Introduction

The genus Bacillus encompasses the Gram-positive, rod-shaped, aerobic, spore-forming bacteria (Claus and Berkeley 1986). The so-called Bacillus cereus sensu lato group comprises six highly related species: B. cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides and Bacillus weihenstephanensis. The species B. thuringiensis is distinguished from the other Bacillus species by the synthesis, upon sporulation, of a proteinaceous parasporal inclusion body, the crystal. In some strains, this crystal has been shown to be specifically toxic to some insect pests. Over the last century, some B. thuringiensis strains have been developed as biological insecticides for the control of lepidopteran, dipteran or coleopteran pests, respectively (Höfte and Whiteley 1989; Glare and O’Callaghan 2000). Several screening programs have been established in the hope of isolating novel B. thuringiensis strains expressing novel insecticidal, pesticidal and biological activities. As a result, by 1996, 50,000 B. thuringiensis strains were kept in various collections worldwide (Sanchis et al. 1996). H serotyping, the immunological reaction to the bacterial flagellar antigen, has been established as the method of choice for the classification of the wide diversity of B. thuringiensis strains (de Barjac and Bonnefoi 1962). At least 69 B. thuringiensis H serotypes and 80 serological varieties (serovars), have now been characterized (Lecadet et al. 1999). H serotyping, however, is limited in its capability to distinguish strains from the same H serotype by the rarity of antigenic subfactors and will not distinguish strains from the same serovars.

The 16S rRNA gene (which codes for the RNA component of the ribosome small subunit) is the golden standard for single-gene phylogenetic analyses (Woese 1987; Woese et al. 1990). The gyrB gene (a house-keeping gene encoding the subunit B protein of DNA gyrase, topoisomerase type I) (Yamamoto and Harayama 1995) and aroE gene (a house-keeping gene encoding the shikimate dehydrogenase) (Maiden et al. 1998) have also been used to complement the 16S rRNA gene in bacterial phylogenetic analysis. Both house-keeping genes are present in most bacteria, are not transmitted horizontally, and their evolutionary rate is higher than that of the 16S rRNA (Yamamoto and Harayama 1995; Maiden et al. 1998). Recently, the gyrB gene has proven useful at distinguishing between members of the B. cereus sensu lato group (Bavykin et al. 2004; Ko et al. 2004; La Duc et al. 2004; Park et al. 2007).

We set to investigate the capability of each gene, 16S rRNA, gyrB and aroE, to discriminate among, first, B. thuringiensis H serotypes; second, serovars from the same H serotype; and third, strains from the same serovar.

We report here the 16S rRNA, gyrB and aroE genes nucleotide sequences from B. thuringiensis strains which encompass 21 H serotypes. Sequences from 16 additional strains from four species in the Bacillus cereus group sensu lato (B. cereus, B. anthracis, B. mycoides and B. weihenstephanensis) were also included for comparison purposes. These sequences were pair-wise compared and phylogenetic relationships were revealed. The level of resolution of each gene nucleotide sequence in discriminating among B. thuringiensis H serotypes is discussed. The gyrB gene was retained for the subsequent analyses on B. thuringiensis serovars from the same H serotype and strains from the same serovar.

Materials and methods

Bacterial strains, culture conditions and DNA isolation

The 74 bacterial strains used in this study and their sources are listed in Tables 1 and 3. The 40 strains included in Table 1 encompass 24 B. thuringiensis strains from 21 H serotypes, four B. cereus, three B. anthracis, four B. mycoides and five B. weihenstephanensis strains. The 42 B. thuringiensis strains included in Table 3 encompass 12 H serotypes and 25 serovars. A B. thuringiensis serovar is a serological variety that share common flagellar antigens, including, when present, common distinct antigenic subfactors. Hence, whereas most B. thuringiensis H serotypes contain a single serovar, some have been further divided into two or more serovars (Table 3). A B. thuringiensis H serotype is designated for the letter H followed by a number. A serovar is given a latine name (Lecadet et al. 1999). Eight B. thuringiensis strains are common to Tables 1 and 3. The genomes had been fully sequenced for eight of the 74 bacterial strains: the three B. anthracis strains, three of the four B. cereus strains, B. thuringiensis serovar konkukian and B. weihenstephanensis strain KBAB4. Their sequences were available from GenBank (Table 1; http://www.ncbi.nlm.nih.gov/). The total DNAs from two B. weihenstephanensis strains, WSBC 10001 and WSBC 10365, were kindly provided by Dr. Monika Ehling-Schulz, Technical University of Munich, Germany. These ten strains were not cultured in the present study. The other 64 bacterial strains were cultured and their total DNAs isolated as described previously (Xu and Côté 2003, 2006). E. coli strain TOP10 (Invitrogen Canada, Burlington, ON, Canada) was used for cloning PCR fragments. It was cultured as described elsewhere (Xu and Côté 2003, 2006).

