DNA replication of the two Vibrio cholerae chromosomes

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Vibrio cholerae is one of the deadliest pathogens in the history of humankind. It is the causative agent of cholera, a disease characterized by a profuse and watery diarrhea. A growing problem of pandemic cholera is caused by antibiotic resistant strains. The search for novel and specific drug targets requires a deep understanding of the V. cholerae biology and one focus was laid on the peculiar genome arrangement in recent years. While most bacteria carry their genetic information on a single chromosome, the genome of V. cholerae is split on two chromosomes as it is the case for almost all members of the genus Vibrio. The size of the Chr1 and Chr2 of V. cholerae chromosomes is about 3 and 1 Mbps, respectively. DNA replication of each of the two chromosomes is initiated at a single replication origin with ori1 regulation being dependent on the initiator protein DnaA and ori2 on the Vibrio-specific protein RctB. Interestingly, Chr2 replication is initiated after about two thirds of Chr1 are replicated leading to synchronous termination of replication of the two replicons. This timing was recently found to be dependent on a novel DNA sequence designated crtS (Chr2 triggering site) located on Chr1. It was shown that doubling of the crtS by the process of DNA replication triggers the initiation at ori2. However, our knowledge about this mechanism is only incomplete and consequently we mainly focus our work on the crtS site. A set of genetic and genomics experiments is planned to study the crtS-based triggering of Chr2 replication. In addition, we hypothesize that additional unknown factors are involved in V. cholerae replication origin regulation. We intent to find such predicted as-of-yet unknown factors using a high-throughput genetic screen. Identification of a new regulator would open up an exciting new avenue of research focus.