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Chromosome organization mediated by structural maintenance of chromosomes (SMC) complexes is vital in many organisms. SMC complexes act as motors that extrude DNA loops, but it remains unclear what happens when multiple complexes encounter one another on the same DNA in living cells and how these interactions may help to organize an active genome. We therefore created a crash-course track system to study SMC complex encounters in vivo by engineering defined SMC loading sites in the Bacillus subtilis chromosome. Chromosome conformation capture (Hi-C) analyses of over 20 engineered strains show an amazing variety of chromosome folding patterns. Through three-dimensional polymer simulations and theory, we determine that these patterns require SMC complexes to bypass each other in vivo, as recently seen in an in vitro study. We posit that the bypassing activity enables SMC complexes to avoid traffic jams while spatially organizing the genome. Hi-C analyses of engineered Bacillus subtilis strains with defined SMC loading sites and 3D polymer simulations indicate that SMC complex encounters on the same DNA are resolved via bypassing in vivo.

Key Points The bacterial chromosome must be linearly compacted more than 1,000-fold to fit within the bacterial cell. The chromosome is compacted in an orderly and hierarchical fashion in lockstep with DNA replication. This condensation has a central role in organizing replicated sister chromosomes and driving their segregation. The organization of the chromosome within the bacterial cell recapitulates the genetic map. Segregation of bacterial chromosomes can be broken down into three discrete steps: separation of the newly replicated origins; bulk chromosome segregation; and resolution and transport of the replication termini at the division septum. In many bacteria, origin segregation is facilitated by a highly conserved partitioning system. Bulk chromosome segregation is principally driven by the orderly compaction of the replicated sisters along adjacent DNA segments. This lengthwise condensation is mediated by the concerted action of supercoiling, small nucleoid-associated proteins and structural maintenance of chromosome (SMC) condensin complexes. Segregation of replicated termini requires topoisomerase IV to remove catenanes and XerCD recombinase to convert chromosome dimers into monomers. Decatenation and dimer resolution are coordinated and facilitated by a septum-associated DNA translocase. Bacterial chromosomes were originally thought to be unstructured and largely unconstrained, but recent advances have supplemented historical research to reveal a highly structured and dynamic chromosome organization. This Review discusses our latest understanding of bacterial chromosome organization, including how the simultaneous nature of DNA replication and chromosome segregation in bacteria necessitates intricate interplay between these processes. The bacterial chromosome must be compacted more than 1,000-fold to fit into the compartment in which it resides. How it is condensed, organized and ultimately segregated has been a puzzle for over half a century. Recent advances in live-cell imaging and genome-scale analyses have led to new insights into these problems. We argue that the key feature of compaction is the orderly folding of DNA along adjacent segments and that this organization provides easy and efficient access for protein–DNA transactions and has a central role in driving segregation. Similar principles and common proteins are used in eukaryotes to condense and to resolve sister chromatids at metaphase.

Genome organization is important for DNA replication, gene expression, and chromosome segregation. In bacteria, two large families of proteins, nucleoid-associated proteins (NAPs) and SMC complexes, play important roles in organizing the genome. NAPs are highly abundant DNA-binding proteins that can bend, wrap, bridge, and compact DNA, while SMC complexes load onto the chromosome, translocate on the DNA, and extrude DNA loops. Although SMC complexes are capable of traversing the entire chromosome bound by various NAPs in vivo, it is unclear whether SMC translocation is influenced by NAPs. In this study, using Bacillus subtilis as a model system, we expressed a collection of representative bacterial and archaeal DNA-binding proteins that introduce distinct DNA structures and potentially pose different challenges for SMC movement. By fluorescence microscopy and chromatin immunoprecipitation, we observed that these proteins bound to the genome in characteristic manners. Using genome-wide chromosome conformation capture (Hi-C) assays, we found that the SMC complex traversed these DNA-binding proteins without slowing down. Our findings revealed that the DNA-loop-extruding activity of the SMC complex is unaffected by exogenously expressed DNA-binding proteins, which highlights the robustness of SMC motors on the busy chromatin.

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 the Bacillus subtilis genome, 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. Replisomes duplicate genomes while SMC complexes organize the DNA. Replisomes and SMC collide frequently but it is unclear how the collisions are resolved. Here, the authors find that in Bacillus subtilis cells, SMC does not affect replisome progression, but replisomes restrict SMC movement by blocking and unloading it.

Bacterial chromosomes have been found to possess one of two distinct patterns of spatial organization. In the first, called “ ori - ter ” and exemplified by Caulobacter crescentus , the chromosome arms lie side-by-side, with the replication origin and terminus at opposite cell poles. In the second, observed in slow-growing Escherichia coli (“left- ori -right”), the two chromosome arms reside in separate cell halves, on either side of a centrally located origin. These two patterns, rotated 90° relative to each other, appear to result from different segregation mechanisms. Here, we show that the Bacillus subtilis chromosome alternates between them. For most of the cell cycle, newly replicated origins are maintained at opposite poles with chromosome arms adjacent to each other, in an ori-ter configuration. Shortly after replication initiation, the duplicated origins move as a unit to midcell and the two unreplicated arms resolve into opposite cell halves, generating a left- ori -right pattern. The origins are then actively segregated toward opposite poles, resetting the cycle. Our data suggest that the condensin complex and the parABS partitioning system are the principal driving forces underlying this oscillatory cycle. We propose that the distinct organization patterns observed for bacterial chromosomes reflect a common organization–segregation mechanism, and that simple modifications to it underlie the unique patterns observed in different species.