Table 1 Bacillus species and strains used in Figs. 3–5
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PCR amplification

16S rRNA gene

The 16S rRNA gene was amplified with primer pair 16S-F1/-R1: 16S-F1 (5′-GAACGCTGGCGGCGTGCCTAA-3′) and 16S-R1 (5′-GCACCTTCCGATACGGCTACC-3′). Primers were designed based on the consensus sequence of the 16S rRNA gene in B. thuringiensis serovar konkukian, B. halodurans, B. licheniformis, B. cereus E33L and B. anthracis Ames Ancestor, all publicly available from GenBank. DNA was amplified for 30 cycles at a denaturing temperature of 95°C for 60 s, annealing at 63°C for 45 s, and extension at 72°C for 45 s.

gyrB gene

The gyrB gene was amplified with primer pair gyrB-F1/-R1: gyrB-F1 (5′-ATGGAACAAAAGCAAATGCA-3′) and gyrB-R1 (5′-TTAAATATCAAGGTTTTTCA-3′). Primers were designed based on the consensus sequence of the gyrB gene in B. thuringiensis serovar konkukian, and B. cereus strains E33L, ATCC 10987 and ATCC 14579, and B. anthracis strains Ames Ancestor, Ames and Sterne. A second primer pair gyrB-F2/-R2: gyrB-F2 (5′-ACWCGTATGCGTGARYTRGC-3′) and gyrB-R2 (5′-CCTTGYTTTGCWGAWCCDCC-3′) was developed to amplify the central region of gyrB gene. These primers were designed based on the partial sequence of the gyrB gene determined with the gyrB-F1/-R1 primer pair. With the gyrB-F1/-R1 primer pair, DNA was amplified for 30 cycles at a denaturing temperature of 95°C for 30 s, annealing at 46°C for 30 s, and extension at 72°C for 30 s. With the gyrB-F2/-R2 primer pair, DNA was amplified for 30 cycles at a denaturing temperature of 95°C for 45 s, annealing at 50°C for 30 s, and extension at 72°C for 60 s.

aroE gene

The aroE gene was amplified with primer pair aroE-F1/-R1: aroE-F1 (5′-ATCGGAAATCCAATTGGACA-3′) and aroE-R1 (5′-CCTGTCCACATTTCAAAYGC-3′). Primers were designed based on the consensus sequence of the aroE gene in B. thuringiensis serovar konkukian, B. cereus strains E33L, ATCC 10987 and ATCC 14579, and B. anthracis strains Ames Ancestor, Ames and Sterne. DNA was amplified for 30 cycles at denaturing temperature of 95°C for 30 s, annealing at 47°C for 30 s, and extension at 72°C for 60 s.

Positions of primers respective to the B. thuringiensis serovar konkukian 16S rRNA, gyrB and aroE genes are shown in Fig. 1a, b and c, respectively. All amplified products were visualized on a 0.7% agarose gel and stored at −20°C.

