Entry Exclusion of Conjugative Plasmids of the IncA, IncC, and Related Untyped Incompatibility Groups

Abstract

Conjugative plasmids of incompatibility group C (IncC), formerly known as A/C 2 , disseminate antibiotic resistance genes globally in diverse pathogenic species of Gammaproteobacteria. Salmonella genomic island 1 (SGI1) can be mobilized by IncC plasmids and was recently shown to reshape the conjugative type IV secretion system (T4SS) encoded by these plasmids to evade entry exclusion. Entry exclusion blocks DNA translocation between cells containing identical or highly similar plasmids. Here, we report that the protein encoded by the entry exclusion gene of IncC plasmids (eexC) mediates entry exclusion in recipient cells through recognition of the IncC-encoded TraG C protein in donor cells. Phylogenetic analyses based on EexC and TraG C homologs predicted the existence of at least three different exclusion groups among IncC-related conjugative plasmids. Mating assays using Eex proteins encoded by representative IncC and IncA (former A/C 1 ) and related untyped plasmids confirmed these predictions and showed that the IncC and IncA plasmids belong to the C exclusion group, thereby explaining their apparent incompatibility despite their compatible replicons. Representatives of the two other exclusion groups (D and E) are untyped conjugative plasmids found in Aeromonas sp. Finally, we determined through domain swapping that the carboxyl terminus of the EexC and EexE proteins controls the specificity of these exclusion groups. Together, these results unravel the role of entry exclusion in the apparent incompatibility between IncA and IncC plasmids while shedding light on the importance of the TraG subunit substitution used by SGI1 to evade entry exclusion. IMPORTANCE IncA and IncC conjugative plasmids drive antibiotic resistance dissemination among several pathogenic species of Gammaproteobacteria due to the diversity of drug resistance genes that they carry and their ability to mobilize antibiotic resistance-conferring genomic islands such as SGI1 of Salmonella enterica. While historically grouped as "IncA/C," IncA and IncC replicons were recently confirmed to be compatible and to abolish each other's entry into the cell in which they reside during conjugative transfer. The significance of our study is in identifying an entry exclusion system that is shared by IncA and IncC plasmids. It impedes DNA transfer to recipient cells bearing a plasmid of either incompatibility group. The entry exclusion protein of this system is unrelated to any other known entry exclusion proteins.

Crucial Role ofSalmonellaGenomic Island 1 Master Activator in Parasitism of IncC plasmids

“…Although probably unable to activate self-transfer, entry of SGI1 in the host could resuscitate such "zombie" plasmids that would transiently regain their capacity to transfer, and mediate mobilisation of SGI1 (Supplementary Figure S6). This process would be facilitated by the ability of SGI1 to escape entry exclusion exerted by IncA and IncC plasmids that normally prevents or strongly reduces redundant transfer between cells that contain plasmids of the same entry exclusion group (31,38). Figure 1A), we propose that an IncC plasmid-encoded factor, triggers a positive feed-back loop in response to low levels of SgaCD likely via activation of sgaDC expression.…”

“…Oligonucleotides used in this study are listed in Supplementary Table S1. New sequencing data obtained from pVCR94 (NZ_CP033514.1) revealed that pVCR94 Sp (38) was missing eight conserved IncC genes. Therefore a new derivative, pVCR94 Sp2 , was constructed using the one-step chromosomal gene inactivation technique with pMS1, primer pair pVCR94delY2.f/94DelXnoFRT.rev and pVI36 as the template (39).…”

Exploration of the propagation of transpovirons within Mimiviridae reveals a unique example of commensalism in the viral world

“…All transpovirons encode a short predicted membrane protein although their primary sequence does appear to be conserved. The dominance effect is reminiscent of plasmids incompatibility or entry exclusion for related plasmids [60][61][62][63] or superinfection immunity [64] and superinfection exclusion [65] used by prophages to prevent superinfection by other bacteriophages. For the latter, DNA injection is often inhibited by a membrane-bound protein [66][67][68], paralleling the eventual role of the transpoviron-encoded membrane protein in giant virus particles carrying a transpoviron.…”

Commensalism in theMimiviridaegiant virus family

“…The sequence of this short protein is not conserved between transpovirons associated to different host virus clades but all transpovirons encode such predicted membrane protein. The dominance effect is reminiscent of plasmids incompatibility or entry exclusion for related plasmids (35–38) or superinfection immunity (39) and superinfection exclusion (40) used by prophages to prevent superinfection by other bacteriophages. For the latter, DNA-injection is inhibited by a membrane-bound protein (41–43), paralleling the eventual role of the transpoviron-encoded membrane protein in giant virus particles carrying a transpoviron.…”