CTP and parS coordinate ParB partition complex dynamics and ParA-ATPase activation for ParABS-mediated DNA partitioning

ParABS partition systems, comprising the centromere-like DNA sequence parS, the parS-binding ParB-CTPase and the nucleoid-binding ParA-ATPase, ensure faithful segregation of bacterial chromosomes and low-copy-number plasmids. F-plasmid partition complexes containing ParBF and parSF move by generating and following a local concentration gradient of nucleoid-bound ParAF. However, the process through which ParBF activates ParAF-ATPase has not been defined. We studied CTP- and parSF-modulated ParAF-ParBF complex assembly, in which DNA-bound ParAF-ATP dimers are activated for ATP hydrolysis by interacting with two ParBF N-terminal domains. CTP or parSF enhances the ATPase rate without significantly accelerating ParAF-ParBF complex assembly. Together, parSF and CTP accelerate ParAF-ParBF assembly without further significant increase in ATPase rate. Magnetic-tweezers experiments showed that CTP promotes multiple ParBF loading onto parSF-containing DNA, generating condensed partition complex-like assemblies. We propose that ParBF in the partition complex adopts a conformation that enhances ParBF-ParBF and ParAF-ParBF interactions promoting efficient partitioning.

CTP and parS control ParB partition complex dynamics and ParA-ATPase activation for ParABS-mediated DNA partitioning

ParABS partition systems, comprising the centromere-like DNA sequence parS, the parS-binding ParB-CTPase and the nucleoid-binding ParA-ATPase, ensure faithful segregation of bacterial chromosomes and low-copy-number plasmids. F-plasmid partition complexes containing ParBF and parSF move by generating and following a local concentration gradient of nucleoid-bound ParAF. However, the process of ParBF activation of ParAF-ATPase has not been defined. We studied CTP- and parSF-stimulated ParAF—ParBF complex assembly leading to ParAF-ATPase activation. Activation of DNA-bound ParAF-ATP dimers for ATP hydrolysis requires binding of two ParBF N-terminal domains. CTP or parSF enhances the ATPase rate without increasing ParAF—ParBF assembly kinetics. Together, parSF and CTP accelerate ParAF—ParBF assembly without further increasing the ATPase rate. Magnetic-tweezers experiments showed that CTP promotes multiple ParBF loading onto parSF-containing DNA, generating condensed partition complex-like assemblies. We propose that ParBF in the partition complex adopts a conformation that enhances ParBF—ParBF and ParAF—ParBF interactions promoting efficient partitioning.

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.

Chromosome organization by a conserved condensin-ParB system in the actinobacterium Corynebacterium glutamicum

Higher-order chromosome folding and segregation is tightly regulated in all domains of life. In bacteria, details on nucleoid organization regulatory mechanisms and function remains poorly characterized, especially in non-model species. Here, we investigate the role of DNA partitioning protein ParB and condensin complexes, two key players in bacterial chromosome structuring, in the actinobacterium Corynebacterium glutamicum. Chromosome conformation capture reveals SMC-mediated long-range interactions around ten centromere-like parS sites clustered at the replication origin (oriC). At least one oriC-proximal parS site is necessary for a reliable chromosome segregation. Using a combination of chromatin immunoprecipitation and photoactivated single molecule localization microscopy evidences the formation of distinct ParB-nucleoprotein subclusters in dependence of parS numbers. We further identified and functionally characterized two condensin paralogs. Whereas SMC/ScpAB complexes are loaded via ParB at parS sites mediating chromosomal inter-arm contacts like in Bacillus subtilis, the MukBEF-like SMC complex MksBEFG does not contribute to chromosomal DNA-folding. Rather, the MksBEFG complex is involved in plasmid maintenance and interacts with the polar oriC-tethering factor DivIVA. These data complement current models of ParB-SMC/ScpAB crosstalk, while showing that some condensin complexes evolved functions uncoupled from chromosome folding.

Robust and conserved stochastic self-assembly mechanism for dynamic ParB-parS partition complexes on bacterial chromosomes and plasmids

SummaryChromosome and plasmid segregation in bacteria are mostly driven by ParABS systems. These DNA partitioning machineries rely on large nucleoprotein complexes assembled on centromere sites (parS). However, the mechanism of how a few parS-bound ParB proteins nucleate the formation of highly concentrated ParB clusters remains unclear despite several proposed physico-mathematical models. We discriminated between these different models by varying some key parameters in vivo using the plasmid F partition system. We found that ‘Nucleation & caging’ is the only coherent model recapitulating in vivo data. We also showed that the stochastic self-assembly of partition complexes (i) does not directly involve ParA, (ii) results in a dynamic structure of discrete size independent of ParB concentration, and (iii) is not perturbed by active transcription but is by protein complexes. We refined the ‘Nucleation & Caging’ model and successfully applied it to the chromosomally-encoded Par system of Vibrio cholerae, indicating that this stochastic self-assembly mechanism is widely conserved from plasmids to chromosomes.

Intrinsic disorder in the partitioning protein KorB persists after co-operative complex formation with operator DNA and KorA

The ParB protein, KorB, from the RK2 plasmid is required for DNA partitioning and transcriptional repression. It acts co-operatively with other proteins, including the repressor KorA. Like many multifunctional proteins, KorB contains regions of intrinsically disordered structure, existing in a large ensemble of interconverting conformations. Using NMR spectroscopy, circular dichroism and small-angle neutron scattering, we studied KorB selectively within its binary complexes with KorA and DNA, and within the ternary KorA/KorB/DNA complex. The bound KorB protein remains disordered with a mobile C-terminal domain and no changes in the secondary structure, but increases in the radius of gyration on complex formation. Comparison of wild-type KorB with an N-terminal deletion mutant allows a model of the ensemble average distances between the domains when bound to DNA. We propose that the positive co-operativity between KorB, KorA and DNA results from conformational restriction of KorB on binding each partner, while maintaining disorder.