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The evolutionary path of bacteria is mainly influenced by horizontal gene transfers (HGT) since mobile genetic elements carry a panel of key metabolic traits to adapt to changing environments. Among these HGT, conjugation and mobilization have been described for over fifty years, in laboratory liquid media. However, it is now generally admitted that, in nature, bacteria are subjected to various stresses and are mostly found in interface-associated communities. This study focuses on members of the Bacillus cereus group which are food contaminants and characterized by a large extra-chromosomal gene pool. One of these elements, pAW63 which resembles the Bacillus anthracis virulence plasmid pXO2, is used as a model to study conjugation in Gram-positive species. This 72kb-plasmid is a self-transmissible element originating from Bacillus thurigiensis sv. kurstaki and displays an ability to mobilize other plasmids. In this work, the dynamics of pAW63 transfer in the B. cereus group was investigated under different conditions. On one hand, mating partners in liquid medium were submitted to various chemical and physical stresses including extreme osmotic environment and microgravity. On the other hand, capabilities of plasmid transfer were estimated in biofilms by performing an in situ analysis based on the detection of fluorescent proteins. Moreover, bacterial populations were analysed for their conjugation abilities on simulated food microcosms increasing the relevance of the study with regard to ecological niches of B. cereus and other food borne pathogens. The results showed that pAW63 transfer efficiency is largely affected by the chemical composition of the mating medium and was found to increase under specific stress conditions. Biofilms were also proved to be a favored environment for conjugation among members of the B. cereus group. Moreover, it appeared that the bacterial behavior inside a structured community impacts on pAW63 transfer ability resulting in “hotspot

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., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Members of the Bacillus genus include potential pathogens. They are ubiquitous in the environment and contribute substantially to the bacterial communities found in confined places such as the ISS and the Antarctic Concordia station. In a context of planetary and crew health protection, it is necessary to monitor bacterial contamination and adaptation inside the confined stations designed for the exploration of new environments. The first part of this thesis consisted on the study of the Bacillus population diversity found in ISS and Concordia and to the comparison with the situation in environment, with a specific focus on members of the B. cereus group. The contribution of plasmids related to B. anthracis virulence elements to the extrachromosomal gene pool was assessed. The ability of several plasmids with a pXO2-like replicon to sustain conjugative transfer demonstrated the putative importance of horizontal gene transfer in bacterial adaptation. This was further confirmed by the isolation of a new conjugative element in a Concordia B. cereus s.l. isolate and by the detection of plasmid transfer among the Bacillus strains from the confined stations. The actual role of conjugation on bacterial adaptation was further assessed in real situation. The choice of food matrices was relevant since putative food pathogens were detected in the Bacillus population of the ISS and Concordia. Conjugative transfer of the reference conjugative plasmids pAW63 and pXO16 was successfully demonstrated in dairy products. Interestingly, transfer frequencies in foodstuffs turned out to be higher by up to 10 to 100 fold compared to those obtained in laboratory medium. Finally, the specific features of pXO16 make that it was an interesting Gram-positive model to define the mechanisms of conjugation, mobilization and retromobilization, i.e. plasmid capture. Kinetics experiments have shown that retromobilisation followed a two-step mechanism, pXO16 having first to establish in the cell harb, (AGRO 3) -- UCL, 2010

Conjugation, mobilisation and retromobilisation are three related mechanisms of horizontal gene transfer in bacteria. They have been extensively studied in Gram-negative species, where retromobilisation, 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 mobilisable 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, mobilisation and retromobilisation abilities of the conjugative plasmid pXO16 from Bacillus thuringiensis sv israelensis were studied using the mobilisable plasmids pUB110 and pE194 and the "non-mobilisable" element pC194 lacking the mob and oriT features (all from Staphylococcus aureus). Experimental data suggested a successive model since different retromobilisation frequencies were observed between the small plasmids. More importantly, retromobilisation 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, mobilisation and retromobilisation 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 in 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.

The presence of pXO1- and/or pXO2-like plasmid(s) in clinical isolates of B. cereus (sensu stricto) and in strains of the biopesticide Bacillus thuringiensis have been reported recently, and the pXO2-like plasmid pBT9727 was found conjugative as well as another pXO2-like plasmid pAW63. 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 pXO2-related conjugative modules. The occurrence frequency of pXO1- and pXO2-like replicons in random environmental samples was ca. 6.6% and 7.7%, 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 pXO1- and/or pXO2-like replicon(s), large plasmid(s) with a size similar to that of pXO1-like and/or pXO2-like plasmids were also observed, but none of these isolates were found to carry the B. anthracis toxin or capsule virulence genes. Furthermore, 17 out of 22 pXO2-like plasmids containing the transfer modules displayed the capability of self-transfer and of mobilizing small plasmids. No pXO1-, or pXO2-like plasmid lacking the cognate transfer modules has been found to have transfer potential. Among the strains possessing the putative pXO2-like conjugative apparatus, variations on the presence of the group II introns B.th.I.1 and B.th.I.2 were observed, suggesting an important flexibility of the conjugation modules and their regulation. There was no consistent correlation between pXO2-like repA dendrogram and presence/absence of tra region, or between virB4 dendrogram and transfer capabilities. Discrepancies between pXO2-like repA and virB4 dendrograms were also observed, indicating that the evolution of pXO2 is an active process.

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.

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.