Conjugative plasmids play a major role in the spread of antibiotic resistance which is currently a global health concern. Plasmids that belong to the incompatibility P group (IncP-1), are conjugative, broad host range and are stably maintained in phylogenetically diverse organisms. These features make IncP-1 plasmids major contributors in the dissemination of antibiotic resistance genes. RP1 is an early example of an IncP-1α plasmid that was identified in Pseudomonas aeruginosa in a Birmingham hospital in 1969. The IncP-10 plasmid R91 was identified from another P. aeruginosa strain which was also isolated from the same hospital. Interestingly, R91 reduced the conjugal transfer of RP1, a phenomenon known as fertility inhibition (FI). Several FI gene products in other plasmids have been shown to inhibit the transfer of broad host range conjugative plasmids, providing an alternative strategy to combat antibiotic resistance.
In this study, we sequenced and characterised R91-5 (a derepressed mutant of R91) and used transposon mutagenesis to identify the region responsible for FI. Three genes (fipB1, fipB2 and fipB3) were found to be responsible for the FI phenotype. To understand how these FI genes exert their inhibition effects on RP1, we developed an artificial conjugation system. Using this artificial conjugation system, we determined that the RP1 coupling protein, TraG, was the target of the FI proteins. To further elucidate this mechanism of inhibition, we used the pETDuet-1 co-expression vector system and analysed the interactions between FipB1, FipB2 and FipB3 proteins as well as TraG using co-immunoprecipiation. The results revealed that the three fipB genes and traG interact at the protein level. A thorough understanding of the FI mechanism in IncP-1 plasmids can significantly contribute to reducing the burden against the rise of antibiotic resistant bacteria.