Distribution, diversity, and potential mobility of extrachromosomal elements related to the Bacillus anthracis pXO1 and pXO2 virulence plasmids

Abstract

The presence of a pXO1- and/or pXO2-like plasmid(s) in clinical isolates of Bacillus cereus sensu stricto and in strains of the biopesticide Bacillus thuringiensis has been reported recently, and the pXO2-like plasmid pBT9727 and another pXO2-like plasmid, pAW63, were found to be conjugative. In this study, a total of 1,000 B. cereus group isolates were analyzed for the presence of pXO1- and pXO2-like replicons and for the presence of pXO2-related conjugative modules. pXO1- and pXO2-like replicons were present in ca. 6.6% and 7.7% of random environmental samples, respectively, and ca. 1.54% of the strains were positive for pXO2-like transfer module genes. Only the strains harboring a pXO2-like replicon also contained the corresponding transfer genes. For the strains which contained a pXO1- and/or pXO2-like replicon(s), a large plasmid(s) whose size was similar to that of pXO1-like and/or pXO2-like plasmids was also observed, but none of these isolates were found to carry the Bacillus anthracis toxin or capsule virulence genes. Furthermore, 17 of 22 pXO2-like plasmids containing the transfer modules were able to self-transfer and to mobilize small plasmids. No pXO1- or pXO2-like plasmid lacking the cognate transfer modules has been found to have transfer potential. In the strains possessing the putative pXO2-like conjugative apparatus, variations in the presence of the group II introns B.th.I.1 and B.th.I.2 were observed, suggesting that there is important flexibility in the conjugation modules and their regulation. There was no consistent correlation between a pXO2-like repA dendrogram and the presence of the tra region or between a virB4 dendrogram and transfer ability. Discrepancies between pXO2-like repA and virB4 dendrograms were also observed, indicating that the evolution of pXO2 is an active process.

FIG. 1.

Positions of the conjugation-related primers designed for the pXO2-like tra region. The numbers indicate positions in the pAW63 sequence (accession number DQ025752). The positions of three major coding sequences encoding T4SS-like proteins (VirB4, D4, and B11) are indicated, as is the position of the sequence encoding the IEP associated with the group II intron B.th.I.1. Three primer pairs, MB4_F2/MB4_R2, MD4_F2/MD4_F2R2, and MB11_F1/MB11_R1, were used to detect the occurrence of the T4SS-like genes virB4, virD4, and virB11, respectively. Primer pairs B.th.I1_F1/B.th.I1_R2 and MD4_F1/MD4_R1 were used to detect the occurrence of the introns B.th.I.1 and B.th.I.2, respectively. The solid bars indicate predicted PCR products; the broken lines indicate introns.

FIG. 2.

Strategy used for conjugation in triparental mating and biparental mating. (A) Triparental mating was performed using a donor strain containing a pXO1- and/or pXO2-like plasmid(s), the recipient strain B. thuringiensis subsp. israelensis GBJ002, and the helper strain B. thuringiensis subsp. israelensis GBJ001 containing a mobilizable plasmid (pUB110 or pBC16). The antibiotic markers carried by the host strain and plasmids are indicated. Two configurations of potential transconjugants were screened by using their resistance to Nal and Km (pUB110) or to Nal and Tet (pBC16) and by using PCR (PCR positive for pUB110 or pBC16 and then PCR performed to detect the presence of pXO1- or pXO2-like replication genes in transconjugants). (B) Biparental mating was performed using the transconjugants from the triparental mating experiments, which contained both the mobilizable plasmid and pXO1- or pXO2-like plasmid, and the recipient strain B. thuringiensis subsp. israelensis 4Q7-Rif. Bacteria are indicated by thin, medium, and thick lines to distinguish different host strains. Curved arrows indicate the possible transfers.

FIG. 3.

Percentages of the B. cereus group strains with pXO1- and pXO2-like plasmids and T4SS-like transfer modules for environmental and lab collection isolates. Results are shown for environmental isolates, lab collection isolates, and isolates containing the pXO2-like tra region, based on the presence of the virB4 and virD4 transfer genes, which encode the core apparatus of the T4SS.