Fig. 1
figure 1

Map of the Bacillus thuringiensis serovar konkukian 16S rRNA (a), gyrB (b) and aroE (c) genes. The number 1 refers to the first nucleotide of the three gene coding regions. Arrows indicate the orientations and positions of the primer pairs used for amplification. For the 16S rRNA gene, the primer pair 16S-F1/-R1 is at nucleotide positions 4-24/1487-1507. For the gyrB gene, the primer pairs gyrB-F1/-R1 and gyrB-F2/-R2 are at nucleotide positions 1-20/1904-1923 and 580-599/1303-1322, respectively. For the aroE gene, the primer pair aroE-F1/-R1 is at nucleotide positions 22-41/756-776

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DNA cloning and sequencing

Cloning of the amplified products, selection of transformants, isolation and analyses of recombinant plasmids, and determination of the nucleotide sequences were done as described previously (Xu and Côté 2006). The 16S rRNA, gyrB and aroE genes sequences of B. thuringiensis serovar konkukian; B. cereus strains ATCC 14579, ATCC 10987, E33L; B. weihenstephanensis strain KBAB4 and B. anthracis strains Ames Ancestor, Ames and Sterne were retrieved directly from GenBank.

Percentage sequence similarity among B. thuringiensis H serotypes

The multiple alignments of the nucleotide sequences of the orthologous 16S rRNA, gyrB and aroE genes for 21 B. thuringiensis H serotypes listed in Table 1 were done using Clustal W (Thompson et al. 1994). Pairwise comparisons revealed respective percentage sequence similarities. The discriminatory power (DP) was defined here based on the median value of percentage sequence similarities among B. thuringiensis at the H serotype level. Lower median values reflected lower percentage sequence similarities and, consequently, higher discriminating power among B. thuringiensis serovars.

Phylogenetic analysis

The DNA sequences were analyzed using MEGA (Molecular Evolutionary Genetics Analysis; version 3.1) (Kumar et al. 2004). The 16S rRNA, gyrB, and aroE nucleotide sequences from 24 B. thuringiensis strains, four B. cereus, four B. mycoides, five B. weihenstephanensis and three B. anthracis strains were aligned using Clustal W (Thompson et al. 1994), a neighbor-joining tree was constructed (Saitou and Nei 1987), bootstrapped using 1,000 random samples of sites from the alignment, displayed and printed with Tree Explorer, all using MEGA.

Nucleotide sequence accession number

Sequence data from this study have been deposited with GenBank under accession no. EF210214 to EF210315 and EU761164 to EU761197.

Results

16S rRNA, gyrB and aroE genes polymorphism

The nucleotide sequences of the 16S rRNA, gyrB and aroE genes were analyzed in the 40 bacterial strains listed in Table 1. These included 24 B. thuringiensis strains from 21 H serotypes, and 16 strains from four other species in the Bacillus cereus group sensu lato. Although most B. thuringiensis strains were selected from different H serotypes, some serovars were, however, selected purposely from same H serotype (i.e. H3 serovars alesti and fukuokaensis, and H4 serovars sotto and kenyae), and some strains from same serovar (i.e. H2 serovar finitimus, strains BGSC 4B1 and BGSC 4B2) in the hope of obtaining a first indication of the level of resolution each gene nucleotide sequence could provide. The nucleotide sequences of the entire genomes were publicly available for 8 of the 40 bacterial strains: B. thuringiensis serovar konkukian, three B. cereus strains (ATCC 14579, ATCC 10987 and E33L), B. weihenstephanensis strain KBAB4 and the three B. anthracis under study. Their 16S rRNA, gyrB and aroE gene nucleotide sequences were retrieved directly from GenBank. All three genes were amplified, cloned and their nucleotide sequences determined for the 32 other bacterial strains.

A single pair of primers, 16S-F1/-R1 (Fig 1a) proved sufficient to amplify the 16S rRNA gene from all 32 bacterial strains. All strains yielded a single amplicon of 1.5 kbp (later determined at 1,461 bp) in length, primers not included. Likewise, a first pair of primers, gyrB-F1/-R1 (Fig 1b), proved sufficient to amplify the gyrB gene from all 32 bacterial strains. Here also, all strains yielded a single amplicon of 1.9 kbp (1,881 bp) in length, primers not included. To facilitate the determination of the nucleotide sequence of the amplicon central region, a second primer pair, gyrB-F2/-R2 (Fig 1b), was designed for the amplification of the central region. Finally, a single pair of primers, aroE-F1/-R1 (Fig 1c), proved sufficient to amplify the aroE gene from all 32 bacterial strains. Here also, all strains yielded a single amplicon of 0.7 kbp (714 bp) in length, primers not included. Subsets of the amplification products for the 16S rRNA, gyrB and aroE genes for selected bacterial strains are shown in Fig. 2a, b and c, respectively.

