THE ROLE OF A CONSERVED DNA-BINDING PROTEIN IN PRECISE POSITIONING OF THE POLAR SEPTUM DURING BACILLUS SUBTILIS SPORULATION

Bacterial chromosomes are compacted in the cytoplasm into a membrane-less structure called the nucleoid. The nucleoid is condensed and organized by a number of DNA-binding proteins that work in concert to establish its overall 3D structure. Some proteins exploit this spatial organization to localize their activities to specific subcellular regions. In this study, we focused on a developmentally-regulated DNA-binding protein, RefZ (Regulator of FtsZ), and its role in tuning septum placement during sporulation in the model bacterium, Bacillus subtilis. In response to starvation, B. subtilis initiate a developmental program called sporulation, during which the cell division protein, FtsZ, is redeployed from midcell to a polar position. Septation then occurs over one of the cell’s two chromosomes, generating a transient period of genetic asymmetry critical for sporulation. Artificial expression of refZ during growth disrupts FtsZ-ring assembly and blocks cell division, and during sporulation, refZ mutants are delayed in Z-ring shifting. We demonstrate that artificial expression of a RefZ homolog also blocks cell division, indicating that this function is conserved in other Bacillus species. RefZ binds five sites, RBMs, arranged symmetric about the chromosomal origin. The outermost sites on the left and right chromosome arms lie at the boundary of the region reproducibly captured by the sporulation septum. In addition to the refZ gene, we show the position of the RBMs on the chromosome is also conserved across the Bacillus genus. Using a single cell-based fluorescence trapping assay, we find that RefZ and the RBMs are required for precise capture of the chromosome in the future spore compartment. To delineate the role of RefZ’s division regulation function in chromosome capture, we performed a genetic selection-screen to isolate RefZ variants loss-offunction for inhibiting division. Analysis of the variants using our trapping assay indicates that RefZ’s role in chromosome capture is mediated through modulation of cell division. In addition, we find that RefZ acts redundantly with the nucleoid occlusion protein, Noc, to prevent aberrant midcell divisions during sporulation. We propose RefZ acts as a developmentally-regulated nucleoid occlusion protein, helping to maintain the fidelity of division site selection in the early stages of sporulation