Systematic evaluation of the host range and host cell requirements for Gram-negative conjugative plasmids with biotechnological potential

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The current age of antibiotic resistance calls for new strategies to combat bacterial infections. Conjugation is a natural mechanism by which microorganisms exchange genetic material. Repurposing the conjugation process could help the development of novel strategies aimed at fighting antibiotic resistance. Our group has previously described a conjugative plasmid, TP114, showing impressive transfer rates in vivo in the mouse intestinal tract. The ability of such plasmids to transfer in vivo at high rates provides new avenues to fight bacterial infections and develop microbiome editing tools. However, several questions remain regarding the host range of conjugative plasmids and their interactions with donor and recipient cells. We propose to develop an innovative high-throughput conjugation assay to characterize the host range of TP114 and investigate the genetic requirements of donor or recipient bacteria. In our high-throughput assay, we will systematically transfer TP114 into a vast collection of strains that includes pathogens and commensals from the human and animal microbiota, as well as environmental strains. We will further transfer TP114 into the Keio collection, an Escherichia coli mutant library comprising all possible single gene deletions, to assess the influence of every dispensable gene on the conjugation process. To further identify other plasmids with properties similar to TP114, we will investigate the host range and required genetic determinants of a representative set of conjugative plasmids belonging to the most prevalent incompatibility groups in Gram-negative bacteria. Finally, we will test the efficacy of conjugative plasmids as a microbiome editing tool. To do this, we will use a donor bacterium containing a modified TP114 expressing CRISPR/Cas9 functions to deplete Adherent-Invasive Escherichia coli (AIEC) in a Crohn's disease mouse model.Our study is expected to provide the first comprehensive map of host ranges for conjugative plasmids with great biotechnological potential. In addition, we anticipate identifying the contribution of the host's physiology and metabolism to the conjugation process. Hence our results will be helpful to design novel microbial engineering tools that can be used in innovative approaches to manipulate microbiomes. Our results could also lead to new strategies to prevent horizontal gene transfer and the dissemination of antibiotic resistance genes. In addition, this project will provide a highly stimulating and competitive training opportunity, in which trainees will use state-of-the-art equipment and technologies to perform cutting-edge research. This project will build on and significantly improve the expertise at UdeS on bacterial conjugation that is already recognized internationally. It will position UdeS as a leader in bacterial conjugation, horizontal gene transfer and microbiome editing.