SummaryIn vibrios and enterobacteria lateral gene transfer is often facilitated by integrating conjugative elements (ICEs) of the SXT/R391 family. SXT/R391 ICEs integrate by site-specific recombination into prfC and transfer by conjugation, a process that is initiated at a specific locus called the origin of transfer (oriTSXT). We identified genomic islands (GIs) harbouring a sequence that shares > 63% identity with oriTSXT in three species of Vibrio. Unlike SXT/R391 ICEs, these GIs are integrated into a gene coding for a putative stress-induced protein and do not appear to carry any gene coding for a conjugative machinery or for mobilization proteins. Our results show that SXT/R391 ICEs trigger the excision and mediate the conjugative transfer in trans of the three Vibrio GIs at high frequency. GIs' excision is independent of the ICEencoded recombinase and is controlled by the ICEencoded transcriptional activator SetCD, which is expressed during the host SOS response. Both mobI and traI, two ICE-borne genes involved in oriT recognition, are essential for GIs' transfer. We also found that SXT/R391 ICEs mobilize in trans over 1 Mb of chromosomal DNA located 5Ј of the GIs' integration site. Together these results support a novel mechanism of mobilization of GIs by ICEs of the SXT/R391 family.

Integrating conjugative elements (ICEs) are self-transmissible, mobile elements that are widespread among bacteria. Following their excision from the chromosome, ICEs transfer by conjugation, a process initiated by a single-stranded DNA break at a specific locus called the origin of transfer (oriT). The SXT/R391 family of ICEs includes SXT MO10 , R391, and more than 25 related ICEs found in gammaproteobacteria. A previous study mapped the oriT locus of SXT MO10 to a 550-bp intergenic region between traD and s043. We suspected that this was not the correct oriT locus, because the identical traD-s043 region in R391 and other SXT/R391 family ICEs was annotated as a gene of an unknown function. Here, we investigated the location and structure of the oriT locus in the ICEs of the SXT/R391 family and demonstrated that oriT SXT corresponds to a 299-bp sequence that contains multiple imperfect direct and inverted repeats and is located in the intergenic region between s003 and rumB. The oriT SXT locus is well conserved among SXT/R391 ICEs, like R391, R997, and pMERPH, and cross-recognition of oriT SXT and oriT R391 by R391 and SXT MO10 was demonstrated. Furthermore, we identified a previously unannotated gene, mobI, located immediately downstream from oriT SXT , which proved to be essential for SXT MO10 transfer and SXT MO10 -mediated chromosomal DNA mobilization. Deletion of mobI did not impair the SXT MO10 -dependent transfer of the mobilizable plasmid CloDF13, suggesting that mobI has no role in the assembly of the SXT MO10 mating pair apparatus. Instead, mobI appears to be involved in the recognition of oriT SXT .Integrating conjugative elements (ICEs) are a large family of self-transmissible mobile genetic elements that are widespread among bacteria (8,12). These elements confer a range of properties upon the host bacteria, including resistance to antibiotics and heavy metals, virulence, symbiosis establishment, and alternative metabolic pathways. ICEs are made of a core set of genes ensuring their maintenance, mobility, and regulation (8,12,44,50).Following their excision from the chromosome of the host cell as a circular intermediate, ICEs transfer by conjugation, using a mechanism similar to that of conjugative plasmids (8,15,19,23,32,39). Integration into and excision from the chromosome occur by recombination mediated by an ICEencoded site-specific recombinase called integrase (Int) (10). Several ICEs are able to mobilize nonconjugative plasmids in cis and in trans as well as chromosomal DNA in an Hfr-like manner (27).Conjugative DNA transfer takes place in two key steps: (i) biochemical processing of the DNA molecule for transfer and (ii) assembly of a mating apparatus bridging the donor and recipient cells to allow DNA transfer. Typically conjugative DNA transfer is initiated at a specific cis-acting site called the origin of transfer (oriT), required for efficient translocation of the DNA to the recipient cell (23,39). A DNA relaxase encoded by the conjugative element recognizes the oriT locus and cleaves o...

Mobilizable genomic islands (MGIs) are small genomic islands that are mobilizable by SXT/R391 integrating conjugative elements (ICEs) due to similar origins of transfer. Their site-specific integration and excision are catalyzed by the integrase that they encode, but their conjugative transfer entirely depends upon the conjugative machinery of SXT/R391 ICEs. In this study, we report the mechanisms that control the excision and integration processes of MGIs. We found that while the MGI-encoded integrase Int MGI is sufficient to promote MGI integration, efficient excision from the host chromosome requires the combined action of Int MGI and of a novel recombination directionality factor, RdfM. We determined that the genes encoding these proteins are activated by SetCD, the main transcriptional activators of SXT/R391 ICEs. Although they share the same regulators, we found that unlike rdfM, int MGI has a basal level of expression in the absence of SetCD. These findings explain how an MGI can integrate into the chromosome of a new host in the absence of a coresident ICE and shed new light on the cross talk that can occur between mobilizable and mobilizing elements that mobilize them, helping us to understand part of the rules that dictate horizontal transfer mechanisms.

Mobilizable genomic islands (MGIs) are small genomic islands of less than 35 kbp containing an integrase gene and a sequence that resembles the origin of transfer (oriT) of an integrating conjugative element (ICE). MGIs have been shown to site-specifically integrate and excise from the chromosome of bacterial hosts and hijack the conjugative machinery of a coresident ICE to disseminate. To date, MGIs have been described in three strains belonging to three different Vibrio species. In this study, we report the discovery of 11 additional putative MGIs found in various species of Vibrio, Alteromonas, Pseudoalteromonas, and Methylophaga. We designed an MGI capture system that allowed us to relocate chromosomal MGIs onto a low-copy-number plasmid and facilitate their isolation and sequencing. Comparative genomics and phylogenetic analyses of these mobile genetic elements revealed their mosaic structure and their evolution through recombination and acquisition of exogenous DNA. MGIs were found to belong to a larger family of genomic islands (GIs) sharing a similar integrase gene and often integrated into the same integration site yet exhibiting a different mechanism of regulation of excision and mobilization. We found that MGIs can excise only when an ICE of the SXT/R391 family is coresident in the same cell, while GIs still excise regardless.