Genome-wide binding of RctB initiator reveals the mechanism coordinating the replication of Vibrio cholerae two chromosomes.

Bacterial genomes can expand through the acquisition of megaplasmids, which eventually become secondary chromosomes encoding vital functions. In Vibrio cholerae, the replication of its secondary chromosome (Chr2) is initiated by RctB, a replication factor evolved from ancestral plasmid initiators. The hallmark of this system is the synchronization of Chr2 replication termination with the one of the main chromosome (Chr1) via a unique RctB-binding sequence (crtS). The molecular mechanisms underlying this coordination are not fully understood. Here we conducted a comprehensive genome-wide analysis of V. cholerae RctB binding pattern at different cell cycle stages. Our results show that RctB is primarily bound to sites that prevent the initiation of Chr2 replication. This effect is abolished with crtS replication which is coupled to a shift in the RctB binding pattern towards replication-activating sites, allowing Chr2 replication. Furthermore, our structural work supports the formation of diverse oligomeric states of RctB coupled to the allosteric effect of DNA that define the accessibility of ori2. We propose a synchronization model in which, upon replication, crtS can locally destabilize the RctB inhibition complex that otherwise locks the origin of Chr2 replication.