Replication of the Salmonella Genomic Island 1 (SGI1) triggered by helper IncC conjugative plasmids promotes incompatibility and plasmid loss

Author summary TheSalmonellagenomic island 1 (SGI1) and its variants propagate multidrug resistance in several species of human and animal pathogens with the help of IncA and IncC conjugative plasmids that are absolutely required for SGI1 dissemination. These helper plasmids are known to trigger the excision of SGI1 from the chromosome. Here, we found that IncC plasmids also trigger the replication of the excised, circular form of SGI1 by enabling the expression of an SGI1-borne replication initiator gene. In return, high-copy replication of SGI1 interferes with the persistence of the IncC plasmid and prevents its cotransfer into a recipient cell, thereby allowing integration and stabilization of SGI1 into the chromosome of the new host. This finding is important to better understand the complex interactions between SGI1-like elements and their helper plasmids that lead to widespread and highly efficient propagation of multidrug resistance genes to a broad range of human and animal pathogens. The mobilizable resistance islandSalmonellagenomic island 1 (SGI1) is specifically mobilized by IncA and IncC conjugative plasmids. SGI1, its variants and IncC plasmids propagate multidrug resistance in pathogenic enterobacteria such asSalmonella entericaserovars andProteus mirabilis. SGI1 modifies and uses the conjugation apparatus encoded by the helper IncC plasmid, thus enhancing its own propagation. Remarkably, although SGI1 needs a coresident IncC plasmid to excise from the chromosome and transfer to a new host, these elements have been reported to be incompatible. Here, the stability of SGI1 and its helper IncC plasmid, each expressing a different fluorescent reporter protein, was monitored using fluorescence-activated cell sorting (FACS). Without selective pressure, 95% of the cells segregated into two subpopulations containing either SGI1 or the helper plasmid. Furthermore, FACS analysis revealed a high level of SGI1-specific fluorescence in IncC(+)cells, suggesting that SGI1 undergoes active replication in the presence of the helper plasmid. SGI1 replication was confirmed by quantitative PCR assays, and extraction and restriction of its plasmid form. Deletion of genes involved in SGI1 excision from the chromosome allowed a stable coexistence of SGI1 with its helper plasmid without selective pressure. In addition, deletion ofS003(rep) or of a downstream putative iteron-based origin of replication, while allowing SGI1 excision, abolished its replication, alleviated the incompatibility with the helper plasmid and enabled its cotransfer to a new host. Like SGI1 excision functions,repexpression was found to be controlled by AcaCD, the master activator of IncC plasmid transfer. Transient SGI1 replication seems to be a key feature of the life cycle of this family of genomic islands. Sequence database analysis revealed that SGI1 variants encode either a replication initiator protein with a RepA_C domain, or an alternative replication protein with N-terminal replicase and primase C terminal 1 domains.