Fig. 2
figure 2

Agarose gel electrophoresis of 16S rRNA (a), gyrB (b) and aroE (c) gene amplification products in selected strains using specific primers. a Amplification products using the 16S-F1/-R1 primer pair. Lanes: 1, molecular weight marker, 100-bp DNA ladder; 2, Bacillus thuringiensis serovar tolworthi; 3, B. thuringiensis serovar tohokuensis; 4, B. thuringiensis serovar vazensis; 5, B. thuringiensis serovar graciosensis; 6, B. thuringiensis serovar roskildiensis; 7, B. thuringiensis serovar polonensis; 8, B. mycoides ATCC 6462; 9, B. mycoides BGSC 6A13; 10, B. mycoides BGSC 6A19; 11, B. cereus BGSC 6A17; 12, B. weihenstephanensis CCM 4965; 13, B. weihenstephanensis CCM 4966. b Amplification products using the gyrB-F1/-R1 primer pair. Lanes: 1, Molecular weight marker, 100-bp DNA ladder; 2, Bacillus thuringiensis serovar tolworthi; 3, B. thuringiensis serovar tohokuensis; 4, B. thuringiensis serovar vazensis; 5, B. thuringiensis serovar roskildiensis; 6, B. thuringiensis serovar polonensis; 7, B. mycoides ATCC 6462; 8, B. mycoides BGSC 6A13; 9, B. cereus BGSC 6A17; 10, B. weihenstephanensis CCM 4965; 11, B. weihenstephanensis CCM 4966. c Amplification products using the aroE-F1/-R1 primer pair. Lanes: 1, Molecular weight marker, 100-bp DNA ladder; 2, Bacillus thuringiensis serovar tolworthi; 3, B. thuringiensis serovar tohokuensis; 4, B. thuringiensis serovar vazensis; 5, B. thuringiensis serovar roskildiensis; 6, B. thuringiensis serovar polonensis; 7, B. mycoides ATCC 6462; 8, B. weihenstephanensis CCM 4965; 9, B. weihenstephanensis CCM 4966

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16S rRNA, gyrB and aroE genes nucleotide sequences-inferred phylogenetic analyses

Three bootstrapped neighbor-joining trees were generated from the alignment of the nucleotide sequences of the 16S rRNA, gyrB and aroE gene amplicons. They are presented in Figs. 3–5, respectively. Figure 3 shows the phylogenetic relationships between the 16S rRNA gene nucleotide sequences for the 40 bacterial species and strains shown in Table 1. Two clusters, I and II, were revealed at the 99% nucleotide sequence identities. Species-wise, the three B. anthracis strains are grouped together in cluster I and appear undistinguishable from each other. The four B. cereus strains are in cluster I. Two of the four B. mycoides strains, BGSC 6A13 and BGSC 6A19, are located in cluster I whereas the other two, BGSC 6A20 and ATCC 6462, are located in cluster II. The five B. weihenstephanensis strains are found in cluster II. All B. thuringiensis H serotypes are found in cluster I, with the exceptions of H63 (serovar bolivia) and H67 (serovar vazensis) which are found in cluster II alongside the B. weihenstephanensis strains. Two H serotypes, H3 and H4, contained two serovars each. The two H3 serovars, fukuokaensis and alesti, and the two H4 serovars, kenyae and sotto, are all found in cluster I, on different locations. Not only can B. thuringiensis H serotypes be distinguished based on 16S rRNA gene nucleotide sequences, but the same is true for both pairs of serovars from the same H serotype studied here. Interestingly enough, the two strains from the same serovar studied here (serovar finitimus, strains BGSC 4B1 and BGSC 4B2) are, as expected, found next to each other in cluster I, yet different enough as to be distinguishable.

