Vibrio cholerae chromosome 2 copy number is controlled by the methylation-independent binding of its monomeric initiator to the chromosome 1 crtS site

Bacteria contain a primary chromosome and, frequently, either essential secondary chromosomes or dispensable megaplasmids of plasmid origin. Incoming plasmids are often poorly adapted to their hosts and their stabilization requires integration with the host's cellular mechanisms in a process termed domestication. All Vibrio, including pathogenic species, carry a domesticated secondary chromosome (Chr2) where replication is coordinated with that of the primary chromosome (Chr1). Chr2 replication is triggered by the replication of an intergenic sequence (crtS) located on Chr1. Yet, the molecular mechanisms by which crtS replication controls the initiation of Chr2 replication are still largely unknown. In this study, we show that crtS not only regulates the timing of Chr2 initiation but also controls Chr2 copy number. We observed and characterized the direct binding of the Chr2 initiator (RctB) on crtS. RctB binding to crtS is independent of its methylation state. RctB molecules, which naturally form dimers, preferentially bind to crtS as monomers, with DnaK/J protein chaperones shown to stimulate binding of additional RctB monomers on crtS. In this study, we addressed various hypothesis of how replication of crtS could trigger Chr2 replication and provide new insights into its mode of action.