Caractérisation de deux éléments génétiques mobiles de Bacteroides abritant des rétroéléments générateurs de diversité (DGR)

Bacteroides are prominent human gut symbiont anaerobes particularly well adapted to the intestinal environment via their vast polysaccharide utilization capacity and adhesion potential to gut surfaces. This genus also harbors many of ill-understood genetic elements that might contribute to their survival and persistence in the gut. One of such elements are diversity generating retroelements (DGRs), which mutate target regions of bacterial and bacteriophage DNA at a high frequency through a reverse transcriptase-dependent process. Whereas bacteriophage-associated DGRs have been shown to mutate receptor binding proteins, allowing adaptation to host ligand changes, bacterial-associated DGRs have been mostly unexplored. The main objective of my PhD thesis was to study structures potentially targeted by DGRs located within mobile elements. I first explored the lysogenic-lytic transition of the Hankyphage, a remarkably widespread, yet ill-understood Bacteroides phage containing a DGR. I characterized a collection of B. thetaiotaomicron Hankyphages and contributed to determine that the Hankyphage and Bacteroides phylogenies are divergent, indicative of Hankyphage activity and horizontal transmission. Moreover, I developed a digital-droplet PCR-based method to quantify non-lytic virions and constructed different variants of B. thetaiotaomicron strain carrying Hankyphages to study the phage lytic-lysogenic cycle. I showed that Hankyphage virions were produced in absence of inducers and identified a CI-like repressor responsible for inhibiting the B. thetaiotaomicron Hankyphage lytic cycle. Additionally, I observed a generalized lack of plaque formation and secondary lysogenization that could be partly explained by phage structural defects leading to the production of defective particles to a high frequency. Altogether, this study provided new biological insights on the regulation of the lytic cycle a broad-range Hankyphages widespread in Bacteroides. During my PhD thesis, I also studied a chromosomally-associated DGR targeting the tip protein of a type V pilus located within a potential integrative conjugative element (ICE) region. Whereas I showed that this region is transcriptionally silent and non-mobile in laboratory conditions, even upon addition of different inducers, in trans expression experiments showed that this type V pilus could be properly assembled and exposed at the bacterial surface and I investigated potential binding partners or ligand preferences of the pilus, aiming at providing a better understanding of the role of the still ill-characterized type V pili in Bacteroides. Lastly, I also studied the determinant of B. thetaiotaomicron biofilm formation by contributing to show that bile induced biofilm formation and that an extracellular DNase and the efflux of magnesium ions were required for the response to bile. Overall, this work has provided new insights on the regulation of DGR-associated Bacteroides mobile elements and contributed to the mechanistic understanding of biofilm formation in response to a relevant gut cue. This hopefully paves the way for future studies investigating the role of DGRs and community-like behaviour in the intestinal environment.