrB gene sequences and details of fatty acid profiles and differential phenotypic characteristics of the studied strains are available with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Nakamura et al., 1999), B. licheniformis (Skerman et al., 1980), B. amyloliquefaciens (Priest et al., 1987), B. atrophaeus (Nakamura, 1989), B. mojavensis (Roberts et al., 1994), B. vallismortis (Roberts et al., 1996), B. sonorensis (Palmisano et al., 2001), B. velezensis (Ruiz-García et al., 2005a), B. axarquiensis and B. malacitensis (Ruiz-García et al., 2005b), Gram-positive, rod-shaped, endospore-forming, aerobic and fermentative bacteria, are difficult to distinguish from each other by phenotypic characteristics alone, but can be differentiated from one another reliably by using DNA–DNA hybridization. In the present study, we provide evidence, on the basis of sequence similarities of the 16S rRNA and gyrB genes and DNA–DNA hybridization data, that the type strains of B. axarquiensis and B. malacitensis are very closely related to the type strain of B. mojavensis, suggesting that the three species represent the same species.
The bacterial strains used in this study were cultivated in nutrient agar or broth (Difco) and incubated aerobically at 30 °C. Genomic DNA was isolated and purified using the Qiagen Blood & Cell Culture DNA kit. The complete sequence of a 1.5 kb 16S rRNA gene fragment was determined using the MicroSeq Full Gene 16S rDNA bacterial identification kit (Applied Biosystems). Sequencing was performed using an ABI 3730 DNA sequencer (Applied Biosystems and Hitachi). Sequence assembly was performed using the ABI PRISM DNA Sequencing Analysis software (PE Applied Biosystems). Sequence similarity was calculated using the programs of the Wisconsin package version 10.1 (Accelrys Inc.). The sequences obtained in this study and from GenBank were aligned using the CLUSTAL_X program, version 1.8 (Thompson et al., 1997), and phylogenetic analysis was performed using the PHYLIP computer program package (Felsenstein, 2002). Phylogenetic trees were constructed using the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) methods. Bootstrap analysis was made with 1000 replicates except in maximum-likelihood, where only 100 replicates were generated. Tree figures were drawn by TreeView software (Page, 1996).
Sequences of highly conserved housekeeping or other protein-encoding genes provide higher resolution than 16S rRNA gene sequences and may supplant DNA–DNA reassociation or 16S rRNA gene sequence data at the species level for taxonomic analysis (Stackebrandt et al., 2002; Zeigler, 2003). The partial sequence for the gene encoding the subunit B protein of DNA gyrase (gyrB) was included in the phylogenetic analysis for comparative purposes. An approximately 1.2 kb fragment of the gyrB gene was amplified by PCR and sequenced using a set of primers described by Yamamoto & Harayama (1995). Using the method described above, the gyrB gene sequences were also aligned and phylogenetic trees were constructed. Phylogenetic analysis by the neighbour-joining (Fig. 1), maximum-parsimony and maximum-likelihood methods (see Supplementary Figs S1 and S2 available in IJSEM Online) produced similar trees with the exception of minor differences in the tree topology of the basal branches.
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). Sequences of the B. axarquiensis and B. malacitensis type strains shared 99.6 and 99.5 % similarity, respectively, with those of B. subtilis subsp. subtilis BCRC 10255T and B. amyloliquefaciens BCRC 11601T. The 16S rRNA gene sequences (positions 35–1431) of B. axarquiensis LMG 22476T and CIP 108772T and B. axarquiensis CR-119T (GenBank accession no. AY603657) shared 99.9, 99.9 and 99.5 % similarity, respectively, with that of B. mojavensis BCRC 17124T. In contrast, the similarity between B. axarquiensis CR-119T and B. mojavensis reported by Ruiz-García et al. (2005b) was noticeably lower, at 97.4 %. The gyrB gene sequences (positions 316–1480; E. coli K-12 numbering) for B. axarquiensis LMG 22476T and CIP 108772T and B. malacitensis LMG 22477T and CECT 5687T showed high similarities (>97 %) with the investigated B. mojavensis strains and significantly lower similarities (ranging from 80 to 90 %) with their nearest neighbour (Fig. 1b). The similarities of the 16S rRNA and gyrB gene sequences indicate that B. axarquiensis, B. malacitensis and B. mojavensis cannot be differentiated and may represent the same species.
