Characterization of RefZ, a Developmentally Controlled Regulator of Bacillus Subtilis FtsZ

Abstract

Bacillus subtilis is a soil bacterium capable of differentiating into a spore form resistant to both desiccation and heat. Sporulation begins with a single cell possessing two copies of a single, circular genome arranged with an oriC at each cell pole and the termini near midcell. Cell division occurs at the cell quarter over one chromosome, producing two disproportionately sized compartments and capturing approximately one quarter of one chromosome in the newly formed forespore compartment. While it is known that a specific region of the chromosome is reproducibly captured in the forespore, the mechanism underlying the precision of capture is unknown. Here we describe a role for a DNA-binding protein called RefZ and its cognate binding motifs (RBMs) in the precise capture of DNA in the forespore through regulation of FtsZ. RefZ is conserved across the Bacillus genus and remains an inhibitor of cell division in a species-swapping experiment. The RBMs are also conserved in their positioning relative to the oriC across the Bacillus, suggesting that the function of the RBMs is both important and position-dependent in the genus. In B. subtilis, the RBMs flank the region of the chromosome captured at the time of cell division, and we find that RefZ binds the five oriC-proximal RBMs with similar apparent affinity in units of two and four. In the absence of RefZ or when the RBMs are mutated, chromosomal regions normally excluded from the forespore are captured, suggesting that RefZ-RBM complexes play a role in regulating the position of cell division relative to the chromosome during sporulation. Misexpression of RefZ during vegetative growth disrupts FtsZ rings in a manner that requires DNA binding. We investigate the hypothesis that RefZ-RBM complexes mediate precise chromosome capture by regulating FtsZ dynamics. We identified and characterized 10 RefZ loss-of-function variants (rLOFs) capable of binding RBMs, yet unable to inhibit cell division. Using single-cell analysis, we show that the rLOFs do not capture a wildtype complement of DNA in the forespore, instead phenocopying a ΔrefZ mutant. These results suggest that RefZ acts through FtsZ to accomplish chromosome capture. To better understand the molecular basis of RefZ's activity, the structure of RefZ was solved and RefZ and the rLOFs were further characterized. Our data suggest RefZ can exist as a monomer or dimer, and that RefZ’s oligomerization state both on and off DNA, likely control its capacity to influence FtsZ dynamics in vivo.