Vibrio cholerae, the pathogenic bacterium that causes cholera, has two chromosomes (Chr1, Chr2) that replicate in a well-orchestrated sequence. Chr2 initiation is triggered only after the replication of the crtS site on Chr1. The initiator of Chr2 replication, RctB, displays activities corresponding with its different binding sites: initiator at the iteron sites, repressor at the 39m sites, and trigger at the crtS site. The mechanism by which RctB relays the signal to initiate Chr2 replication from crtS is not well-understood. In this study, we provide new insights into how Chr2 replication initiation is regulated by crtS via RctB. We show that crtS (on Chr1) acts as an anti-inhibitory site by preventing 39m sites (on Chr2) from repressing initiation. The competition between these two sites for RctB binding is explained by the fact that RctB interacts with crtS and 39m via the same DNA-binding surface. We further show that the extreme C-terminal tail of RctB, essential for RctB self-interaction, is crucial for the control exerted by crtS. This subregion of RctB is conserved in all Vibrio, but absent in other Rep-like initiators. Hence, the coordinated replication of both chromosomes likely results from the acquisition of this unique domain by RctB.

Integrons are genetic elements found exclusively in bacteria. They are well known for their role in disseminating antibiotic resistance genes among pathogens and more generally for enabling bacteria to rapidly adapt to changing environmental conditions. Integrons constitute a natural system to capture, stockpile, shuffle, express and disseminate genes embedded in cassettes. All these events are governed by the integron integrase through site-specific recombination between integron att sites (attC and attI sites). Here, we demonstrate that integron integrase can efficiently catalyze the insertion of cassettes in bacterial genomes, outside the att sites carried by the integron system. Surprisingly, analysis of more than 500,000 independent clones revealed that the genome recombination sites differ greatly, in terms of sequence and structure, from both classical attC and attI recombination sites. We named these new sites attG. Notably, among these a few are driving integration at very high rates. We also showed that, once inserted in genomes, cassettes can be expressed if located near a bacterial promoter. Moreover, even if occurring at low frequency, genome inserted cassettes can be excised precisely or imprecisely, inducing in this latter case, chromosomal modifications. These results unveil a new route for antibiotic resistance dissemination and expand the role of integrons in bacterial genome evolution.