Comparative analysis of mobilizable genomic islands.

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.

Dynamics of the SetCD-regulated integration and excision of genomic islands mobilized by integrating conjugative elements of the SXT/R391 family.

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.

Integrating conjugative elements of the SXT/R391 family trigger the excision and drive the mobilization of a new class of Vibrio genomic islands.

In 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 (oriT(SXT) ). We identified genomic islands (GIs) harbouring a sequence that shares >63% identity with oriT(SXT) 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 ICE-encoded recombinase and is controlled by the ICE-encoded 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 5' of the GIs' integration site. Together these results support a novel mechanism of mobilization of GIs by ICEs of the SXT/R391 family.

Identification of the origin of transfer (oriT) and a new gene required for mobilization of the SXT/R391 family of integrating conjugative elements.

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).