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.

Coevolution of the Organization and Structure of Prokaryotic Genomes

“…Linear chromosomes can also be circularized (Volff et al 1997). Laboratory manipulations have shown that chromosomes can double in size in a small number of events (Itaya et al 2005), be split in multiple chromosomes (Itaya and Tanaka 1997), and multiple chromosomes can be merged into one (Val et al 2012). The effects of these dramatic structural modifications are as small as the rules of genome organization are respected.…”

“…This has led to suggestions that secondary replicons might favor evolvability . Additionally, it has been observed that replicon fusions in Vibrio lead to lower growth rates and more frequent dimer formation (Val et al 2012). The systematic presence of two chromosomes in the genus of Vibrio, typically very fast-growing bacteria, led to suggestions that multiple chromosomes facilitate rapid bacterial growth and the management of chromosome dimerization in large genomes.…”

The tRNA arg Gene and engA Are Essential Genes on the 1.7-Mb pSymB Megaplasmid of Sinorhizobium meliloti and Were Translocated Together from the Chromosome in an Ancestral Strain

“…Other examples in which essential genes appear to have been transferred to second replicons include p42e of Rhizobium etli (73), pNRC100 of Halobacterium sp. strain NRC-1 (74), chromosome III of Burkholderia cenocepacia AU-1054 (4), pSmeSM11d of S. meliloti SM11 (42), the linear chromosome of A. tumefaciens (17), and the second chromosome of Vibrio cholerae (5,75). However, we are aware of few studies in which postulated essential genes on second replicons have been verified experimentally.…”

DNA Methylation

“…This suggests that the genome organization around the origin may influence function and that care should be taken when analyzing origin function in a non-native context. However, in a V. cholerae strain carrying a fusion between chromosome I and II and with oriCI as sole replication origin, the dam gene could be deleted, which clearly shows that Dam methylation is not required for oriCI replication or any other processes essential for growth [191]. …”