Lai, N. K. | Search bioRxiv

bioRxiv2026

Efficient septum formation is essential for chromosome segregation in Bacillus subtilis when SMC function is impaired

Structural maintenance of chromosomes (SMC) complexes play conserved roles in chromosome organization, segregation, and repair in all domains of life. In Bacillus subtilis, SMC is required for segregation of newly replicated origins. To investigate whether other proteins function with SMC in this process, we performed a synthetic lethal screen with an smc hypomorphic allele (smc*) that is mildly defective in chromosome segregation. In addition to recovering previously reported interactions of smc with parB and spoIIIE, our screen identified minJ and divIVA as essential in the smc* background. We show that the synthetic lethality between smc* and{Delta} minJ or{Delta} divIVA arises from defects in segregating the replication terminus. Importantly, deletion of minD, which suppresses the cell division defects of{Delta} minJ and{Delta} divIVA, restored terminus segregation and viability in the smc* background. These findings support a model in which proper septum formation promotes chromosome terminus resolution and segregation by enabling SpoIIIE-mediated DNA clearance from the division septum during cytokinesis. These findings highlight the interdependence between chromosome segregation and cell division. ImportanceThe SMC complex plays a central role in chromosome organization and segregation in Bacillus subtilis, but the cellular functions that become important when SMC activity is reduced are not well understood. Using a hypomorphic smc allele, we discovered that mutations affecting cell division become essential when chromosome organization and segregation are impaired. Our findings support a model in which efficient septum formation enables proper localization of the SpoIIIE DNA translocase, which in turn resolves and segregates the chromosome terminus region. These results highlight the critical role of cell division in supporting chromosome segregation.

microbiology↗

bioRxiv2025

The B. subtilis replicative polymerases bind the sliding clamp with different strengths to tune replication processivity and fidelity

Ring-shaped sliding clamp proteins are essential components of the replication machinery, the replisome, across all domains of life. In bacteria, DNA polymerases bind the sliding clamp, DnaN, through conserved short peptide sequences called clamp-binding motifs. Clamp binding increases the processivity and rate of DNA synthesis and is generally required for polymerase activity. The current understanding of clamp-polymerase interactions was elucidated in the model bacterium Escherichia coli, which has a single replicative polymerase, Pol III. However, many bacteria have two essential replicative polymerases, such as PolC and DnaE in Bacillus subtilis. PolC performs the bulk of DNA synthesis whereas the error-prone DnaE only synthesizes short stretches of DNA on the lagging strand. How the clamp interacts with the two polymerases and coordinates their activity is unknown. We investigated this question by combining in vivo single-molecule fluorescence microscopy with biochemical and microbiological assays. We found that PolC-DnaN binding is essential for replication, although weakening the interaction is tolerated with only minimal effects. In contrast, the DnaE-DnaN interaction is dispensable for replication. Altering the clamp-binding strength of DnaE produces only subtle effects on DnaE cellular localization and dynamics, but it has a substantial impact on mutagenesis. Our results support a model in which DnaE acts distributively during replication but can be stabilized on the DNA template by clamp binding. This study provides new insights into the coordination of multiple replicative polymerases in bacteria and the role of the clamp in polymerase processivity, fidelity, and exchange.

biochemistry↗