Fig. 3
figure 3

Bootstrapped neighbor-joining tree of Bacillus thuringiensis (Bt), B. cereus, B. mycoides and B. weihenstephanensis generated from the alignment of 16S rRNA nucleotide sequences. Bootstrap values higher than 30% are indicated. Roman numerals, I and II, refer to the two clusters revealed at the 99% nucleotide sequence identities. The horizontal bar represents 0.005% differences in nucleotide identities. Abbreviations: Bt: Bacillus thuringiensis; Bc: B. cereus; Bm: B. mycoides; Bw: B. weihenstephanenesis

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Figure 4 shows the phylogenetic relationships between the gyrB gene nucleotide sequences for the same 40 bacterial species and strains (Table 1). Seven clusters, I–VII, were revealed at the 95% nucleotide sequence identities. Species-wise, here also the three B. anthracis strains are grouped together in cluster I and appear undistinguishable from each other. The four B. cereus strains are found in three clusters. Strains ATCC 14579 and BGSC 6A17 are grouped together in cluster III while the other two, ATCC 10987 and E33L, are located in clusters I and IV, respectively. The four B. mycoides strains are found in three clusters. Strains BGSC 6A20 and ATCC 6462 are grouped together in cluster VII while the other two, BGSC 6A13 and BGSC 6A19, are located in cluster II and VI, respectively. The five B. weihenstephanensis strains are found in cluster VII. All B. thuringiensis H serotypes are distributed in clusters I–V with the exception of B. thuringiensis H67 (serovar vazensis) which is found in cluster VII alongside the B. weihenstephanensis strains. Cluster VI contains no B. thuringiensis H serotype. The two H3 serovars, fukuokaensis and alesti, are both found in cluster V. The two H4 serovars, kenyae and sotto, are found in different clusters, III and V, respectively. Here also, B. thuringiensis H serotypes can be distinguished based on gyrB gene nucleotide sequences. This is true also for serovars from the same H serotype and for strains from the same serovar as exemplified here again by both B. thuringiensis serovar finitimus strains found next to each other, in cluster I, yet different enough as to be distinguishable.

Fig. 4
figure 4

Bootstrapped neighbor-joining tree of Bacillus thuringiensis (Bt), B. cereus, B. mycoides and B. weihenstephanensis generated from the alignment of gyrB nucleotide sequences. Bootstrap values higher than 30% are indicated. Roman numerals, I–VII, refer to the seven clusters revealed at the 95% nucleotide sequence identities. The horizontal bar represents 0.005% differences in nucleotide identities. Abbreviations are as indicated in Fig. 3

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Figure 5 shows the phylogenetic relationships between the aroE gene nucleotide sequences for the same 40 bacterial species and strains (Table 1). Five clusters, I–V, were revealed at the 95% nucleotide sequence identities. Species-wise, here also, the three B. anthracis strains are grouped together in cluster III and appear undistinguishable from each other. The four B. cereus strains are found in two clusters. Strains BGSC 6A17 and ATCC 14579 are located in cluster I whereas the other two, ATCC 10987 and E33L are located in cluster III. The four B. mycoides strains are found in three different clusters. Strains BGSC 6A13, BGSC 6A20, ATCC 6462 and BGSC 6A19 are found in clusters III, IV, IV and V, respectively. The five B. weihenstephanensis strains are found in cluster IV. The B. thuringiensis H serotypes are distributed in each of the five clusters. Interestingly, here also, B. thuringiensis H67 (serovar vazensis) is found alongside the B. weihenstephanensis strains. B. thuringiensis H63 (serovar bolivia) is also found in this same cluster. The two H3 serovars, fukuokaensis and alesti, are found in cluster I close to each other. The two H4 serovars, kenyae and sotto, are found on separate locations in cluster I. Here also, B. thuringiensis H serotypes can be distinguished based on aroE gene nucleotide sequences. This is true also for serovars from the same H serotype. Interestingly enough, however, strains from the same serovar, here the two B. thuringiensis serovar finitimus strains, are found next to each other in cluster III and appear undistinguishable.

