title: Genome engineering in Vibrio cholerae: a feasible approach to address biological issues.
abstract: Although bacteria with multipartite genomes are prevalent, our knowledge of the mechanisms maintaining their genome is very limited, and much remains to be learned about the structural and functional interrelationships of multiple chromosomes. Owing to its bi-chromosomal genome architecture and its importance in public health, Vibrio cholerae, the causative agent of cholera, has become a preferred model to study bacteria with multipartite genomes. However, most in vivo studies in V. cholerae have been hampered by its genome architecture, as it is difficult to give phenotypes to a specific chromosome. This difficulty was surmounted using a unique and powerful strategy based on massive rearrangement of prokaryotic genomes. We developed a site-specific recombination-based engineering tool, which allows targeted, oriented, and reciprocal DNA exchanges. Using this genetic tool, we obtained a panel of V. cholerae mutants with various genome configurations: one with a single chromosome, one with two chromosomes of equal size, and one with both chromosomes controlled by identical origins. We used these synthetic strains to address several biological questions--the specific case of the essentiality of Dam methylation in V. cholerae and the general question concerning bacteria carrying circular chromosomes--by looking at the effect of chromosome size on topological issues. In this article, we show that Dam, RctB, and ParA2/ParB2 are strictly essential for chrII origin maintenance, and we formally demonstrate that the formation of chromosome dimers increases exponentially with chromosome size.
authors.affiliation: Institut Pasteur, Unité Plasticité du Génome Bactérien, Département Génomes et Génétique, Paris, France.
authors.forename: Marie-Eve
authors.initials: ME
authors.lastname: Val
authors.forename: Ole
authors.initials: O
authors.lastname: Skovgaard
authors.forename: Magaly
authors.initials: M
authors.lastname: Ducos-Galand
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Bland
authors.forename: Didier
authors.initials: D
authors.lastname: Mazel
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002771:Cholera / Q000382:microbiology; D002876:Chromosomes, Bacterial / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics; D045566:DNA, Cruciform / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D016680:Genome, Bacterial / Q000235:genetics*; D059765:Homologous Recombination / Q000235:genetics; D006801:Humans; D018741:Replication Origin / Q000235:genetics*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics* / Q000378:metabolism; D014734:Vibrio cholerae / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D045566:DNA, Cruciform; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)