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Agriculture and Agri-Food Canada, Research Centre, 430 Gouin Blvd, St-Jean-sur-Richelieu, Quebec, Canada J3B 3E6
Correspondence
Jean-Charles Côté
cotejc{at}agr.gc.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Published online ahead of print on 19 September 2002 as DOI 10.1099/ijs.0.02346-0.
The GenBank accession numbers for the 16S rDNA and 16S–23S ITS of different strains of Bacillus and related genera used in this work are AF478062–AF478111.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), the genus Bacillus has encompassed a variety of phenotypically heterogeneous species exhibiting a wide range of nutritional requirements, physiological and metabolic diversity and DNA base composition. Numerical classification based on a series of phenetic characters has been used for the classification of 368 Bacillus strains into 79 clusters (Priest et al., 1988). At about the same time, rRNA sequences were being established as a most useful molecular chronometer to infer phylogenetic relationships because they are present in all organisms and changes in the nucleotide sequences were deemed to occur in a clocklike manner (Woese, 1987). Soon, several Bacillus species were reclassified based on 16S rDNA sequence alignment. Rössler et al. (1991) grouped nine Bacillus species into four clusters. Ash et al. (1991) separated 51 Bacillus species into five phylogenetically distinct clusters. Further characterizations at the genotypic and phenotypic levels of selected Bacillus species have led to the creation of several new genera: Amphibacillus (Niimura et al., 1990), Alicyclobacillus (Wisotzkey et al., 1992), Paenibacillus (Ash et al., 1993), Aneurinibacillus and Brevibacillus (Shida et al., 1996a), Virgibacillus (Heyndrickx et al., 1998), Gracilibacillus and Salibacillus (Wainø et al., 1999), Filobacillus (Schlesner et al., 2001), Geobacillus (Nazina et al., 2001), Ureibacillus (Fortina et al., 2001), and Jeotgalibacillus and Marinibacillus (Yoon et al., 2001). Recently, partial 16S rDNA sequence (Goto et al., 2000) and rRNA gene restriction patterns (Joung & Côté, 2002) have been used for the rapid identification or classification of Bacillus species and related genera, respectively.
The 16S–23S internal transcribed spacer (ITS) region has been widely studied for the presence of functional motifs (Berg et al., 1989; Nodwell & Greenblatt, 1993; Pfeiffer & Hartmann, 1997), specific processing sites (Apirion & Miczak, 1993) and secondary structures (Nour, 1998; Liiv et al., 1998). Because the 16S–23S ITS region is hypervariable, as opposed to the more conserved 16S rRNA coding region, it has also been used in the study of prokaryotic diversity at the species and subspecies levels (Gürtler & Stanisich, 1996; García-Martínez et al., 1999). The ITS-PCR fingerprints have been used to reveal length polymorphisms between Bacillus species (Daffonchio et al., 1998a) and at the intra-specific level (Daffonchio et al., 1998b). Part of the ITS has been amplified by PCR and used as a probe for the detection, identification and phylotyping of Bacillus species (de Silva et al., 1998).
The current classification of species within the genus Bacillus and correlated genera is well established and is based on a combination of numerous experimental approaches. In the present study, we aimed to determine whether or not a combination of part of the 16S rRNA conserved sequence with part of the 16S–23S ITS hypervariable sequence could be informative enough to be used as a simple, useful marker for the classification of Bacillus species and related genera. A nucleotide sequence containing the last 150 bp located at the 3' end of the 16S rRNA coding region and the first 70 bp located at the 5' end of the 16S–23S ITS was used to infer the phylogenetic relationships between 46 Bacillaceae species and eight more distant bacterial species. The robustness of this classification will be assessed by comparison with the current Bacillaceae classifications.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). They were grown following ATCC guidelines (Pienta et al., 1996).
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DNA extraction.
For the total DNA isolation, bacterial cells were washed with TESS buffer [10 mM Tris/HCl, 1 mM Na2EDTA, 0·1 M NaCl and 0·1 % Sarkosyl (N-lauroylsarcosine)] and resuspended in TE buffer (10 mM Tris/HCl, 1 mM Na2EDTA). Cells were lysed with 50 mg lysozyme ml-1 and 0·1 % SDS. The subsequent phenol/chloroform extractions and ethanol precipitation were carried out as described by Sambrook et al. (1989).