Fig. 5
figure 5

Bootstrapped neighbor-joining tree of Bacillus thuringiensis (Bt), B. cereus, B. mycoides and B. weihenstephanensis generated from the alignment of aroE nucleotide sequences. Bootstrap values higher than 30% are indicated. Roman numerals, I–V, refer to the five clusters revealed at the 95% nucleotide sequence identities. The horizontal bar represents 0.005% differences in nucleotide identities. Abbreviations are as indicated in Fig. 3

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Percentage sequence similarity among B. thuringiensis H serotypes

Pairwise 16S rRNA, gyrB and aroE gene nucleotide sequence comparisons among the 21 B. thuringiensis H serotypes listed in Table 1 revealed respective percentage sequence similarities (supplementary data). The discriminatory power (DP) of the 16S rRNA gene was compared with the DP of the gyrB and aroE genes. The 16S rRNA gene showed the highest median value of percentage sequence similarity of 99%. The gyrB and aroE genes both showed lower median value of percentage sequence similarities of 91% (Table 2). The gyrB and aroE genes showed a median percentage of nucleotide sequence divergence among B. thuringiensis H serotypes up to nine fold greater than the one of the 16S rRNA gene. Clearly, both genes can be used to achieve deeper levels of discrimination among B. thuringiensis H serotypes than the 16S rRNA gene. The gyrB gene, however, present some advantages over the aroE gene as a gene of choice for discriminating among B. thuringiensis lower classification ranks, serovars from same H serotype and strains from same serovar. Here, the gyrB gene nucleotide sequence could distinguish strains from same serovar as exemplified with the two B. thuringiensis serovar finitimus strains under study. The aroE gene nucleotide sequence could not. But, perhaps more importantly, the gyrB gene is increasingly used as a macromolecule of choice for bacterial phylogenetic analysis and a database of gyrB sequences was implemented a few years ago (Watanabe et al. 2001). The latter will prove invaluable as more B. thuringiensis sequences are added.

Table 2 Percentage nucleotide sequence similarity between 16S rRNA, gyrB and aroE genes among Bacillus thuringiensis H serotypes
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gyrB gene polymorphism among serovars from same H serotype and among strains from same serovar

The capability of the gyrB gene nucleotide sequences to distinguish B. thuringiensis serovars from the same H serotype and strains from the same serovar was further studied. Table 3 lists the 42 B. thuringiensis strains used in this analysis. This includes eight B. thuringiensis strains from Table 1 and 34 additional ones. Together, they encompass 12 H serotypes and 25 serovars. Amplifications were carried out as described before using both pairs of primers, gyrB-F1/-R1 and gyrB-F2/-R2. Amplicon sizes were as described previously.

Table 3 Bacillus thuringiensis serovars and strains used in Fig. 6
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gyrB nucleotide sequences-inferred phylogenetic analyses

Figure 6 shows the phylogenetic relationships between the gyrB gene nucleotide sequences for the 42 B. thuringiensis strains shown in Table 3. Four clusters, I–IV, were revealed at the 96% nucleotide sequence identities.

Fig. 6
figure 6

Bootstrapped neighbor-joining tree of Bacillus thuringiensis serovars and strains generated from the alignment of gyrB nucleotide sequences. Bootstrap values higher than 30% are indicated. Roman numerals, I–IV, refer to the four clusters revealed at the 96% nucleotide sequence identities. The horizontal bar represents 0.005% differences in nucleotide identities

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Although some serovars from the same H serotype are found in the same clusters, this is not necessarily true for all serovars from the same H serotype. Indeed, serovars from the same H serotype do not necessarily cluster together. For example, H5 serovars galleriae and canadensis are both found in cluster I. H8 serovars ostriniae and morrisoni are in cluster IV. H11 serovars toumanoffi and kyushuensis are in cluster IV. H18 serovars yosoo and kumamotoensis are in cluster I. For H3, however, whereas serovar kurstaki is found in cluster I, serovars alesti, sumiyoshiensis and fukuokaensis are found in cluster IV. Likewise, H4 serovars kenyae and sotto are found in clusters I and IV, respectively. H10 serovars londrina and darmstadiensis are found in clusters I and IV. H20 serovars yunnanensis and pondicheriensis are found in clusters I and II. H24 serovars novosibirsk and neoleonensis are found in clusters III and IV. H28 serovars jegathesan and monterrey are found in clusters I and II. The gyrB gene nucleotide sequences could distinguish B. thuringiensis serovars from the same H serotype. No gyrB gene nucleotide sequences from B. thuringiensis serovars from the same H serotype were identical.

