Replication of a specific site on the main chromosome of V. cholerae triggers the replication initiation of its secondary chromosome.

The Génolevures Consortium 1 Our knowledge of yeast genomes remains largely dominated by the extensive studies on Saccharomyces cerevisiae and the consequences of its ancestral duplication, leaving the evolution of the entire class of hemiascomycetes only partly explored. We concentrate here on five species of Saccharomycetaceae, a large subdivision of hemiascomycetes, that we call ''protoploid'' because they diverged from the S. cerevisiae lineage prior to its genome duplication. We determined the complete genome sequences of three of these species: Kluyveromyces (Lachancea) thermotolerans and Saccharomyces (Lachancea) kluyveri (two members of the newly described Lachancea clade), and Zygosaccharomyces rouxii. We included in our comparisons the previously available sequences of Kluyveromyces lactis and Ashbya (Eremothecium) gossypii. Despite their broad evolutionary range and significant individual variations in each lineage, the five protoploid Saccharomycetaceae share a core repertoire of approximately 3300 protein families and a high degree of conserved synteny. Synteny blocks were used to define gene orthology and to infer ancestors. Far from representing minimal genomes without redundancy, the five protoploid yeasts contain numerous copies of paralogous genes, either dispersed or in tandem arrays, that, altogether, constitute a third of each genome. Ancient, conserved paralogs as well as novel, lineage-specific paralogs were identified.

A major determinant of Vibrio cholerae pathogenicity, the cholera enterotoxin, is encoded in the genome of an integrated phage, CTXvarphi. CTXvarphi integration depends on two host-encoded tyrosine recombinases, XerC and XerD. It occurs at dif1, a 28 bp site on V. cholerae chromosome 1 normally used by XerCD for chromosome dimer resolution. The replicative form of the phage contains two pairs of binding sites for XerC and XerD in inverted orientations. Here we show that in the single-stranded genome of the phage, these sites fold into a hairpin structure, which creates a recombination target for XerCD. In the presence of XerD, XerC can catalyze a single pair of strand exchanges between this target and dif1, resulting in integration of the phage. This integration strategy explains why the rules that normally apply to tyrosine recombinase reactions seemed not to apply to CTXvarphi integration and, in particular, why integration is irreversible.

Unlike most bacteria, Vibrio cholerae harbors two distinct, nonhomologous circular chromosomes (chromosome I and II). Many features of chromosome II are plasmid-like, which raised questions concerning its chromosomal nature. Plasmid replication and segregation are generally not coordinated with the bacterial cell cycle, further calling into question the mechanisms ensuring the synchronous management of chromosome I and II. Maintenance of circular replicons requires the resolution of dimers created by homologous recombination events. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover at a specific site, dif, by two tyrosine recombinases, XerC and XerD. The process is coordinated with cell division through the activity of a DNA translocase, FtsK. Many E. coli plasmids also use XerCD for dimer resolution. However, the process is FtsK-independent. The two chromosomes of the V. cholerae N16961 strain carry divergent dimer resolution sites, dif1 and dif2. Here, we show that V. cholerae FtsK controls the addition of a crossover at dif1 and dif2 by a common pair of Xer recombinases. In addition, we show that specific DNA motifs dictate its orientation of translocation, the distribution of these motifs on chromosome I and chromosome II supporting the idea that FtsK translocation serves to bring together the resolution sites carried by a dimer at the time of cell division. Taken together, these results suggest that the same FtsK-dependent mechanism coordinates dimer resolution with cell division for each of the two V. cholerae chromosomes. Chromosome II dimer resolution thus stands as a bona fide chromosomal process.

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.

International audienceCholera toxin is encoded in the genome of CTXvarphi, a lysogenic filamentous phage of Vibrio cholerae. CTXvarphi variants contribute to the genetic diversity of cholera epidemic strains. It has been shown that the El Tor variant of CTXvarphi hijacks XerC and XerD, two host-encoded tyrosine recombinases that normally function to resolve chromosome dimers, to integrate at dif1, the dimer resolution site of the larger of the two V. cholerae chromosomes. However, the exact mechanism of integration of CTXvarphi and the rules governing its integration remained puzzling, with phage variants integrated at either or both dimer resolution sites of the two V. cholerae chromosomes. We designed a genetic system to determine experimentally the tropism of integration of CTXvarphi and thus define rules of compatibility between phage variants and dimer resolution sites. We then showed in vitro how these rules are explained by the direct integration of the single-stranded phage genome into the double-stranded bacterial genome. Finally, we showed how the evolution of phage attachment and chromosome dimer resolution sites contributes to the generation of genetic diversity among cholera epidemic strains

The effects on cell physiology of gene order within the bacterial chromosome are poorly understood. In silico approaches have shown that genes involved in transcription and translation processes, in particular ribosomal protein (RP) genes, localize near the replication origin (oriC) in fast-growing bacteria suggesting that such a positional bias is an evolutionarily conserved growth-optimization strategy. Such genomic localization could either provide a higher dosage of these genes during fast growth or facilitate the assembly of ribosomes and transcription foci by keeping physically close the many components of these macromolecular machines. To explore this, we used novel recombineering tools to create a set of Vibrio cholerae strains in which S10-spec-α (S10), a locus bearing half of the ribosomal protein genes, was systematically relocated to alternative genomic positions. We show that the relative distance of S10 to the origin of replication tightly correlated with a reduction of S10 dosage, mRNA abundance and growth rate within these otherwise isogenic strains. Furthermore, this was accompanied by a significant reduction in the host-invasion capacity in Drosophila melanogaster. Both phenotypes were rescued in strains bearing two S10 copies highly distal to oriC, demonstrating that replication-dependent gene dosage reduction is the main mechanism behind these alterations. Hence, S10 positioning connects genome structure to cell physiology in Vibrio cholerae. Our results show experimentally for the first time that genomic positioning of genes involved in the flux of genetic information conditions global growth control and hence bacterial physiology and potentially its evolution.

SummaryDam methylates GATC sequences in γ-proteobacteria genomes, regulating several cellular functions including replication. In Vibrio cholerae, which has two chromosomes, Dam is essential for viability, owing to its role in chr2 replication initiation. In this study, we isolated spontaneous mutants of V. cholerae that were able to survive the deletion of dam. In these mutants, homologous recombination and chromosome dimer resolution are essential, unless DNA mismatch repair is inactivated. Furthermore, the initiator of chr2 replication, RctB, is no longer required. We show that, instead, replication of chr2 is insured by spontaneous fusion with chr1 and piggybacking its replication machinery. We report that natural fusion of chr1 and chr2 occurred by two distinct recombination pathways: homologous recombination between repeated IS elements and site-specific recombination between dif sites. Lastly, we observed a preferential fusion of the two chromosomes in their terminus of replication.