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

Abstract

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 C58 lab model, Ch1 and Ch2 were fused into a linear dicentric chromosome. Here we analyzed Agrobacterium natural isolates from the French Collection for Plant-Associated Bacteria and identified two strains distinct from C58 with fused chromosomes. Chromosome conformation capture identified integration junctions that were different from the C58 fusion strain. Genome-wide DNA replication profiling showed that both replication origins remained active. Transposon sequencing revealed that partitioning systems of both chromosome centromeres were essential. Importantly, the site-specific recombinase XerCD is 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. However, 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 geographically separated environmental isolates of A. tumefaciens containing fused chromosomes with integration junctions different from the C58 fusion chromosome, 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 reveal how multipartite genomes function across different bacterial species and the role of genomic plasticity in bacterial genetic diversification.

Keywords: Agrobacterium tumefaciens; Hi-C; chromosome fusion; multipartite genome; natural isolates.

Fig 1

Phylogenetic analysis of natural A. tumefaciens isolates. (A) Maximum likelihood phylogeny based on 4,192 genes conserved in the six analyzed strains. The tree is midpoint rooted. Branches with ultrafast bootstrap >95% and SH-aLRT >80% are black; branches with other support are gray. Branch scale is indicated by a bar. (B) Genome synteny between 47-2, CFBP_2407, and CFBP_2642. Blue arrows indicate individual replicons and their direction in the assembly. Colored bars linking replicons of different strains indicate regions of similarity. Copper links indicate the same orientation; green links indicate inversions. Darker colors indicate greater sequence similarity. Red boxes indicate the position of rrn regions. Cyan rounded rectangles indicate the position of integrative and conjugative elements (ICEs).

Fig 2

Natural A. tumefaciens isolates CFBP_2407 and CFBP_2642 exhibit linear dicentric chromosomes. (A–C) CFBP_2407 and CFBP_2642 contain a fused chromosome, while 47-2 serves as a binary control. (Top) Schematics of the genome composition of the indicated strains. J1 and J2 mark the junctions of chromosome fusion. (Middle) Normalized Hi-C interaction maps of the respective genomes. Red circles indicate the terminus regions of the circular Ch1 (A) or the linear fusion chromosomes (B and C). (Bottom) Marker frequency analysis of exponentially growing cells. The y-axis depicts the number of reads.

Fig 3

Identifying the junction of chromosome fusion. (Left) Hi-C reads from fusion isolates (CFBP_2407 and CFBP_2642) were mapped to the genome of the binary 47-2 strain. White spaces indicate unmapped regions on the 47-2 genome. (Middle) The breakpoints of the Hi-C maps were highlighted with red dashed lines. The maps were cut and reassembled to best match the confluent maps generated using sequenced genomes (right). Based on this approach, we identified genetic loci of the probable fusion junctions (red arrows).

Fig 4

Sites of chromosome fusion in natural isolates and the lab C58 strain. (A) (Upper panel) Schematic illustration of chromosome fusion events in the natural isolates of CFBP_2407 and CFBP_2642 compared with the binary control 47-2. CFBP fusion isolates have a 77 kb insertion (maroon) resembling an integrative conjugative element. (Lower panel) Genetic context of the fusion junctions in the binary and fusion strains. The 77 kb ICE region is not drawn to scale. (B) Schematic illustration of chromosome fusion events in C58 (13).

Fig 5

Gene essentiality in the three natural isolates. Transposon sequencing profiles of Ch1 partitioning system parAB (A) and Ch2 partitioning system repABC (B) and genes encoding for tyrosine recombinases xerC (C) and xerD (D). The x-axis depicts genome position, and the y-axis represents the number of transposon insertions. (E) Distribution of the FtsK orienting polar sequences (KOPS) (GGGNAGGG) on the chromosomes of the indicated strains. Black and blue bars indicate KOPS sequences on the top and bottom DNA strands, respectively. The dif site is illustrated by a black triangle.