FIG. 4.

Large-plasmid and Southern blotting of strains containing pXO1- and/or pXO2-like plasmid(s). (A) Plasmid profiles; (B) Southern blot patterns obtained with the partial pXO2-like repA fragment as the probe; (C) Southern blot patterns obtained with the partial pXO1-like repX fragment as the probe. Lane 1, AND508 (1) containing four large plasmids (used as references; the position of the second large plasmid, pBtoxis, is indicated by an arrow); lane 2, AW06 harboring pAW63 (72 kb; position indicated by an arrow); lane 3, VD142; lane 4, VD022; lane 5, VD023; lane 6, IS075; lane 7, ISP2954; lane 8, Schrouff. VD142 and ISP2954 are PCR positive for pXO2-like repA; VD022, VD023, IS075, and Schrouff are PCR positive for both pXO2-like repA and pXO1-like repX. chr, chromosome. The scale to the left of each blot was used as a ladder to compare the positions of plasmids and hybridization signals. The arrowheads and arrows indicate the positions of pXO2- and pXO1-like plasmids, respectively.

FIG. 5.

Plasmid profiles of strains involved in tri- and biparental mating. Lane 1, AND508 containing four large plasmids (the position of the reference element, pBtoxis, is indicated by an arrow); lane 2, T03001; lane 3, TT03001-1 [transconjugant from triparental mating of T03001, GBJ001(pBC16), and GBJ002]; lane 4, TT03001-2 (transconjugant from biparental mating of TT03001-1 and 4Q7-Rif); lane5, VD023; lane 6, TK023-1 [transconjugant from triparental mating of VD023, GBJ001(pUB110), and GBJ002]; lane 7, TT023-1 [transconjugant from triparental mating of VD023, GBJ001(pBC16), and GBJ002]; lane 8, VD148; lane 9, TVD148-1 [transconjugant from triparental mating of VD148, GBJ001(pBC16), and GBJ002]; lane 10, TVD148-2 (transconjugant from biparental mating of TVD148-1 and 4Q7-Rif); lane 11, AW06 harboring pAW63 (72 kb; position indicated by an arrow); lanes 12 and 13, GBJ001(pUB110) and GBJ001(pBC16) containing the mobilizable plasmids pUB110 and pBC16, used as helper strains in triparental mating. Chr., chromosome. The arrowheads and arrows indicate the positions of pXO2- and pXO1-like plasmids, respectively, as identified by Southern blot hybridization using partial pXO2-like repA and pXO1-like repX fragments as probes (data not shown).

FIG. 6.

pXO2-like repA dendrogram (A) and alignment of partial repA sequences (B). The levels of identity of the 36 sequences ranged from 77% to 100% based on DNA sequences (and from 79% to 100% based on amino acid sequences). The pBT9727, pAW63, and pXO2 reference plasmids are enclosed in boxes. Filled triangles indicate strains containing transfer modules, while open triangles indicate strains missing the cognate transfer modules. Stars indicate strains carrying a B.th.I1 and/or B.th.I2 insertion(s). ++, good mobilization efficiency (similar to that of pAW63); +, low mobilization efficiency (10² to 10³ times lower than that of pAW63); −, no mobilization. The scale bar indicates genetic distance (0.02 nucleotide substitution per site). The boxes in panel B indicate the two indels consisting of seven and eight nucleotides present in the repA sequences. All bootstrap support values of >70% are indicated at the appropriate nodes.

FIG. 7.

Comparison of pXO2-like repA (A) and virB4 (B) dendrograms. The levels of identity for the 22 sequences ranged from 77% to 100% based on repA sequences (and from 80% to 100% based on amino acid sequences) and from 73% to 100% based on virB4 sequences (and from 72% to 100% based on amino acid sequences). For an explanation of the symbols and scale bar see the legend to Fig. 6. All bootstrap support values of >70% are indicated at the appropriate nodes.