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Abstract Structural maintenance of chromosomes (SMC) complexes organize genomes by extruding DNA loops, while replisomes duplicate entire chromosomes. These essential molecular machines must collide frequently in every cell cycle, yet how such collisions are resolved in vivo remains poorly understood. Taking advantage of the ability to load SMC complexes at defined sites in theBacillus subtilisgenome, we engineered head-on and head-to-tail collisions between SMC complexes and the replisome. Replisome progression was monitored by genome-wide marker frequency analysis, and SMC translocation was monitored by time-resolved ChIP-seq and Hi-C. We found that SMC complexes do not impede replisome progression. By contrast, replisomes restrict SMC translocation regardless of collision orientations. Combining experimental data with simulations, we determined that SMC complexes are blocked by the replisome and then released from the chromosome. Occasionally, SMC complexes can bypass the replisome and continue translocating. Our findings establish that the replisome is a barrier to SMC-mediated DNA-loop extrusion in vivo, with implications for processes such as chromosome segregation, DNA repair, and gene regulation that require dynamic chromosome organization in all organisms. 

ABSTRACT Multipartite bacterial genome organization can confer advantages, including coordinated gene regulation and faster genome replication, but is challenging to maintain.Agrobacterium tumefacienslineages 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 analyzedAgrobacteriumnatural 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. IMPORTANCEMost bacterial genomes are monopartite with a single, circular chromosome. However, some species, likeAgrobacterium 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 ofA. tumefaciensstrain C58 in which the circular and linear chromosomes fused to form a single dicentric chromosome. Here we discovered two geographically separated environmental isolates ofA. tumefacienscontaining 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. 

ABSTRACT Histone-like nucleoid structuring (H-NS) and H-NS-like proteins serve as global gene silencers and work with antagonistic transcriptional activators (counter-silencers) to properly coordinate the expression of virulence genes in pathogenic bacteria. InBrucella, MucR has been proposed as a novel H-NS-like gene silencer, but direct experimental evidence is lacking. Here, we show that MucR serves as an H-NS-like silencer of theBrucella abortusgenes encoding the polar autotransporter adhesins BtaE and BmaC, the c-di-GMP-specific phosphodiesterase BpdB, and the quorum-sensing regulator BabR. We also demonstrate that the MarR-type transcriptional activator MdrA can displace MucR from thebtaEpromoter, supporting the existence of MucR counter-silencers inBrucella. Moreover, our chromatin immunoprecipitation (ChIP)-seq analysis identified 546 MucR enrichment peaks along the genome, including in the promoters of the genes encoding the Type IV secretion machinery and effectors and the quorum-sensing regulator VjbR. Importantly, MucR ChIP-seq peaks overlap with the previously described binding sites for the transcriptional activators VjbR, BvrR, and CtrA suggesting that these regulators serve as MucR counter-silencers and work in concert with MucR to coordinate virulence gene expression inBrucella. In addition, using chromosome conformation capture (Hi-C), we show that like H-NS inEscherichia coli, MucR alters the global structure of theBrucellanucleoid. Finally, a copy of theE. coli hnsrescues the distinctive growth defect and elevatedbtaEexpression of aB. abortus mucRmutant. Together, these findings solidify the role of MucR as a novel type of H-NS-like protein and suggest that MucR’s gene-silencing properties play a key role in virulence inBrucella. IMPORTANCEHistone-like nucleoid structuring (H-NS) and H-NS-like proteins coordinate host-associated behaviors in many pathogenic bacteria, often through forming silencer/counter-silencer pairs with signal-responsive transcriptional activators to tightly control gene expression.Brucellaand related bacteria do not encode H-NS or homologs of known H-NS-like proteins, and it is unclear if they have other proteins that perform analogous functions during pathogenesis. In this work, we provide compelling evidence for the role of MucR as a novel H-NS-like protein inBrucella. We show that MucR possesses many of the known functions attributed to H-NS and H-NS-like proteins, including the formation of silencer/counter-silencer pairs to control virulence gene expression and global structuring of the nucleoid. These results uncover a new role for MucR as a nucleoid structuring protein and support the importance of temporal control of gene expression inBrucellaand related bacteria. 

Borrelia burgdorferi, a causative agent of Lyme disease, contains the most segmented bacterial genome known to date, with one linear chromosome and over twenty plasmids. How this unusually complex genome is organized, and whether and how the different replicons interact are unclear. We recently demonstrated thatB.burgdorferiis polyploid and that the copies of the chromosome and plasmids are regularly spaced in each cell, which is critical for faithful segregation of the genome to daughter cells. Regular spacing of the chromosome is controlled by two separate partitioning systems that involve the protein pairs ParA/ParZ and ParB/Smc. Here, using chromosome conformation capture (Hi-C), we characterized the organization of theB.burgdorferigenome and the interactions between the replicons. We uncovered that although the linear chromosome lacks contacts between the two replication arms, the two telomeres are in frequent contact. Moreover, several plasmids specifically interact with the chromosomeoriCregion, and a subset of plasmids interact with each other more than with others. We found that Smc and the Smc-like MksB protein mediate long-range interactions on the chromosome, but they minimally affect plasmid-chromosome or plasmid-plasmid interactions. Finally, we found that disruption of the two partition systems leads to chromosome restructuring, correlating with the mis-positioning of chromosomeoriC. Altogether, this study revealed the conformation of a complex genome and analyzed the contribution of the partition systems and SMC family proteins to this organization. This work expands the understanding of the organization and maintenance of multipartite bacterial genomes.