Recombinant plasmid from E. coli strain TOP10 was isolated using the alkaline-lysis method (Stephen et al., 1990) with some modifications. Sodium acetate (pH 5·2) was used to replace potassium acetate for renaturing DNA. After removing cell debris and chromosomal DNA by centrifugation, an equal volume of 7·5 M ammonium sulfate was added to precipitate RNA. The RNA was removed by centrifugation at 13 000 r.p.m. for 20 min. Plasmid DNA was precipitated with 2 vols ethanol, and the pellet was air-dried and resuspended in sterile water.
Amplification of the 3' end 16S rDNA and the 16S–23S ITS region.
The 3' end of 16S rDNA, the 16S–23S ITS region and the 5' end of 23S rDNA was amplified with a pair of primers: L516SF (5'-TCGCTAGTAATCGCGGATCAGC-3') and L523SR (5'-GCATATCGGTGTTAGTCCCGTCC-3'). Amplification was performed in a Thermal Cycler 9600 (Perkin Elmer) in a total volume of 50 µl containing about 50 ng DNA, 0·25 µM each primer, 200 µM dNTP, 1·5 mM MgCl2 and 1·25 U Taq DNA polymerase (Qiagen). PCR was performed under the following conditions: 45 s at 95 °C and then 30 cycles of 15 s at 94 °C, 30 s at 53 °C and 90 s at 72 °C. Amplification products were visualized on agarose gels.
Cloning and sequencing methods.
The amplified DNAs were cloned into a pCRII-TOPO cloning vector using the TOPO TA cloning kit (Invitrogen), following the manufacturer's instructions. Transformants were selected on LB agar plates containing kanamycin (50 µg ml-1), X-Gal (40 µg ml-1) and IPTG (0·5 mM). A single clone was selected for each Bacillaceae species. The recombinant plasmids were isolated using the alkaline-lysis method, digested with EcoRI and visualized on agarose gels to confirm the presence of an inserted fragment.
The dideoxynucleotide chain-termination method (Sanger et al., 1977), using the near-infrared fluorescence automated DNA sequencer (LI-COR model 4200), was used to sequence the DNA fragments.
Sequence analysis.
The 3' end of the 16S rDNA and the 16S–23S ITS of the 40 Bacillaceae species sequenced in this study, as well as six other Bacillus sequences (Table 1
), and eight other sequences from more distant species available in the GenBank database (Clostridium perfringens, AB045290; Sarcina ventriculi, AF110272; Lactobacillus pantheris, AF413523; Desulfotomaculum kuznetsovii, AY036903; Thermoactinomyces vulgaris, AF138739; Streptococcus gallolyticus, AF323911; Deinococcus radiodurans, NC_001263; ‘Salmonella enterica’, NC_003198), were used for comparison. The sequences were aligned using the CLUSTAL W program (Thompson et al., 1994) and the most parsimonious phylogenetic trees were constructed using the DNAPARS program of the PHYLIP package, version 3.6a2 (Felsenstein, 1989, 2001). The order of the input sequences was randomized by DNAPARS. Stability of the groupings was estimated by bootstrap analysis on 100 trees using SEQBOOT in the same package. Trees were visualized using TREEVIEW software, version 1.6.1 (Page, 1996).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). A multiple alignment of the nucleotide sequences from these 46 Bacillaceae species was performed.
Conservation of the 3' end 16S rRNA coding region
Comparative analysis of the 3' end 16S rRNA coding region reveals that at least the last 157 bp share extensive nucleotide identities with all 46 Bacillaceae species (alignment available as supplementary data in IJSEM Online at http://ijs.sgmjournals.org). This sequence encompasses the 16S rRNA gene highly conserved regions 2, 3 and 4 (Lane et al., 1985; Weisburg et al., 1991; Gürtler & Stanisich, 1996) located at nucleotide positions -157 to -140, -52 to -38, and -20 to -4, respectively. Region 2 is believed to be the most highly conserved sequence in eubacteria, archaea and eukaryotes (Lane et al., 1985). Regions 3 and 4 are present in many eubacteria (Weisberg et al., 1991). Region 2 was identical for all 46 Bacillaceae species analysed, except for Bacillus anthracis for which the corresponding sequence was not available in full. Only Bacillus cereus showed a nucleotide substitution in region 3, at position -38, where deoxyadenosine is replaced by deoxyguanosine. All six Brevibacillus species, Brevibacillus agri, Brevibacillus borstelensis, Brevibacillus brevis, Brevibacillus choshinensis, Brevibacillus formosus and Brevibacillus parabrevis shared the same nucleotide substitution: C to A in region 4, at nucleotide position -18.
