In all living organisms, it is essential to transmit genetic information faithfully to the next generation. The SMC-ParAB-parS system is widely employed for chromosome segregation in bacteria. A DNA-binding protein ParB nucleates on parS sites and must associate with neighboring DNA, a process known as spreading, to enable efficient chromosome segregation. Despite its importance, how the initial few ParB molecules nucleating at parS sites recruit hundreds of further ParB to spread is not fully understood. Here, we reconstitute a parS-dependent ParB spreading event using purified proteins from Caulobacter crescentus and show that CTP is required for spreading. We further show that ParB spreading requires a closed DNA substrate, and a DNA-binding transcriptional regulator can act as a roadblock to attenuate spreading unidirectionally in vitro. Our biochemical reconstitutions recapitulate many observed in vivo properties of ParB and opens up avenues to investigate the interactions between ParB-parS with ParA and SMC.

Proper chromosome segregation during cell division is essential in all domains of life. In the majority of bacterial species, faithful chromosome segregation is mediated by the tripartite ParABS system, consisting of an ATPase protein ParA, a CTPase and DNA-binding protein ParB, and a centromere-like parS site. The parS site is most often located near the origin of replication and is segregated first after chromosome replication. ParB nucleates on parS before binding to adjacent non-specific DNA to form a multimeric nucleoprotein complex. ParA interacts with ParB to drive the higher-order ParB–DNA complex, and hence the replicating chromosomes, to each daughter cell. Here, we review the various models for the formation of the ParABS complex and describe its role in segregating the origin-proximal region of the chromosome. Additionally, we discuss outstanding questions and challenges in understanding bacterial chromosome segregation.

Proper chromosome segregation is essential in all living organisms. In Caulobacter crescentus, the ParA–ParB–parS system is required for proper chromosome segregation and cell viability. The bacterial centromere-like parS DNA locus is the first to be segregated following chromosome replication. parS is bound by ParB protein, which in turn interacts with ParA to partition the ParB-parS nucleoprotein complex to each daughter cell. Here, we investigated the genome-wide distribution of ParB on the Caulobacter chromosome using a combination of in vivo chromatin immunoprecipitation (ChIP-seq) and in vitro DNA affinity purification with deep sequencing (IDAP-seq). We confirmed two previously identified parS sites and discovered at least three more sites that cluster ∼8 kb from the origin of replication. We showed that Caulobacter ParB nucleates at parS sites and associates non-specifically with ∼10 kb flanking DNA to form a high-order nucleoprotein complex on the left chromosomal arm. Lastly, using transposon mutagenesis coupled with deep sequencing (Tn-seq), we identified a ∼500 kb region surrounding the native parS cluster that is tolerable to the insertion of a second parS cluster without severely affecting cell viability. Our results demonstrate that the genomic distribution of parS sites is highly restricted and is crucial for chromosome segregation in Caulobacter.

In all living organisms, it is essential to transmit genetic information faithfully to the next generation. The SMC-ParAB-parS system is widely employed for chromosome segregation in bacteria. A DNA-binding protein ParB nucleates on parS sites and must associate with neighboring DNA, a process known as spreading, to enable efficient chromosome segregation. Despite its importance, how the initial few ParB molecules nucleating at parS sites recruit hundreds of further ParB to spread is not fully understood. Here, we reconstitute a parS-dependent ParB spreading event using purified proteins from Caulobacter crescentus and show that CTP is required for spreading. We further show that ParB spreading requires a closed DNA substrate, and a DNA-binding transcriptional regulator can act as a roadblock to attenuate spreading unidirectionally in vitro. Our biochemical reconstitutions recapitulate many observed in vivo properties of ParB and opens up avenues to investigate the interactions between ParB-parS with ParA and SMC.Faithful chromosome segregation is essential in all domains of life if daughter cells are each to inherit the full set of genetic information. The SMC-ParAB-parS complex is widely employed for chromosome segregation in bacteria 1-13 . The centromere parS is the first DNA locus to be segregated following chromosome replication 8,9,14,15 . ParB specifically nucleates on parS before spreading outwards to the flanking DNA and bridges/cages DNA together to form a nucleoprotein network in vivo [16][17][18][19][20][21][22][23] . This nucleoprotein complex recruits SMC to disentangle and organize replicated DNA 3,11,13,24,25 . ParB-parS also interacts with an ATPase ParA to power the segregation of replicated chromosomes [26][27][28][29][30] . Engineered strains harboring a nucleation-competent but spreading-defective mutant of parB are either unviable 10 or have elevated number of anucleate cells 4,7,8,15,[31][32][33][34] . Despite the importance of spreading for proper chromosome segregation, the mechanism by which a few parS-bound ParB can recruit hundreds more ParB molecules to the vicinity of parS to assemble a high molecular-weight nucleoprotein complex is not fully understood.Since the first report in 1995 35 , ParB spreading has been observed in vivo by chromatin immunoprecipitation in multiple bacterial species 12,[15][16][17]19,36 . The nucleation of ParB on parS has also been demonstrated in vitro 4,10,16,17,20,[37][38][39] , however parS-dependent ParB spreading has resisted biochemical reconstitution [17][18][19]40,41 . Unsuccessful attempts to reconstitute spreading in vitro suggests that additional factors might be missing. Recently, works by Soh et al (2019) and Osorio-Valeriano et al (2019) on Bacillus subtilis and Myxococcus xanthus ParB, respectively, showed that ParB binds and hydrolyzes cytidine triphosphate (CTP) to cytidine diphosphate (CDP), and that CTP modulates the binding affinity of ParB to parS 42,43 . A co-crystal structure of Bacillus ParB with CDP and that of a Myxococcus ParB-li...

Proper chromosome segregation is essential in all living organisms. The ParA-ParB-parS system is widely employed for chromosome segregation in bacteria. Previously, we showed that Caulobacter crescentus ParB requires cytidine triphosphate to escape the nucleation site parS and spread by sliding to the neighboring DNA (Jalal et al., 2020). Here, we provide the structural basis for this transition from nucleation to spreading by solving co-crystal structures of a C-terminal domain truncated C. crescentus ParB with parS and with a CTP analog. Nucleating ParB is an open clamp, in which parS is captured at the DNA-binding domain (the DNA-gate). Upon binding CTP, the N-terminal domain (NTD) self-dimerizes to close the NTD-gate of the clamp. The DNA-gate also closes, thus driving parS into a compartment between the DNA-gate and the C-terminal domain. CTP hydrolysis and/or the release of hydrolytic products are likely associated with reopening of the gates to release DNA and recycle ParB. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation, spreading, and recycling.