ParB-type DNA Segregation Proteins Are CTP-Dependent Molecular Switches.

During cell division, newly replicated DNA is actively segregated to the daughter cells. In most bacteria, this process involves the DNA-binding protein ParB, which condenses the centromeric regions of sister DNA molecules into kinetochore-like structures that recruit the DNA partition ATPase ParA and the prokaroytic SMC/condensin complex. Here, we report the crystal structure of a ParB-like protein (PadC) that emerges to tightly bind the ribonucleotide CTP. The CTP-binding pocket of PadC is conserved in ParB and composed of signature motifs known to be essential for ParB function. We find that ParB indeed interacts with CTP and requires nucleotide binding for DNA condensation in vivo. We further show that CTP-binding modulates the affinity of ParB for centromeric parS sites, whereas parS recognition stimulates its CTPase activity. ParB proteins thus emerge as a new class of CTP-dependent molecular switches that act in concert with ATPases and GTPases to control fundamental cellular functions.

Connecting the dots: key insights on ParB for chromosome segregation from single-molecule studies.

Bacterial cells require DNA segregation machinery to properly distribute a genome to both daughter cells upon division. The most common system involved in chromosome and plasmid segregation in bacteria is the ParABS system. A core protein of this system - partition protein B (ParB) - regulates chromosome organization and chromosome segregation during the bacterial cell cycle. Over the past decades, research has greatly advanced our knowledge of the ParABS system. However, many intricate details of the mechanism of ParB proteins were only recently uncovered using in vitro single-molecule techniques. These approaches allowed the exploration of ParB proteins in precisely controlled environments, free from the complexities of the cellular milieu. This review covers the early developments of this field but emphasizes recent advances in our knowledge of the mechanistic understanding of ParB proteins as revealed by in vitro single-molecule methods. Furthermore, we provide an outlook on future endeavors in investigating ParB, ParB-like proteins, and their interaction partners.

Distinct architectural requirements for the parS centromeric sequence of the pSM19035 plasmid partition machinery.

Three-component ParABS partition systems ensure stable inheritance of many bacterial chromosomes and low-copy-number plasmids. ParA localizes to the nucleoid through its ATP-dependent nonspecific DNA-binding activity, whereas centromere-like parS-DNA and ParB form partition complexes that activate ParA-ATPase to drive the system dynamics. The essential parS sequence arrangements vary among ParABS systems, reflecting the architectural diversity of their partition complexes. Here, we focus on the pSM19035 plasmid partition system that uses a ParBpSM of the ribbon-helix-helix (RHH) family. We show that parSpSM with four or more contiguous ParBpSM-binding sequence repeats is required to assemble a stable ParApSM-ParBpSM complex and efficiently activate the ParApSM-ATPase, stimulating complex disassembly. Disruption of the contiguity of the parSpSM sequence array destabilizes the ParApSM-ParBpSM complex and prevents efficient ATPase activation. Our findings reveal the unique architecture of the pSM19035 partition complex and how it interacts with nucleoid-bound ParApSM-ATP.

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.

Center Finding in E. coli and the Role of Mathematical Modeling: Past, Present and Future.

We review the key role played by mathematical modeling in elucidating two center-finding patterning systems in Escherichia coli: midcell division positioning by the MinCDE system and DNA partitioning by the ParABS system. We focus particularly on how, despite much experimental effort, these systems were simply too complex to unravel by experiments alone, and instead required key injections of quantitative, mathematical thinking. We conclude the review by analyzing the frequency of modeling approaches in microbiology over time. We find that while such methods are increasing in popularity, they are still probably heavily under-utilized for optimal progress on complex biological questions.

A Bidimensional Segregation Mode Maintains Symbiont Chromosome Orientation toward Its Host.

All living organisms require accurate segregation of their genetic material. However, in microbes, chromosome segregation is less understood than replication and cell division, which makes its decipherment a compelling research frontier. Furthermore, it has only been studied in free-living microbes so far. Here, we investigated this fundamental process in a rod-shaped symbiont, Candidatus Thiosymbion oneisti. This gammaproteobacterium divides longitudinally as to form a columnar epithelium ensheathing its nematode host. We hypothesized that uninterrupted host attachment would affect bacterial chromosome dynamics and set out to localize specific chromosomal loci and putative DNA-segregating proteins by fluorescence in situ hybridization and immunostaining, respectively. First, DNA replication origins (ori) number per cell demonstrated symbiont monoploidy. Second, we showed that sister ori segregate diagonally prior to septation onset. Moreover, the localization pattern of the centromere-binding protein ParB recapitulates that of ori, and consistently, we showed recombinant ParB to specifically bind an ori-proximal site (parS) in vitro. Third, chromosome replication ends prior to cell fission, and as the poles start to invaginate, termination of replication (ter) sites localize medially, at the leading edges of the growing septum. They then migrate to midcell, concomitantly with septation progression and until this is completed. In conclusion, we propose that symbiont ParB might drive chromosome segregation along the short axis and that tethering of sister ter regions to the growing septum mediates their migration along the long axis. Crucially, active bidimensional segregation of the chromosome allows transgenerational maintenance of its configuration, and therefore, it may represent an adaptation to symbiosis. VIDEO ABSTRACT.