Structural basis for TrbM-enhanced conjugation and interbacterial killing in the RP4 plasmid

Bacterial conjugation drives horizontal gene transfer and antibiotic resistance via type IV secretion systems (T4SS) on conjugative plasmids like RP4. Previously, we found that the RP4-T4SS mediates interbacterial killing, a process enhanced by the uncharacterized gene trbM. Here, we characterize RP4-TrbM and identify structural features essential for boosting both conjugation and killing. Computational analyses reveal that the RP4-TrbM shares similarities with known bacterial adhesins in other conjugative systems. Homologs from plasmids R751, R388, and pKM101 could complement RP4-TrbM-knockout strains, restoring and enhancing conjugation (R751-TrbM and pKM101-Pep) and conjugation-associated killing (R751-TrbM, R388-KikA, and pKM101-Pep), while TivB12 from conjugative plasmid R6K could not complement the RP4-T4SS. Furthermore, we identified an essential functional domain in RP4-TrbM that retains activity even when repositioned between a different signal peptide and C terminus. These findings expand our understanding of the RP4 conjugative machinery and highlight TrbM-like proteins as promising targets for inhibiting T4SS-mediated processes.