The alignment of the 157 bp sequences of the 3' end 16S rDNA of the 46 Bacillaceae species (Table 1) and species of five more Gram-positive genera, Clostridium perfringens, Sarcina ventriculi, Lactobacillus pantheris, Desulfotomaculum kuznetsovii and Thermoactinomyces vulgaris, two Gram-positive cocci, Streptococcus gallolyticus and Deinococcus radiodurans, and a Gram-negative, ‘Salmonella enterica’, was used to construct a phylogenetic tree (Fig. 1). All 46 Bacillaceae species listed in Table 1 are present in the main Group A. Lactobacillus pantheris, Sarcina ventriculi and Clostridium perfringens formed the small Group B, whereas Deinococcus radiodurans, ‘Salmonella enterica’, Desulfotomaculum kuznetsovii, Thermoactinomyces vulgaris and Streptococcus gallolyticus remained ungrouped. Within Group A, both Geobacillus species, all nine Paenibacillus species and all six Brevibacillus species can each be traced to respective common nodes, exclusive of other genera. Some closely related species, such as Bacillus atrophaeus and Bacillus mojavensis; Bacillus lentus and Bacillus insolitus; Geobacillus kaustophilus and Geobacillus stearothermophilus; Bacillus halodurans and Bacillus anthracis; Bacillus psychrophilus and Bacillus psychrosaccharolyticus; Brevibacillus borstelensis, Brevibacillus brevis and Brevibacillus formosus; Brevibacillus parabrevis and Brevibacillus agri; Paenibacillus gordonae and Paenibacillus validus; and Paenibacillus alvei, Paenibacillus macerans and Paenibacillus larvae, could not be distinguished from each other. Clearly, the 157 bp sequence at the 3' end 16S rDNA was sufficient to distinguish the bacterial genera under study, separate genera closely related to Bacillus (Group A) from other more distant genera and cluster species within genera for Geobacillus, Paenibacillus and Brevibacillus. It could not, however, distinguish closely related species.
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). The lengths of each 16S–23S ITS copy were 172, 230 and 348 bp for Bacillus amyloliquefaciens; 232, 303 and 344 bp for Bacillus fusiformis; 168, 172 and 334 bp for Bacillus licheniformis; and 222, 273, 304 and 432 bp for Bacillus lentus. Two incomplete copies of 567 and 629 bp were obtained for Brevibacillus borstelensis. In each species, only the longest 16S–23S ITS copies carried two tRNA genes: tRNAile and tRNAala, except Brevibacillus borstelensis for which both ITS copies (ITS 1 and ITS 2, Fig. 2c) harboured the two tRNA genes.
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) available from GenBank and the seven allelic sequences in Bacillus halodurans (data not shown). The conserved 5' end 16S–23S ITS was at least 70 bp long in each bacterial strain studied. For Brevibacillus borstelensis this conserved region between both alleles was nearly 400 bp long (Fig. 2c). In most cases, the central part of the 16S–23S ITS studied was less conserved. Deletions most often occurred in the region harbouring the tRNAile and tRNAala genes. Both tRNA genes were either coexistent or absent on the same allele. In no case was only either of the tRNA genes present. Whether or not the deletion of both tRNA genes on some alleles might be a consequence of gene regulation is debatable.
For some species, like Bacillus subtilis and Bacillus amyloliquefaciens, the 3' end of the 16S–23S ITS region was also highly conserved between alleles. For the other species under study, this region was either less conserved or the conserved region was short.
To determine whether or not the conservation of the first 70 bp of the 5' end of 16S–23S ITS allelic sequences within strains could be extended to other bacteria, copies of the 16S–23S ITS allelic sequences of seven Gram-positive bacteria available from GenBank were compared (alignment data not shown). These included Streptococcus pyogenes MGAS8232 (GenBank NC_003485), Streptococcus pyogenes M1 GAS (NC_002737), Streptococcus pneumoniae (NC_003098), Staphylococcus aureus (NC_003923), Listeria monocytogenes (NC_003210), Clostridium acetobutylicum (NC_003030) and Clostridium perfringens (NC_003366). They contained 6, 6, 4, 5, 6, 11 and 10 16S–23S ITS allelic sequences, respectively. The nucleotide sequence alignment of the 16S–23S ITS alleles showed that the first 70 bp were highly conserved between alleles of the same strain. They were also highly conserved between alleles of strains belonging to the same species, as exemplified by Streptococcus pyogenes MGAS8232 and Streptococcus pyogenes M1 GAS. The first 70 bp were not, however, conserved between alleles of different species of the same genus as exemplified by Streptococcus pyogenes and Streptococcus pneumoniae and by Clostridium acetobutylicum and Clostridium perfringens. This is in agreement with our results on selected species of Bacillus and related genera.