The G+C content was determined using reversed-phase HPLC according to Tamaoka & Komagata (1984) with slight modifications. The nucleoside mixture was separated using a Cosmosil 5C18 column (Waters, 4.0x150 mm; Nacalai Tesque) in a mobile phase composed of 0.2 M (NH4)H2PO4/acetonitrile (20 : 1, v/v) at a flow rate of 1 ml min–1 at room temperature. DNA relatedness values between B. axarquiensis LMG 22476T and CIP 108772T, B. malacitensis LMG 22477T and CECT 5687T, B. mojavensis strains BCRC 17124T and BCRC 17531, B. subtilis subsp. subtilis BCRC 10255T and B. amyloliquefaciens BCRC 11601T were determined using the fluorometric hybridization method in microdilution wells as described previously (Ezaki et al., 1989; Goris et al., 1998; Chern et al., 2004). The hybridization temperature was 42 °C. DNA G+C contents of B. axarquiensis (LMG 22476T and CIP 108772T), B. malacitensis (LMG 22477T and CECT 5687T) and B. mojavensis strains BCRC 17124T and BCRC 17531 were respectively 43, 44, 44 and 44 mol%. These values were close to the value of 43 mol% determined for the type strain of B. mojavensis by Roberts et al. (1994). The relatedness values of genomic DNA of the strains tested are showed in the Table 1. B. axarquiensis LMG 22476T and CIP 108772T, B. malacitensis LMG 22477T and CECT 5687T and B. mojavensis BCRC 17124T and BCRC 17531 showed high DNA–DNA relatedness values, in the range 83–98 %, indicating clearly that these taxa belong to the same genospecies. These strains showed less than 40 % relatedness with B. subtilis subsp. subtilis BCRC 10255T, B. vallismortis BCRC 17183T and B. amyloliquefaciens BCRC 11601T. The results contradict those obtained by Ruiz-García et al. (2005b), who reported a low hybridization value of less than 47 % between the type strains of these taxa.
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, who reported a significant quantity (11.5 %) of 16 : 0 fatty acid in B. axarquiensis CR-119T and no unsaturated fatty acids in B. axarquiensis CR-119T or B. malacitensis CR-95T.
Additional biochemical tests were performed using the API CH50 and 20E kits (bioMérieux) according to the manufacturer's instructions. Supplementary Table S2 shows differential biochemical features between the tested Bacillus strains. Very few of these characteristics could be used to differentiate B. axarquiensis and B. malacitensis from one another or from the other species. Only a negative oxidase test and positive reactions for acid production from gentiobiose and inulin differentiated B. axarquiensis from B. malacitensis and B. mojavensis. Additionally, B. axarquiensis LMG 22476T and CIP 108772T are both negative for arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase activities (not shown in Supplementary Table S2), which differed from the results obtained by Ruiz-García et al. (2005b).
Although RAPD analysis with primers OPA3 and OPL12 could be used to distinguish B. axarquiensis and B. malacitensis from one another and from B. mojavensis (Ruiz-García et al., 2005b), genetic heterogeneity in the same species has also been reported by some authors (Tynkkynen et al., 1999; Matarante et al., 2004; Naser et al., 2006).
Previous data reported by Ruiz-García et al. (2005b) gave DNA–DNA relatedness values for B. axarquiensis and B. malacitensis strains with strains of other closely related species that were lower than 46.9 %, which is much lower than the values (83–99 %) determined in our study. Our results show that the strains submitted to the culture collections are pure, and our DNA–DNA hybridization experiments clearly identify the B. axarquiensis and B. malacitensis type strains, as deposited in the culture collections, as B. mojavensis. The DNA–DNA hybridization experiments confirmed the results of our other investigation methods (16S rRNA and gyrB gene sequencing) used to differentiate between the species. In addition, these methods could clearly distinguish between type or reference strains of other closely related species. Our investigation shows clearly that the B. axarquiensis and B. malacitensis type strains can not be distinguished from the B. mojavensis type strain or another B. mojavensis reference strain at the species level. The data from the present study show that no significant phenotypic or genotypic differences exist to justify the separation of the type strains of B. axarquiensis and B. malacitensis and that of B. mojavensis. Consequently, it is proposed that Bacillus axarquiensis Ruiz-García et al. 2005 and Bacillus malacitensis Ruiz-García et al. 2005 should be considered as later heterotypic synonyms of Bacillus mojavensis Roberts et al. 1994.
Emended description of Bacillus mojavensis Roberts et al. 1994
The description is the same as that given by Roberts et al. (1994) except for the following traits. Oxidase, citrate utilization and nitrite reduction are variable. Does not hydrolyse L-rhamnose or D-galactose. Acid production from D-xylose, inositol, inulin, D-raffinose, gentiobiose, amygdalin and methyl 
-D-glucoside is strain-dependent. Major fatty acids are 15 : 0 anteiso, 15 : 0 iso, 17 : 0 iso and 17 : 0 anteiso. The DNA G+C content ranges from 43 to 44 mol%. Known habitats are desert soils and brackish sediments. Additional chemotaxonomic data and results of biochemical tests are given in Supplementary Tables S1 and S2.
