Deciphering interbacterial killing mediated by a conjugative plasmid in Vibrio cholerae

Mobile genetic elements (MGEs) and bacteria are engaged in an evolutionary arms race, with conjugative plasmids promoting horizontal gene transfer (HGT) and bacterial defence systems restricting their spread. The 7th Pandemic El Tor (7PET) lineage of Vibrio cholerae, responsible for the ongoing cholera pandemic, rarely carry plasmids compared with environmental strains. This low plasmid prevalence is attributed to two 7PET-specific defence systems, DdmABC and DdmDE: DdmABC triggers abortive infection (Abi), causing host cell sacrifice to prevent phage and plasmid replication, whereas DdmDE directly degrades plasmid DNA. Because Abi imposes a substantial burden on cells acquiring large conjugative plasmids, plasmid carriage is strongly selected against in 7PET strains. Unexpectedly, we identified a conjugative plasmid from an environmental V. cholerae strain, pSA7G1, that shows the opposite phenotype. Despite being targeted by both defence systems, pSA7G1 provides a fitness advantage to its 7PET host when competed against plasmid-free cells. This thesis aimed to define the genetic determinants and molecular mechanisms underlying this phenotype. Fluorescence microscopy revealed that plasmid-free cells exhibit rounding, membrane permeabilization and cytoplasmic loss, supporting that pSA7G1-carrying cells outcompete plasmid-free cells through killing. Using serial deletion, we found that both the mating pair formation (MPF) and mobility (mob) modules, typically required for plasmid transfer, are essential for the phenotype, demonstrating that killing is coupled to conjugation. After engineering a two-plasmid system in which an unrelated oriT-containing plasmid is mobilized by co-residing pSA7G1 deleted from its oriT, we observed that transfer of the unrelated plasmid was sufficient for killing. This suggests that donor cells may deliver an effector protein together with transferred DNA. Beyond the conjugation machinery, two additional plasmid regions are required for the phenotype. One contributes to plasmid stability, and its deletion causes plasmid loss and thus abolishes killing. The second contributes directly to the phenotype, as its deletion disrupts killing without altering plasmid stability, host fitness or conjugation. Within the second region, we identified two key genes. One encodes an exclusion protein that blocks pSA7G1 transfer when artificially expressed into plasmid-free cells, thereby protecting them. The second encodes a putative effector protein whose deletion eliminates killing in both pSA7G1 and the two-plasmid system. Although toxicity could not be demonstrated upon induction, the putative effector contains a signal sequence directing its periplasmic localisation, suggesting compartment-based protection in host cells. Together, these findings support a model in which pSA7G1 encodes a novel plasmid addiction system relying on sustained transfer of an effector protein along with plasmid DNA. This mechanism efficiently eliminates