Intra-serovar strains, i.e. strains from the same serovar, are found in the same cluster in close proximity. This is true for strains in the kurstaki, galleriae, kenyae and entomocidus serovars in cluster I, finitimus in cluster II, and alesti, sotto, morrisoni in cluster IV. Although strains from the same serovar were found in the same cluster, none was undistinguishable. The gyrB gene nucleotide sequence was sufficient to distinguish each intra-serovar strain. This is also true for the pathovars and biovars studied here as exemplified by B. thuringiensis serovar morrisoni pathovars tenebrionis and sandiego, by B. thuringiensis serovar entomocidus and its biovar subtoxicus, and by B. thuringiensis serovar sotto and its biovar dendrolimus.

Discussion

Our aim, at the onset of this work, was to investigate the capability of each of the three genes under study, 16S rRNA, gyrB and aroE, to discriminate among B. thuringiensis H serotypes, serovars from the same H serotype and strains from the same serovar. First, these genes were analyzed in 21 B. thuringiensis H serotypes and four other B. cereus sensu lato species (B. cereus, B. anthracis, B. mycoides and B. weihenstephanensis). Nucleotide sequences were pair-wise compared and phylogenetic relationships were inferred. The groups were analyzed at the species level. Each of the three genes grouped all three B. anthracis together. All three genes also grouped all five B. weihenstephanensis strains together. Perhaps surprisingly, the four B. mycoides were found in different clusters suggesting a higher level of heterogeneity. The 16S rRNA, gyrB and aroE gene nucleotide sequences grouped the four B. cereus in a single or in three or two different clusters, respectively. The 16S rRNA gene grouped all B. thuringiensis strains in cluster I with two exceptions: serovars bolivia and vazensis, both found in cluster II along with the B. weihenstephanensis strains suggesting a closer relationship. The gyrB gene saw the B. thuringiensis strains distributed in five clusters with serovar vazensis in a sixth cluster along with the B. weihenstephanensis strains. The aroE gene saw the B. thuringiensis strains distributed in all five clusters. Interestingly enough, here also, serovars bolivia and vazensis were found in cluster IV along with the B. weihenstephanensis strains adding support to the closer relationship between these two B. thuringiensis serovar with this B. cereus sensu lato species. On a side note, the presence of a parasporal inclusion body is the single most discriminating criterion for the identification of B. thuringiensis among Bacillus. Whether B. thuringiensis serovar vazensis, or serovar bolivia, might share common additional features with B. weihenstephanensis remains to be confirmed. We are currently pursuing studies of this type. Each of the three genes under study could discriminate among B. thuringiensis H serotypes and the discriminatory power of each was calculated. Both house-keeping genes, gyrB and aroE, showed a median percentage of nucleotide sequence divergence among B. thuringiensis H serotypes up to nine fold greater than the one of the 16S rRNA gene. The gyrB gene was retained for subsequent analyses: the discrimination of B. thuringiensis serovars from the same H serotype, and the discrimination of strains from the same serovar. A total of 42 B. thuringiensis strains which encompassed 25 serovars from 12 H serotypes were analyzed. The gyrB gene nucleotide sequences could distinguish B. thuringiensis serovars from the same H serotype. In addition, the gyrB gene nucleotide sequence was sufficient to distinguish B. thuringiensis strains from the same serovar. Clearly, whereas all three genes under study, 16S rRNA, gyrB and aroE could discriminate among B. thuringiensis H serotypes, the gyrB gene has proven capable of discriminating serovars from the same H serotype and strains from the same serovar.