Deschenes, F. | bioRxiv Science

bioRxiv2025

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

Broad-host-range IncA and IncC (A/C) conjugative plasmids propagate multidrug resistance in bacteria and mobilize chromosomal resistance islands, including Salmonella Genomic Island 1 (SGI1) across genera. A/C plasmids usually mobilize SGI1 at very high frequencies while being inhibited by SGI1. Here, we identified a broadly conserved fertility inhibitor, which we named CtiC (for Conjugative Transfer Inhibition of IncC), encoded by SGI1 and kin, that hampers A/C plasmid transfer. ctiC expression is both constitutive and activated during conjugative transfer of A/C plasmids. Our results indicate that the suppression of ctiC significantly enhances plasmid transfer, suggesting it counters a general improvement in conjugation mediated by the two genes traHG, located upstream of ctiC in SGI1. CtiC specifically prevents the cotransfer of the helper plasmid, which destabilizes SGI1 in transconjugants. Structural predictions revealed that CtiC resembles the C-terminus of the MOBH12-family relaxase TraI of A/C plasmids. Bacterial two-hybrid assays and relaxase domain substitutions show that CtiC interferes with relaxosome assembly by binding to the plasmid-encoded mobilization factor MobI, which 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 MOBH12 relaxases.

microbiology↗

bioRxiv2021

Genomic islands targeting dusA in Vibrio species are distantly related to Salmonella Genomic Island 1 and mobilizable by IncC conjugative plasmids

Salmonella Genomic Island 1 (SGI1) and its variants are significant contributors to the spread of antibiotic resistance among Gammaproteobacteria. All known SGI1 variants integrate at the 3 end of trmE, a gene coding for a tRNA modification enzyme. SGI1 variants are mobilized specifically by conjugative plasmids of the incompatibility groups A and C (IncA and IncC). Using a comparative genomics approach based on genes conserved among members of the SGI1 group, we identified diverse integrative elements distantly related to SGI1 in several species of Vibrio, Aeromonas, Salmonella, Pokkaliibacter, and Escherichia. Unlike SGI1, these elements target two alternative chromosomal loci, the 5 end of dusA and the 3 end of yicC. Although they share many features with SGI1, they lack antibiotic resistance genes and carry alternative integration/excision modules. Functional characterization of IMEVchUSA3, a dusA-specific integrative element, revealed promoters that respond to AcaCD, the master activator of IncC plasmid transfer genes. Quantitative PCR and mating assays confirmed that IMEVchUSA3 excises from the chromosome and is mobilized by an IncC helper plasmid from Vibrio cholerae to Escherichia coli. IMEVchUSA3 encodes the AcaC homolog SgaC that associates with AcaD to form a hybrid activator complex AcaD/SgaC essential for its excision and mobilization. We identified the dusA-specific recombination directionality factor RdfN required for the integrase-mediated excision of dusA-specific elements from the chromosome. Like xis in SGI1, rdfN is under the control of an AcaCD-responsive promoter. Although the integration of IMEVchUSA3 disrupts dusA, it provides a new promoter sequence and restores the reading frame of dusA for proper expression of the tRNA-dihydrouridine synthase A. Phylogenetic analysis of the conserved proteins encoded by SGI1-like elements targeting dusA, yicC, and trmE gives a fresh perspective on the possible origin of SGI1 and its variants. Author summaryWe identified integrative elements distantly related to the Salmonella Genomic Island 1 (SGI1), a key vector of antibiotic resistance genes in Gammaproteobacteria. SGI1 and its variants reside at the 3 end of trmE, share a large, highly conserved core of genes, and carry a complex integron that confers multidrug resistance phenotypes to their hosts. Unlike members of the SGI1 group, these novel genomic islands target the 5 end dusA or the 3 end of yicC, lack multidrug resistance genes, and seem much more diverse. We showed here that, like SGI1, these elements are mobilized by conjugative plasmids of the IncC group. Based on comparative genomics and functional analyses, we propose a hypothetical model of the evolution of SGI1 and its siblings from the progenitor of IncA and IncC conjugative plasmids via an intermediate dusA-specific integrative element through gene losses and gain of alternative integration/excision modules.

microbiology↗