Mechanisms for Chromosome Segregation in Bacteria

The process of DNA segregation, the redistribution of newly replicated genomic material to daughter cells, is a crucial step in the life cycle of all living systems. Here, we review DNA segregation in bacteria which evolved a variety of mechanisms for partitioning newly replicated DNA. Bacterial species such as Caulobacter crescentus and Bacillus subtilis contain pushing and pulling mechanisms that exert forces and directionality to mediate the moving of newly synthesized chromosomes to the bacterial poles. Other bacteria such as Escherichia coli lack such active segregation systems, yet exhibit a spontaneous de-mixing of chromosomes due to entropic forces as DNA is being replicated under the confinement of the cell wall. Furthermore, we present a synopsis of the main players that contribute to prokaryotic genome segregation. We finish with emphasizing the importance of bottom-up approaches for the investigation of the various factors that contribute to genome segregation.

SMC and the bactofilin/PadC scaffold have distinct yet redundant functions in chromosome segregation and organization in Myxococcus xanthus

In bacteria, ParABS systems and structural maintenance of chromosome (SMC) condensin-like complexes are important for chromosome segregation and organization. The rod-shaped Myxococcus xanthus cells have a unique chromosome arrangement in which a scaffold composed of three bactofilins (BacNOP) and PadC positions the essential ParB·parS segregation complexes and the DNA segregation ATPase ParA in the subpolar regions. Here, we identify the Smc and ScpAB subunits of the SMC complex in M. xanthus and demonstrate that SMC is conditionally essential with mutants containing smc or scpAB deletions being temperature sensitive. Lack of SMC caused defects in chromosome segregation and organization. Lack of the BacNOP/PadC scaffold caused chromosome segregation defects but was not essential. Inactivation of SMC was synthetic lethal with lack of the BacNOP/PadC scaffold. Lack of SMC interfered with formation of the BacNOP/PadC scaffold while lack of this scaffold did not interfere with chromosome association by SMC. Altogether, our data support that three systems cooperate to enable chromosome segregation in M. xanthus, whereby ParABS constitutes the basic machinery and SMC and the BacNOP/PadC scaffold have distinct yet redundant roles in this process with SMC supporting individualization of daughter chromosomes and BacNOP/PadC making the ParABS system operate more robustly

Rules and Exceptions: The Role of Chromosomal ParB in DNA Segregation and Other Cellular Processes

The segregation of newly replicated chromosomes in bacterial cells is a highly coordinated spatiotemporal process. In the majority of bacterial species, a tripartite ParAB-parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB), and its target(s) parS sequence(s), facilitates the initial steps of chromosome partitioning. ParB nucleates around parS(s) located in the vicinity of newly replicated oriCs to form large nucleoprotein complexes, which are subsequently relocated by ParA to distal cellular compartments. In this review, we describe the role of ParB in various processes within bacterial cells, pointing out interspecies differences. We outline recent progress in understanding the ParB nucleoprotein complex formation and its role in DNA segregation, including ori positioning and anchoring, DNA condensation, and loading of the structural maintenance of chromosome (SMC) proteins. The auxiliary roles of ParBs in the control of chromosome replication initiation and cell division, as well as the regulation of gene expression, are discussed. Moreover, we catalog ParB interacting proteins. Overall, this work highlights how different bacterial species adapt the DNA partitioning ParAB-parS system to meet their specific requirements.

ATP-driven separation of liquid phase condensates in bacteria

Liquid-liquid phase separated (LLPS) states are key to compartmentalise components in the absence of membranes, however it is unclear whether LLPS condensates are actively and specifically organized in the sub-cellular space and by which mechanisms. Here, we address this question by focusing on the ParABS DNA segregation system, composed of a centromeric-like sequence (parS), a DNA-binding protein (ParB) and a motor (ParA). We show that parS-ParB associate to form nanometer-sized, round condensates. ParB molecules diffuse rapidly within the nucleoid volume, but display confined motions when trapped inside ParB condensates. Single ParB molecules are able to rapidly diffuse between different condensates, and nucleation is strongly favoured by parS. Notably, the ParA motor is required to prevent the fusion of ParB condensates. These results describe a novel active mechanism that splits, segregates and localises non-canonical LLPS condensates in the sub-cellular space.

Direct observation of independently moving replisomes in Escherichia coli

The replication and transfer of genomic material from a cell to its progeny are vital processes in all living systems. Here we visualize the process of chromosome replication in E. coli cells with an increased width. Monitoring the replication of single chromosomes yields clear examples of replication bubbles that reveal that the two replisomes move independently from the origin to the terminus of replication along each of the two arms of the circular chromosome, providing direct support for the so-called train-track model, and against a factory model for replisomes. The origin of replication duplicates near midcell, initially splitting to random directions and subsequently towards the poles. The probability of successful segregation of chromosomes significantly decreases with increasing cell width, indicating that chromosome confinement by the cell boundary is an important driver of DNA segregation. Our findings resolve long standing questions in bacterial chromosome organization.