A dicentric bacterial chromosome requires XerC/D site-specific recombinases for resolution.

Unlike eukaryotes and archaea, which have multiple replication origins on their chromosomes, bacterial chromosomes usually contain a single replication origin.1 Here, we discovered a dicentric bacterial chromosome with two replication origins, which has resulted from the fusion of the circular and linear chromosomes in Agrobacterium tumefaciens. The fused chromosome is well tolerated, stably maintained, and retains similar subcellular organization and genome-wide DNA interactions found for the bipartite chromosomes. Strikingly, the two replication origins and their partitioning systems are both functional and necessary for cell survival. Finally, we discovered that the site-specific recombinases XerC and XerD2 are essential in cells harboring the fused chromosome but not in cells with bipartite chromosomes. Analysis of actively dividing cells suggests a model in which XerC/D are required to recombine the sister fusion chromosomes when the two centromeres on the same chromosome are segregated to opposite cell poles. Thus, faithful segregation of dicentric chromosomes in bacteria can occur because of site-specific recombination between the sister chromatids during chromosome partitioning. Our study provides a natural comparative platform to examine a bacterial chromosome with multiple origins and a possible explanation for the fundamental difference in bacterial genome architecture relative to eukaryotes and archaea.1.

Centromere Interactions Promote the Maintenance of the Multipartite Genome in Agrobacterium tumefaciens.

Many pathogens or symbionts of animals and plants contain multiple replicons, a configuration called a multipartite genome. Multipartite genomes enable those species to replicate their genomes faster and better adapt to new niches. Despite their prevalence, the mechanisms by which multipartite genomes are stably maintained are poorly understood. Agrobacterium tumefaciens is a plant pathogen that contains four replicons: a circular chromosome (Ch1), a linear chromosome (Ch2), and two large plasmids. Recent work indicates that their replication origins are clustered at the cell poles in a manner that depends on their ParB family centromeric proteins: ParB1 for Ch1 and individual RepB paralogs for Ch2 and the plasmids. However, understanding of these interactions and how they contribute to genome maintenance is limited. By combining genome-wide chromosome conformation capture (Hi-C) assays, chromatin-immunoprecipitation sequencing (ChIP-seq), and live cell fluorescence microscopy, we provide evidence here that centromeric clustering is mediated by interactions between these centromeric proteins. We further show that the disruption of centromere clustering results in the loss of replicons. Our data establish the role of centromeric clustering in multipartite genome stability. IMPORTANCE About 10% of sequenced bacteria have multiple replicons, also known as multipartite genomes. How these multipartite genomes are maintained is still poorly understood. Here, we use Agrobacterium tumefaciens as a model and show that the replication origins of the four replicons are clustered through direct interactions between the centromeric proteins; disruption of origin clustering leads to the loss of replicons. Thus, our study provided evidence that centromeric clustering is important for maintaining multipartite genomes.