Menikpurage, I. P. | Search bioRxiv

bioRxiv2022

ParA and its functions that go beyond chromosome segregation in Caulobacter crescentus

Maintaining the integrity of the chromosome after the completion of each cell cycle is paramount for bacterial survival. Mechanistic details remain incomplete for how bacteria manage to retain intact chromosomes in each daughter cell after each cell division. In this study, we examined the partitioning protein ParA and its functions on chromosomal maintenance that go beyond triggering the onset of chromosome segregation. Our data demonstrate that ParA can promote the onset of chromosome replication initiation in Caulobacter crescentus cells with sub-physiological levels of the replication initiator protein DnaA. Increasing the cellular levels of ParA results in over-initiation of chromosome replication in this bacterium. We show that the ability of ParA to impact replication initiation is independent from ParAs ability to trigger the onset of chromosome segregation. Surprisingly, our work revealed that perturbing the balance of the components of ParAs nucleotide-dependent cycle can have severe defects in cell cycle coordination and can potentially be lethal to the cell. Increasing the levels of different forms of ParA also impacted cell length independent of their replication initiation frequencies. Our results, together with past observations, suggest a model where ParA can serve as a checkpoint coordinating various cell cycle events involved in maintenance of the chromosome.

microbiology↗

bioRxiv2019

Chromosome dynamics in bacteria: triggering replication at opposite location and segregation in opposite direction

The accurate onset of chromosome replication and segregation are fundamental for the survival of the cell. In bacteria, regulation of chromosome replication lies primarily at the initiation step. The bacterial replication initiator DnaA recognizes the origin of replication (ori) and opens this double stranded site allowing for the assembly of the DNA replication machinery. Following the onset of replication initiation, the partitioning protein ParA triggers the onset of chromosome segregation by direct interactions with ParB-bound to the centromere. The subcellular organization of ori and centromere are maintained after the completion of each cell cycle. It remains unclear what triggers the onset of these key chromosome regulators DnaA and ParA. One potential scenario is that the microenvironment of where the onset of replication and segregation take place hosts the regulators that trigger the activity of DnaA and ParA. In order to address this, we analyzed whether the activity of DnaA and ParA are restricted to only one site within the cell. In non-dividing cells of the alpha proteobacterium Caulobacter crescentus, ori and centromere are found near the stalked pole. To test DnaAs ability to initiate replication away from the stalked pole, we engineered a strain where movement of ori was induced in the absence of chromosome replication. Our data show that DnaA can initiate replication of the chromosome independently of the subcellular localization of ori. Furthermore, we discovered that the partitioning protein ParA was functional and could segregate the replicated centromere in the opposite direction from the new pole toward the stalked pole. We showed that the organization of the ParA gradient can be completely reconstructed in the opposite orientation by rearranging the location of the centromere. Our data reveal the high flexibility of the machineries that trigger the onset of chromosome replication and segregation in bacteria. Our work also provides insights into the coordination between replication and segregation with the cellular organization of specific chromosomal loci.

microbiology↗