Linear dicentric chromosomes in bacterial natural isolates reveal common constraints for replicon fusion

Summary Multipartite bacterial genome organization can confer advantages including coordinated gene regulation and faster genome replication but is challenging to maintain. Agrobacterium tumefaciens lineages often contain a circular chromosome (Ch1), a linear chromosome (Ch2), and multiple plasmids. We previously observed that in some stocks of the lab model strain C58, Ch1 and Ch2 were fused into a linear dicentric chromosome. Here we analyzed Agrobacterium natural isolates from the French Collection for Plant-Associated Bacteria (CFBP) and identified two strains with fused chromosomes. Chromosome conformation capture identified integration junctions that were different from the C58 fusion strain. Genome-wide DNA replication profiling showed both replication origins remain active. Transposon sequencing revealed that partitioning systems of both chromosome centromeres are essential. Importantly, the site-specific recombinases XerCD are required for the survival of the strains containing the fusion chromosome. Our findings show that replicon fusion occurs in natural environments and that balanced replication arm sizes and proper resolution systems enable the survival of such strains. Importance Most bacterial genomes are monopartite with a single, circular chromosome. But some species, like Agrobacterium tumefaciens, carry multiple chromosomes. Emergence of multipartite genomes is often related to adaptation to specific niches including pathogenesis or symbiosis. Multipartite genomes confer certain advantages, however, maintaining this complex structure can present significant challenges. We previously reported a laboratory-propagated lineage of A. tumefaciens strain C58 in which the circular and linear chromosomes fused to form a single dicentric chromosome. Here we discovered two environmental isolates of A. tumefaciens containing fused chromosomes derived from a different route, revealing the constraints and diversification of this process. We found that balanced replication arm sizes and the repurposing of multimer resolution systems enable the survival and stable maintenance of dicentric chromosomes. These findings help us better understand how multipartite genomes function across different bacterial species and the role of genomic plasticity in bacterial genetic diversification.