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1 Bioresource Collection and Research Center, Food Industry Research and Development Institute, PO Box 246, Hsinchu 30099, Taiwan
2 Marine Biotechnology Institute, Heita, Kamaishi, Iwate, 026-0001, Japan
Correspondence
Fwu-Ling Lee
fll{at}firdi.org.tw
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ABSTRACT |
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Details of strains and sequence accession numbers and a table of DNA–DNA reassociation values and gyrB and 16S rRNA gene sequences similarities are available as supplementary material 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), Bacillus licheniformis (Skerman et al., 1980), Bacillus amyloliquefaciens (Priest et al., 1987), Bacillus atrophaeus (Nakamura, 1989), Bacillus mojavensis (Roberts et al., 1994), Bacillus vallismortis (Roberts et al., 1996), Bacillus subtilis subsp. spizizenii (Nakamura et al., 1999) and Bacillus sonorensis (Palmisano et al., 2001). These taxa can be differentiated from one another by fatty acid composition analysis, restriction digest analysis and DNA–DNA hybridization analysis, but are quite difficult to differentiate by phenotypic characteristics (Roberts et al., 1994; Nakamura et al., 1999).
16S rRNA gene sequence analysis is the most commonly used method for identifying bacteria or for constructing bacterial phylogenetic relationships (Woese, 1987; Vandamme et al., 1996; Joung & Cote, 2002); however, its usefulness is limited because of the high percentage of sequence similarity between closely related species (Ash et al., 1991; Martínez-Murcia et al., 1992; Christensen et al., 1998). The use of protein-encoding genes as phylogenetic markers is now a common approach (Yamamoto & Harayama, 1998; Ko et al., 2004; Chelo et al., 2007). Detailed investigations have demonstrated that sequences from protein-encoding genes can accurately predict genome relatedness and may replace DNA–DNA hybridization for species identification and delineation in the future (Stackebrandt et al., 2002; Zeigler, 2003).
The gyrB gene encodes the subunit B protein of DNA gyrase, a type II DNA topoisomerase, which plays an essential role in DNA replication and is distributed universally among bacterial species (Watt & Hickson, 1994; Huang, 1996). The rate of molecular evolution inferred from gyrB gene sequences is faster than that inferred from 16S rRNA gene sequences (Yamamoto & Harayama, 1995). gyrB gene sequences have been used in phylogenetic studies of Pseudomonas (Yamamoto & Harayama, 1998), Acinetobacter (Yamamoto & Harayama, 1996; Yamamoto et al., 1999), Mycobacterium (Kasai et al., 2000; Niemann et al., 2000), Salmonella, Shigella and Escherichia coli (Fukushima et al., 2002), Aeromonas (Yáñez et al., 2003) and the Bacillus anthracis–cereus–thuringiensis group (La Duc et al., 2004); results from these studies have indicated that gyrB is a suitable phylogenetic marker for the study of phylogenetic and taxonomic relationships at the species level. In the present study, it has been shown that direct sequencing of the gyrB gene could be used for identification and phylogenetic analysis of species of the B. subtilis group.
A total of eight Bacillus type strains and 24 Bacillus reference strains were used in this study (see Supplementary Table S1 available in IJSEM Online). They were obtained from the Bioresource Collection and Research Center (BCRC; http://wdcm.nig.ac.jp/CCINFO/CCINFO.xml?59). All strains were cultivated on nutrient agar or in nutrient broth (Difco) at 30 °C for 24 h under aerobic conditions.
Genomic DNA was extracted using the Qiagen Blood & Cell Culture DNA kit. DNA–DNA relatedness values were determined using the fluorometric hybridization method in microdilution wells as described previously (Ezaki et al., 1989; Chern et al., 2004; Tai et al., 2006).
The gyrB gene was amplified by PCR as described previously (Yamamoto & Harayama, 1995). PCR was performed using the Takara Ex Taq kit. PCR products were purified with the PCR-M clean up system (Viogene) and sequenced with a BigDye Terminator v3.1 cycle-sequencing kit on a 3730 DNA sequencer (Applied Biosystems and Hitachi). DNA sequencing was determined using gyrB degenerate primers UP-1S and UP-2Sr (Yamamoto & Harayama, 1995) and BS-F (5'-GAAGGCGGNACNCAYGAAG-3') and BS-R (5'-CTTCRTGNGTNCCGCCTTC-3') (designed from conserved regions of gyrB nucleotide sequences of members of the B. subtilis group) at 3.2 µM concentration. The DNA sequence was double-checked by sequencing both strands. Approximately 1.5 kb of the 16S rRNA gene was determined using the MicroSeq Full Gene 16S rDNA Bacterial Identification kit (Applied Biosystems).
Sequence similarities were calculated using programs of the Wisconsin Package, version 10.1 (Accelrys). Multiple sequences were aligned using the program CLUSTAL_X, version 1.8 (Thompson et al., 1997). Phylogenetic analysis was performed using PHYLIP (Felsenstein, 1993) and MEGA (Kumar et al., 2004). Evolutionary distances were calculated by Kimura's two-parameter model (Kimura, 1980). Phylogenetic trees were constructed by the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) methods with bootstrap values based on 1000 replications.
