Plasmid capture by the Bacillus thuringiensis conjugative plasmid pXO16.

Conjugation, mobilization, and retromobilization are three related mechanisms of horizontal gene transfer in bacteria. They have been extensively studied in gram-negative species, where retromobilization, the capture of DNA from a recipient by a donor cell, was shown to result from two successive steps: the transfer of the conjugative plasmid from the donor to the recipient followed by the retrotransfer of the mobilizable plasmid to the donor. This successive model was established for gram-negative bacteria but was lacking experimental data from the gram-positive counterparts. In the present work, the mobilization and retromobilization abilities of the conjugative plasmid pXO16 from Bacillus thuringiensis subsp. israelensis were studied using the mobilizable plasmids pUB110 and pE194 and the "nonmobilizable" element pC194 lacking the mob and oriT features (all from Staphylococcus aureus). Experimental data suggested a successive model, since different retromobilization frequencies were observed between the small plasmids. More importantly, retromobilization was shown to be delayed by 50 and 150 min for pUB110 and pE194, respectively, compared to pXO16 conjugation. Natural liquid foods (cow milk, soy milk, and rice milk) were used to evaluate the putative ecological impact of these transfers. In cow and soy milk, conjugation, mobilization, and retromobilization were shown to occur at frequencies of 8.0 x 10(-1), 1.0 x 10(-2), and 1.2 x 10(-4) transconjugants per recipient, respectively. These data are comparable to those obtained with LB medium and about 10-fold lower than in the case of rice milk. Taken together, these results emphasize the potential role of plasmid capture played by B. thuringiensis in natural environments.

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

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.

Plasmid exchanges among members of the Bacillus cereus group in foodstuffs.

The Bacillus cereus sensu lato group is genetically very close and possesses a remarkable plasmid gene pool that encodes a variety of functions such as virulence and self-transfer capabilities. The potential for horizontal transfer among the various subspecies of this group, which includes the human opportunistic pathogens B. cereus sensu stricto and B. anthracis as well as the biopesticide B. thuringiensis, has led to growing concerns regarding food safety and public health. In this study, the conjugative behaviour of B. thuringiensis strains was compared in LB medium, milk and rice pudding using the pXO16 and pAW63 conjugative systems, as well as the mobilisable plasmid pC194, in bi- and triparental matings. Conjugation and mobilisation of these plasmids were shown to occur at significant levels in both food products, attaining the highest transfer frequencies in milk, with an approximately ten-fold increase in conjugative transfer in this growth medium as compared to liquid LB. Furthermore, the ability of an emetic strain of B. cereus to function as either plasmid donor or recipient partner in heterologous biparental matings with B. thuringiensis was demonstrated in these food matrices.

Characterization of Bacilli isolated from the confined environments of the Antarctic Concordia station and the International Space Station.

Bacillus and related genera comprise opportunist and pathogen species that can threaten the health of a crew in confined stations required for long-term missions. In this study, 43 Bacilli from confined environments, that is, the Antarctic Concordia station and the International Space Station, were characterized in terms of virulence and plasmid exchange potentials. No specific virulence feature, such as the production of toxins or unusual antibiotic resistance, was detected. Most of the strains exhibited small or large plasmids, or both, some of which were related to the replicons of the Bacillus anthracis pXO1 and pXO2 virulence elements. One conjugative element, the capacity to mobilize and retromobilize small plasmids, was detected in a Bacillus cereus sensu lato isolate. Six out of 25 tested strains acquired foreign DNA by conjugation. Extremophilic bacteria were identified and exhibited the ability to grow at high pH and salt concentrations or at low temperatures. Finally, the clonal dispersion of an opportunist isolate was demonstrated in the Concordia station. Taken together, these results suggest that the virulence potential of the Bacillus isolates in confined environments tends to be low but genetic transfers could contribute to its capacity to spread.

Characterization of a novel temperate phage originating from a cereulide-producing Bacillus cereus strain.

A novel temperate bacteriophage was isolated from a Bacillus cereus cereulide-producing strain and named vB_BceS-IEBH. vB_BceS-IEBH belongs to the Siphoviridae family. The complete genome sequence (53 kb) was determined and annotated. Eighty-seven ORFs were detected and for 28, a putative function was assigned using the ACLAME database. vB_BceS-IEBH replicates as a plasmid in the prophage state. Accordingly, a 9-kb plasmid-like region composed of 13 ORFs was identified. A fragment of around 2000 bp comprising an ORF encoding a putative plasmid replication protein was shown to be self-replicating in Bacillus thuringiensis. Mass spectrometry analysis of the purified vB_BceS-IEBH particle identified 8 structural proteins and enabled assignment of a supplementary ORF as being part of the morphogenesis module. Genome analysis further illustrates the diversity of mobile genetic elements and their plasticity within the B. cereus group.

Sympatric soil communities of Bacillus cereus sensu lato: population structure and potential plasmid dynamics of pXO1- and pXO2-like elements.

Eighty soil-borne Bacillus cereus group isolates were collected from two neighbouring geographical sites in Belgium. Their genetic relationships and population structure were assessed using Multilocus sequence typing analysis of five chromosomal genes, while the contribution of extrachromosomal elements to the population dynamics was gauged by the presence, diversity and transfer capacity of pXO1- and pXO2-like plasmids. Globally, the bacterial population displayed a broad diversity, including an important subpopulation of psychrotolerant isolates related to Bacillus weihenstephanensis. pXO1- and pXO2-like replicons were present in 12% and 21% of the isolates, but no Bacillus anthracis-related toxin genes were found. Furthermore, only one of the isolates containing a pXO2-related plasmid was shown to be able to mobilize small non-self-conjugative plasmids. Interestingly, several B. cereus sensu lato isolates displaying the same sequence type were observed to have different plasmid contents, suggesting the occurrence of horizontal gene exchange. Similarly, a number of pXO2-like replicons with identical sequences were found in distinct bacterial isolates, therefore strongly arguing for lateral transfers among sympatric bacteria.

Self-transfer and mobilisation capabilities of the pXO2-like plasmid pBT9727 from Bacillus thuringiensis subsp. konkukian 97-27.

Recent characterisations of plasmids related to the anthrax virulence plasmids pXO1 and pXO2 in clinical isolates of Bacillus cereus and Bacillus thuringiensis have contributed to the emerging picture of a virulence-associated plasmid pool in the B. cereus sensu lato group. The family of pXO2-like plasmids includes the conjugative plasmid pAW63 from the biopesticide strain B. thuringiensis subsp. kurstaki HD73 and the heretofore cryptic plasmid pBT9727 from the clinical strain B. thuringiensis subsp. konkukian 97-27. Comparative sequence analysis of these three plasmids suggested that they were derived from an ancestral conjugative plasmid, with pAW63 retaining its self-transfer capabilities, and pXO2 having lost them through genetic drift. Such properties had not been investigated in pBT9727, but sequence homologies led us to predict that it may possess self-transfer capabilities. Here, we report that pBT9727 is indeed conjugative, and is able to promote its own transfer as well as that of small mobilisable plasmids.