The type strain is strain RO-H-1T (=NRRL B-14698T=DSM 9205T=LMG 17797T=CIP 104095T=ATCC 51516T=BCRC 17124T=NBRC 15718T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376.[CrossRef][Medline]
Felsenstein, J. (2002). PHYLIP (phylogeny inference package), version 3.6a. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, USA.
Fitch, W. M. (1971). Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20, 406–416.
Goris, J., Suzuki, K., De Vos, P., Nakase, T. & Kersters, K. (1998). Evaluation of a microplate DNA-DNA hybridization method compared with the initial renaturation method. Can J Microbiol 44, 1148–1153.[CrossRef]
Matarante, A., Baruzzi, F., Cocconcelli, P. S. & Morea, M. (2004). Genotyping and toxigenic potential of Bacillus subtilis and Bacillus pumilus strains occurring in industrial and artisanal cured sausages. Appl Environ Microbiol 70, 5168–5176.
Nakamura, L. K. (1989). Taxonomic relationship of black-pigmented Bacillus subtilis strains and a proposal for Bacillus atrophaeus sp. nov. Int J Syst Bacteriol 39, 295–300.
Nakamura, L. K., Roberts, M. S. & Cohan, F. M. (1999). Relationship of Bacillus subtilis clades associated with strains 168 and W23: a proposal for Bacillus subtilis subsp. subtilis subsp. nov. and Bacillus subtilis subsp. spizizenii subsp. nov. Int J Syst Bacteriol 49, 1211–1215.
Naser, S. M., Hagen, K. E., Vancanneyt, M., Cleenwerck, I., Swings, J. & Tompkins, T. A. (2006). Lactobacillus suntoryeus Cachat and Priest 2005 is a later synonym of Lactobacillus helveticus (Orla-Jensen 1919) Bergey et al. 1925 (Approved Lists 1980). Int J Syst Evol Microbiol 56, 355–360.
Page, R. D. M. (1996). TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357–358.
Palmisano, M. M., Nakamura, L. K., Duncan, K. E., Istock, C. A. & Cohan, F. M. (2001). Bacillus sonorensis sp. nov., a close relative of Bacillus licheniformis, isolated from soil in the Sonoran Desert, Arizona. Int J Syst Evol Microbiol 51, 1671–1679.[Abstract]
Priest, F. G., Goodfellow, M., Shute, L. A. & Berkeley, R. C. W. (1987). Bacillus amyloliquefaciens sp. nov., nom. rev. Int J Syst Bacteriol 37, 69–71.
Roberts, M. S., Nakamura, L. K. & Cohan, F. M. (1994). Bacillus mojavensis sp. nov., distinguishable from Bacillus subtilis by sexual isolation, divergence in DNA sequence, and differences in fatty acid composition. Int J Syst Bacteriol 44, 256–264.
Roberts, M. S., Nakamura, L. K. & Cohan, F. M. (1996). Bacillus vallismortis sp. nov., a close relative of Bacillus subtilis, isolated from soil in Death Valley, California. Int J Syst Bacteriol 46, 470–475.
Ruiz-García, C., Bejar, V., Martinez-Checa, F., Llamas, I. & Quesada, E. (2005a). Bacillus velezensis sp. nov., a surfactant-producing bacterium isolated from the river Velez in Malaga, southern Spain. Int J Syst Evol Microbiol 55, 191–195.
Ruiz-García, C., Quesada, E., Martinez-Checa, F., Llamas, I., Urdaci, M. C. & Bejar, V. (2005b). Bacillus axarquiensis sp. nov. and Bacillus malacitensis sp. nov., isolated from river-mouth sediments in southern Spain. Int J Syst Evol Microbiol 55, 1279–1285.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Skerman, V. B. D., McGowan, V. & Sneath, P. H. A. (editors) (1980). Approved lists of bacterial names. Int J Syst Bacteriol 30, 225–420.
Smith, N. R., Gibson, T., Gordon, R. E. & Sneath, P. H. A. (1964). Type cultures and proposed neotype cultures of some species in the genus Bacillus. J Gen Microbiol 34, 269–272.[Medline]
Stackebrandt, E., Frederiksen, W., Garrity, G. M., Grimont, P. A. D., Kämpfer, P., Maiden, M. C., Nesme, X., Rosselló-Mora, R., Swings, J. & other authors (2002). Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52, 1043–1047.[Abstract]
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.[CrossRef]
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Tynkkynen, S., Satokari, R., Saarela, M., Mattila-Sandholm, T. & Saxelin, M. (1999). Comparison of ribotyping, randomly amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis in typing of Lactobacillus rhamnosus and L. casei strains. Appl Environ Microbiol 65, 3908–3914.
Yamamoto, S. & Harayama, S. (1995). PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains. Appl Environ Microbiol 61, 1104–1109.[Abstract]
Zeigler, D. R. (2003). Gene sequences useful for predicting relatedness of whole genomes in bacteria. Int J Syst Evol Microbiol 53, 1893–1900.
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