Bourque et al. (1995) have amplified, cloned and sequenced a single copy of the 16S–23S ITS of seven different Bacillus thuringiensis varieties and 18 Bacillus thuringiensis var. kurstaki strains. The length of the 16S–23S ITS for each Bacillus thuringiensis strain was around 144 bp and its nucleotide sequence was highly conserved throughout the strains. This suggests that the entire 16S–23S ITS region is conserved among alleles of any given Bacillus thuringiensis strains and between Bacillus thuringiensis varieties. Our results indicate that the 16S–23S ITS is not always conserved, even between allelic sequences within a bacterial strain, but the first 70 nt at the 5' end of 16S–23S ITS are conserved between alleles within strains and within species.
Although the sequences of the first 70 nt at the 5' 16S–23S ITS are conserved between the rrn alleles within a strain or within a species, the nucleotide identities between different species were quite variable (alignment data not shown). In some cases, no meaningful similarities in the 5' 16S–23S ITS region were observed between species of the same genus. The 5' end of the 16S–23S ITS nucleotide sequence could be species-specific and might provide a rapid, easy and useful marker for species discrimination and identification.
Phylogenetic analysis
The two highly conserved sequences identified above, the last 150 bp located at the 3' 16S rDNA and the first 70 bp located at the 5' 16S–23S ITS, were combined into a 220 bp sequence. A most parsimonious phylogenetic tree was constructed with the DNAPARS program of the PHYLIP package using the alignment of the 220 bp sequences of the 40 Bacillaceae species sequenced in this study and those of six Bacillus species available in GenBank (Fig. 3).
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. Although each group allowed for the two most distant species to share a Dmax no greater than 0·31, the highest Dmax observed here was 0·24 for species within Group VIII. Clearly each group could contain more distant Bacillaceae species, as long as they share a Dmax up to 0·31. Presumably, other and new Bacillaceae species could be added on this phylogenetic tree and many could fall into one of these 10 existing groups. Seven of the 10 groups were homogeneous. Groups II, V, VI and X contain seven, two, nine and five Bacillus species, respectively, exclusive of other genera. Bacillus subtilis, the Bacillus genus type species was found in Group VI. Group VII contains all six Brevibacillus species, exclusive of other genera. Likewise, Groups VIII and IX only contain six and three Paenibacillus species, respectively. The latter two groups originate from a single node and were later separated into two smaller branches using the grouping threshold value of 0·31. Both branches are within a Dmax value of 0·464.
In addition to Bacillus coagulans, Group I contains two Geobacillus species, Geobacillus stearothermophilus and Geobacillus kaustophilus. Interestingly, Priest et al. (1988), using numerical phenetic data, also classified these three species in the same cluster. Two other clusters, Groups III and IV, each contained two Bacillaceae species from two different genera, Bacillus laevolacticus and Virgibacillus pantothenticus in Group III, and Bacillus badius and Marinibacillus marinus in Group IV. Whether both groups are heterogeneous as suggested by the different genera names is not clear. Bacillus laevolacticus in Group III and Marinibacillus marinus in Group IV were not included in the studies of Priest et al. (1988) and Ash et al. (1991). When the genus Virgibacillus was created (Heyndrickx et al., 1998), Bacillus laevolacticus was not included in the study. Likewise, when the genus Marinibacillus was created (Yoon et al., 2001), Bacillus badius was not included in the analysis. It appears that the relationship between Bacillus laevolacticus and Virgibacillus pantothenticus (Group III), and between Bacillus badius and Marinibacillus marinus (Group IV) could still be open to debate.
Whereas the analysis of nucleotide identities in the 3' end 16S rDNA could distinguish Bacillaceae from other more distant bacterial taxa and distinguish between some Bacillaceae genera, additional analysis of nucleotide identities in the 5' 16S–23S ITS has added complementary information essential for further clustering of more closely related species. The present groupings based only on nucleotide identities in the 3' end 16S rDNA and 5' 16S–23S ITS are, in general, in agreement with current classifications based on series of phenetic and molecular data.
