Bacterial genomes are mainly composed of two types of replicons: chromosomes, which are essential, and plasmids. Although most bacteria have only one chromosome, bacteria with secondary chromosomes have arisen independently in several taxa and represent approximately 10% of all bacterial species. Secondary chromosomes originate from plasmids and possess plasmid-type replication systems. While chromosomes replicate once and during a defined period of the cell cycle, plasmids generally replicate randomly. Vibrio cholerae has its genome divided in two chromosomes (Chr1 and Chr2) that use distinct initiators for replication. Chr1 replication depends on the ubiquitous initiator DnaA, while Chr2 replication is initiated by a Vibrio specific factor, RctB. Despite its plasmid origins, Chr2 replication is tightly controlled and occurs once per cell cycle. Both chromosomes communicate with each other to coordinate their replication through an RctB-binding locus on Chr1, called crtS. We have shown that crtS replication is crucial to trigger Chr2 replication initiation. However, the molecular mechanism by which crtS controls the initiation of Chr2 replication was still obscure. Here we combined in vivo and in vitro approaches to shed light on this question. We have shown that crtS activity is driven by RctB binding in a methylation-independent manner. This appears to be an effective way to integrate the Chr2 replication in the cell cycle.