The interaction of the F-like plasmid-encoded TraN isoforms with their cognate outer membrane receptors

Horizontal gene transfer via conjugation plays a major role in bacterial evolution. In F-like plasmids, efficient DNA transfer is mediated by close association between donor and recipient bacteria. This process, known as mating pair stabilization (MPS), is mediated by interactions between the plasmid-encoded outer membrane (OM) protein TraN in the donor and chromosomally-encoded OM proteins in the recipient. We have recently reported the existence of seven TraN sequence types, which are grouped into four structural types, we named TraN, TraN{beta}, TraN{gamma}, TraN{delta}. Moreover, we have shown specific pairing between TraN and OmpW, TraN{beta} and OmpK36 of Klebsiella pneumoniae, TraN{gamma} and OmpA and TraN{delta} and OmpF. In this study we found that although structurally similar, TraN encoded by the pSLT plasmid (TraN2) binds OmpW in both Escherichia coli and Citrobacter rodentium while TraN encoded by the R100-1 plasmid (TraN1) only binds OmpW in E. coli. AlphaFold2 predictions suggested that this specificity is mediated by a single amino acid difference in loop 3 of OmpW, which we confirmed experimentally. Moreover, we show that single amino acids insertions into loop 3 of OmpK36 affect TraN{beta}-mediated conjugation efficiency of the K. pneumoniae resistance plasmid pKpQIL. Lastly, we report that TraN{beta} can also mediate MPS by binding OmpK35, making it the first TraN variant that can bind more than one OM protein in the recipient. Together, these data show that subtle sequence differences in the OM receptors can impact TraN-mediated conjugation efficiency. ImportanceConjugation plays a central role in the spread of antimicrobial resistance genes amongst bacterial pathogens. Efficient conjugation is mediated by formation of mating pairs via a pilus, followed by mating pair stabilisation (MPS), mediated by tight interactions between the plasmid encoded outer membrane protein (OMP) TraN in the donor (of which there are seven sequence types grouped into the four structural isoforms , {beta}, {gamma}, {delta}) and an OMP receptor in the recipient (OmpW, OmpK36, OmpA and OmpF, respectively). In this study we found that subtle differences in OmpW and OmpK36 have significant consequences on conjugation efficiency and specificity, highlighting the existence of selective pressure affecting plasmid-host compatibility and the flow of horizontal gene transfer in bacteria.