In the dendrogram presented here, the genus Bacillus still appears heterogeneous. Twenty-six Bacillus species are distributed in seven distinct groups (I, II, III, IV, V, VI and X), and a 27th, Bacillus circulans, is ungrouped. In contrast, the newly formed genera Brevibacillus and Paenibacillus are, respectively, more homogeneous. In addition, some Bacillus groups are quite distant from others as exemplified by Groups II and V with a Dmax value of 0·567. This is to be compared with a Dmax value of 0·459 between two different Bacillaceae genera, the Brevibacillus Group VII and the Paenibacillus Group IX. Interestingly, Dmax between both Paenibacillus Groups, VIII and IX, is 0·464. It would be worthwhile to further reassess the actual relatedness between the Bacillus species of the more distant groups to determine whether or not they are similar enough to be rightfully assigned to the same genus or whether the creation of novel genera would be warranted. If the latter holds true, a Dmax of 0·31, as used here, will prove useful not only at clustering Bacillaceae species, but also at suggesting the creation of novel genera.
It is noteworthy that in certain groups, some species were closely related with Dmax values less than 0·05. The species sharing a Dmax of 0·05 are indicated in Table 1. These species share a high proportion of identical nucleotides, not only over their 3' 16S rRNA coding region as expected, but also over their 5' 16S–23S ITS. In Group II, Bacillus psychrophilus and Bacillus psychrosaccharolyticus were within a Dmax of 0·035. The psychrophilic Bacillus species are all phenotypically related (Fox et al., 1992) and their taxonomy is based on a few discriminating properties (Larkin & Stokes, 1967; Abd El-Rahman et al., 2002). Although Bacillus psychrophilus has been unequivocally distinguished from Bacillus globisporus (Nakamura, 1984; Fox et al., 1992), the distinction between Bacillus psychrophilus and Bacillus psychrosaccharolyticus could still be open to debate. Both species, however, were located in different groups based on 16S rRNA sequences (Ash et al., 1991). In Group II, Bacillus simplex and ‘Bacillus maroccanus’, were within a Dmax of 0·011. Interestingly, Bacillus simplex and ‘Bacillus maroccanus’ share an almost identical 16S rRNA gene nucleotide sequence (Ash et al., 1991). ‘Bacillus maroccanus’ is not a validly published name and whether Bacillus simplex and ‘Bacillus maroccanus’ belong to the same species could also still be open to debate. In Group VI, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus mojavensis and Bacillus subtilis were within a Dmax of 0·020. Likewise, these Bacillus subtilis-like bacteria are phenotypically very similar (Chun & Bae, 2000) and share almost identical 16S rRNA gene sequences (Ash et al., 1991). Bacillus fusiformis and Bacillus sphaericus were highly similar with Dmax values as low as 0·007. They are also very similar at the phenotypic level (Priest et al., 1988) and their 16S rRNA gene sequence is very homologous (Ash et al., 1991). In Group X, Bacillus anthracis, Bacillus cereus, Bacillus mycoides and Bacillus thuringiensis were also very similar with a Dmax value of 0·047. The phenotypic and genotypic similarities between all four species has been well documented (Logan & Berkeley, 1984; Claus & Berkeley, 1986; Ash et al., 1991). Recently, Helgason et al. (2000) proposed to regroup Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis in a single species on the basis of genetic evidence. In Group VII, Brevibacillus brevis and Brevibacillus formosus were very close with a Dmax value of 0·025. The similarities between both species is well documented (Shida et al., 1995, 1996a, b). It is worth noting that Brevibacillus parabrevis isolates were originally classified as Brevibacillus brevis. They were later separated into two distinct species (Takagi et al., 1993). Strain ATCC 8264T was retained as the Brevibacillus brevis type strain. Strain ATCC 8186 was assigned to Brevibacillus parabrevis. In our study, the Dmax between both strains was nearly 0·1, in agreement with their separation into two species. In Group VIII, Paenibacillus gordonae and Paenibacillus validus share a Dmax value of 0·049, suggesting they might be the same species. Interestingly, both species have been proposed to be reclassified into a single species under the name Paenibacillus validus (Heyndrickx et al., 1995). As seen here, species separated by Dmax values within 0·05 are often phenotypically and genotypically very similar. The Dmax threshold of 0·05 may prove very useful in identifying species for which further analysis, including DNA–DNA reassociation, may be necessary to clarify whether or not their separation into different species was warranted.
The use of Dmax thresholds of 0·31 and 0·05, as used here for Bacillaceae classification, based only on a combination of a 150 bp sequence located at the 3' end of the 16S rRNA gene and a 70 bp sequence at the 5' end of the ITS, is a simple, rapid approach, suited to larger screening programs and easily accessible to most laboratories. It may reveal, in a different way, Bacillaceae species for which further characterization, including thorough phenotypic comparison, 16S rRNA sequence data and DNA–DNA hybridization, might be warranted for proper classification and/or reassignment at the genus or species level, respectively. Whether these Dmax thresholds may prove useful for other bacterial taxa as well remains to be assessed.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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