The peptidoglycan (PG) sacculus is composed of long glycan strands cross-linked together by short peptides forming a covalently closed meshwork that protects the bacterial cell from osmotic lysis and specifies its shape. PG hydrolases play essential roles in remodeling this three-dimensional network during growth and division but how these autolytic enzymes are regulated remains poorly understood. The FtsEX ABC transporter-like complex has emerged as a broadly conserved regulatory module in controlling cell wall hydrolases in diverse bacterial species. In most characterized examples, this complex regulates distinct PG hydrolases involved in cell division and is intimately associated with the cytokinetic machinery called the divisome. However, in the gram-positive bacterium Bacillus subtilis the FtsEX complex is required for cell wall elongation where it regulates the PG hydrolase CwlO that acts along the lateral cell wall. To investigate whether additional factors are required for FtsEX function outside the divisome, we performed a synthetic lethal screen taking advantage of the conditional essentiality of CwlO. This screen identified two uncharacterized factors (SweD and SweC) that are required for CwlO activity. We demonstrate that these proteins reside in a membrane complex with FtsX and that amino acid substitutions in residues adjacent to the ATPase domain of FtsE partially bypass the requirement for them. Collectively our data indicate that SweD and SweC function as essential co-factors of FtsEX in controlling CwlO during cell wall elongation. We propose that factors analogous to SweDC function to support FtsEX activity outside the divisome in other bacteria.

Borrelia burgdorferi , the tick-transmitted spirochete agent of Lyme disease, has a highly segmented genome with a linear chromosome and various linear or circular plasmids. Here, by imaging several chromosomal loci and 16 distinct plasmids, we show that B. burgdorferi is polyploid during growth in culture and that the number of genome copies decreases during stationary phase. B. burgdorferi is also polyploid inside fed ticks and chromosome copies are regularly spaced along the spirochete’s length in both growing cultures and ticks. This patterning involves the conserved DNA partitioning protein ParA whose localization is controlled by a potentially phage-derived protein, ParZ, instead of its usual partner ParB. ParZ binds its own coding region and acts as a centromere-binding protein. While ParA works with ParZ, ParB controls the localization of the condensin, SMC. Together, the ParA/ParZ and ParB/SMC pairs ensure faithful chromosome inheritance. Our findings underscore the plasticity of cellular functions, even those as fundamental as chromosome segregation. The bacterium Borrelia burgdorferi , which causes Lyme disease and is transmitted by ticks, has a linear chromosome and multiple plasmids. Here, Takacs et al. show that the pathogen is polyploid, the number of genome copies decreases during stationary phase, and chromosome copies are regularly spaced along the cell’s length.

Bacterial species from diverse phyla contain multiple replicons, yet how these multipartite genomes are organized and segregated during the cell cycle remains poorly understood. Agrobacterium tumefaciens has a 2.8-Mb circular chromosome (Ch1), a 2.1-Mb linear chromosome (Ch2), and two large plasmids (pAt and pTi). We used this alpha proteobacterium as a model to investigate the global organization and temporal segregation of a multipartite genome. Using chromosome conformation capture assays, we demonstrate that both the circular and the linear chromosomes, but neither of the plasmids, have their left and right arms juxtaposed from their origins to their termini, generating interarm interactions that require the broadly conserved structural maintenance of chromosomes complex. Moreover, our study revealed two types of interreplicon interactions: “ori-ori clustering” in which the replication origins of all four replicons interact, and “Ch1-Ch2 alignment” in which the arms of Ch1 and Ch2 interact linearly along their lengths. We show that the centromeric proteins (ParB1 for Ch1 and RepBCh2 for Ch2) are required for both types of interreplicon contacts. Finally, using fluorescence microscopy, we validated the clustering of the origins and observed their frequent colocalization during segregation. Altogether, our findings provide a high-resolution view of the conformation of a multipartite genome. We hypothesize that intercentromeric contacts promote the organization and maintenance of diverse replicons.

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 that B . burgdorferi is 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 the B . burgdorferi genome 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 chromosome oriC region, 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 chromosome oriC . 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.

Objective This study aimed to investigate a suitable risk assessment model to predict deep vein thrombosis (DVT) in patients with gynecological cancer. Methods Data from 212 patients with gynecological cancer in the Affiliated Tumor Hospital of Guangxi Medical University were retrospectively analyzed. Patients were risk-stratified with three different risk assessment models individually, including the Caprini model, Wells DVT model, and Khorana model. Results The difference in risk level evaluated by the Caprini model was not different between the DVT and control groups. However, the DVT group had a significantly higher risk level than the control group with the Wells DVT or Khorana model. The Wells DVT model was more effective for stratifying patients in the DVT group into the higher risk level and for stratifying those in the control group into the lower risk level. Receiver operating curve analysis showed that the area under the curve of the Wells DVT, Khorana, and Caprini models was 0.995 ± 0.002, 0.642 ± 0.038, and 0.567 ± 0.039, respectively. Conclusion The Wells DVT model is the most suitable risk assessment model for predicting DVT. Clinicians could also combine the Caprini and Wells DVT models to effectively identify high-risk patients and eliminate patients without DVT.