Approximately 1.2 kb of the gyrB gene was successfully amplified using universal primers UP-1 and UP-2r for all Bacillus species examined in this study. After direct sequencing, 1171 bp gyrB gene sequences, corresponding to nt 316–1480 of the Escherichia coli K-12 sequence with gaps, and 1468 bp 16S rRNA gene sequences, corresponding to nt 30–1489 of the E. coli K-12 sequence with gaps, were used for analysis and the resulting data were deposited in GenBank/EMBL/DDBJ. Accession numbers of gyrB and 16S rRNA gene sequences are listed in Supplementary Table S1.
Species identification and phylogenetic analysis of B. subtilis and related taxa based on gyrB gene sequence analysis
The gyrB gene sequence similarities between the eight type strains were 75.4–95.0 % (mean 81.6 %) (see Supplementary Table S2). The gyrB translated amino acid sequence similarity values were 88.5–99.2 % (mean 92.5 %) (data not shown). In contrast, 16S rRNA gene sequence similarities between the same strains were 98.1–99.8 % (mean 98.9 %). It is clear from comparative sequence analysis that the base substitution rate of the gyrB gene sequence was much faster than that of the 16S rRNA gene sequence and that the gyrB nucleotide sequence showed significantly higher genetic variation than the translated amino acid sequence.
The gyrB gene sequence showed remarkable discrimination (75.4–91.9 %). At the intraspecies level, the nucleotide substitution rates were 0–5 % and were <2 % for most Bacillus species. At the interspecies level, the nucleotide substitution rates were usually >7 %, except for B. vallismortis and B. subtilis subsp. spizizenii, the most similar pair (6.1 % sequence divergence). At the subspecies level, the gyrB gene sequence could be used to discriminate B. subtilis subsp. subtilis from B. subtilis subsp. spizizenii (5 % sequence divergence). This result was consistent with that of gyrA gene sequence analysis (4.8 % sequence divergence) (Chun & Bae, 2000). The 16S rRNA gene sequences of all Bacillus strains tested revealed more than 98 % similarity. These results indicate that the gyrB gene is more useful than the 16S rRNA gene for species and subspecies identification in the B. subtilis group.
The phylogenetic trees constructed from the 16S rRNA and gyrB gene sequences of the 32 Bacillus strains are shown in Fig. 1. The gyrB-based tree clearly delineated four distinct clusters with high bootstrap values (100 %): cluster 1 contained B. subtilis, B. vallismortis and B. mojavensis strains; clusters 2 and 3 contained strains of B. atrophaeus and B. amyloliquefaciens, respectively; and cluster 4 contained B. sonorensis and B. licheniformis strains (Fig. 1). Of the B. subtilis strains, all strains of B. subtilis subsp. subtilis formed a monophyletic clade with 100 % bootstrap support; the gyrB gene sequence similarities among them were 98.1–99.1 % (mean 98.7 %) (data not shown) and the sequence divergence between the B. subtilis subsp. subtilis and B. subtilis subsp. spizizenii strains was 5.0 %. Comparatively, the 16S rRNA gene-based tree yielded two clusters with a bootstrap value of 79 %: cluster 1 contained strains of B. subtilis, B. mojavensis, B. vallismortis, B. atrophaeus and B. amyloliquefaciens and cluster 2 contained strains of B. sonorensis and B. licheniformis. All Bacillus strains among these two cluster groups showed more than 99 % 16S rRNA gene sequence similarity, indicating that the gyrB gene is a better molecular marker than the 16S rRNA gene for the study of phylogenetic and taxonomic relationships at the species level in the B. subtilis group.
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; Stackebrandt et al., 2002). Reassociation values obtained from DNA–DNA hybridization analyses among the type strains and other Bacillus strains examined in this study were determined (data not shown). The DNA–DNA reassociation values between the eight type strains were low, 11–67 % (Supplementary Table S2). Nakamura et al. (1999) divided B. subtilis into B. subtilis subsp. subtilis and Bacillus subtilis subsp. spizizenii mainly using DNA–DNA reassociation data. DNA–DNA relatedness (67 %) and 16S rRNA gene sequence similarity (99.8 %) indicated that these two taxa are closely related; however, the gyrB gene sequence can be used to discriminate between them (95 % sequence similarity). Pairwise analyses indicated that gyrB gene sequences (75.4–95.0 % similarity) are more discriminatory than 16S rRNA gene sequences (98.1–99.8 % similarity) for species differentiation. The gyrB gene sequence-based phylogenetic analysis was consistent with DNA–DNA reassociation data and a linear correlation was observed between gyrB gene sequence similarities and levels of DNA–DNA relatedness (Fig. 2). All strains that exhibited 95.5–100 % gyrB gene sequence similarity showed high DNA–DNA relatedness (70–100 %), suggesting that these strains are conspecific. Strains with approximately 95 % or higher gyrB gene sequence similarity in the cluster exhibited DNA–DNA relatedness of >70 %, an acceptable value for proposal of a single species. Strains with 93–95 % gyrB gene sequence similarity exhibited DNA–DNA relatedness of 60–70 % without exception, indicating that they are grouped at the subspecies level. Exceptionally, B. subtilis subsp. spizizenii BCRC 17366T and B. vallismortis BCRC 17183T showed 93.9 % gyrB gene sequence similarity and 52 % DNA–DNA relatedness. This finding indicated that it might be necessary to use several gene sequences and DNA–DNA hybridization to discriminate species relationships. Nevertheless, based on data obtained from the present study, the gyrB gene sequences have been shown to be a more efficient phylogenetic tool than the 16S RNA gene sequences for discriminating between species of this group.
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ACKNOWLEDGEMENTS |
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