Conjugative transfer inhibition of IncA and IncC plasmids by pervasive SGI1-like elements via relaxosome assembly interference

Broad-host-range IncA and IncC conjugative plasmids propagate multidrug resistance in bacteria and mobilize chromosomal resistance islands, including Salmonella Genomic Island 1 (SGI1) across genera. Mobilization of SGI1 and kin usually occurs at rates orders of magnitude higher than plasmid transfer, which seems inhibited. Here, we identified, using a Tnseq approach, a broadly conserved fertility inhibitor, which we name CtiC (for 'Conjugative Transfer Inhibition of IncC'), encoded by SGI1 and kin that hampers helper plasmid transfer. ctiC expression is constitutive and elicited by the transcriptional activator of the transfer genes of IncA and IncC plasmids. ctiC suppression greatly enhances IncC plasmid transfer, indicating it counters a general improvement of conjugation mediated by SGI1. Our results show that the two genes traHG located upstream of ctiC in SGI1 account for the enhanced transfer properties. CtiC specifically prevents the cotransfer of the helper plasmid, which impairs SGI1 stability in transconjugants. A comparison of structural predictions revealed that the inhibitor resembles the C-terminus of the MOB H12 -family relaxase TraI of IncA and IncC plasmids despite low sequence conservation. Distant CtiC homologues sharing the same predicted structure exhibit similar inhibitory activities. Bacterial two-hybrid assays and relaxase domain substitution show that CtiC interferes with relaxosome assembly that initiates plasmid transfer. We show that CtiC binds to the mobilization factor MobI of IncC plasmids, a key protein that recognizes the origin of transfer, thus preventing its interaction with TraI. Our work reveals a fertility inhibition mechanism that prevents relaxosome assembly and uncovers a functional domain in MOB H12 relaxases.