RefZ and Noc Act Synthetically to Prevent Aberrant Divisions during Bacillus subtilis Sporulation.

During sporulation, Bacillus subtilis undergoes an atypical cell division that requires overriding mechanisms that protect chromosomes from damage and ensure inheritance by daughter cells. Instead of assembling between segregated chromosomes at midcell, the FtsZ-ring coalesces polarly, directing division over one chromosome. The DNA-binding protein RefZ facilitates the timely assembly of polar Z-rings and partially defines the region of chromosome initially captured in the forespore. RefZ binds to motifs (RBMs) located proximal to the origin of replication (oriC). Although refZ and the RBMs are conserved across the Bacillus genus, a refZ deletion mutant sporulates with wild-type efficiency, so the functional significance of RefZ during sporulation remains unclear. To further investigate RefZ function, we performed a candidate-based screen for synthetic sporulation defects by combining ΔrefZ with deletions of genes previously implicated in FtsZ regulation and/or chromosome capture. Combining ΔrefZ with deletions of ezrA, sepF, parA, or minD did not detectably affect sporulation. In contrast, a ΔrefZ Δnoc mutant exhibited a sporulation defect, revealing a genetic interaction between RefZ and Noc. Using reporters of sporulation progression, we determined the ΔrefZ Δnoc mutant exhibited sporulation delays after Spo0A activation but prior to late sporulation, with a subset of cells failing to divide polarly or activate the first forespore-specific sigma factor, SigF. The ΔrefZ Δnoc mutant also exhibited extensive dysregulation of cell division, producing cells with extra, misplaced, or otherwise aberrant septa. Our results reveal a previously unknown epistatic relationship that suggests refZ and noc contribute synthetically to regulating cell division and supporting spore development. IMPORTANCE The DNA-binding protein RefZ and its binding sites (RBMs) are conserved in sequence and location on the chromosome across the Bacillus genus and contribute to the timing of polar FtsZ-ring assembly during sporulation. Only a small number of noncoding and nonregulatory DNA motifs are known to be conserved in chromosomal position in bacteria, suggesting there is strong selective pressure for their maintenance; however, a refZ deletion mutant sporulates efficiently, providing no clues as to their functional significance. Here, we find that in the absence of the nucleoid occlusion factor Noc, deletion of refZ results in a sporulation defect characterized by developmental delays and aberrant divisions.

A DNA-Binding Protein Tunes Septum Placement during Bacillus subtilis Sporulation.

Bacillus subtilis is a bacterium capable of differentiating into a spore form more resistant to environmental stress. Early in sporulation, each cell possesses two copies of a circular chromosome. A polar FtsZ ring (Z ring) directs septation over one of the chromosomes, generating two cell compartments. The smaller "forespore" compartment initially contains only 25 to 30% of one chromosome, and this transient genetic asymmetry is required for differentiation. Timely assembly of polar Z rings and precise capture of the chromosome in the forespore both require the DNA-binding protein RefZ. To mediate its role in chromosome capture, RefZ must bind to specific DNA motifs (RBMs) that localize near the poles at the time of septation. Cells artificially induced to express RefZ during vegetative growth cannot assemble Z rings, an effect that also requires DNA binding. We hypothesized that RefZ-RBM complexes mediate precise chromosome capture by modulating FtsZ function. To investigate, we isolated 10 RefZ loss-of-function (rLOF) variants unable to inhibit cell division yet still capable of binding RBMs. Sporulating cells expressing the rLOF variants in place of wild-type RefZ phenocopied a ΔrefZ mutant, suggesting that RefZ acts through an FtsZ-dependent mechanism. The crystal structure of RefZ was solved, and wild-type RefZ and the rLOF variants were further characterized. Our data suggest that RefZ's oligomerization state and specificity for the RBMs are critical determinants influencing RefZ's ability to affect FtsZ dynamics. We propose that RBM-bound RefZ complexes function as a developmentally regulated nucleoid occlusion system for fine-tuning the position of the septum relative to the chromosome during sporulation.IMPORTANCE The bacterial nucleoid forms a large, highly organized structure. Thus, in addition to storing the genetic code, the nucleoid harbors positional information that can be leveraged by DNA-binding proteins to spatially constrain cellular activities. During B. subtilis sporulation, the nucleoid undergoes reorganization, and the cell division protein FtsZ assembles polarly to direct septation over one chromosome. The TetR family protein RefZ binds DNA motifs (RBMs) localized near the poles at the time of division and is required for both timely FtsZ assembly and precise capture of DNA in the future spore compartment. Our data suggest that RefZ exploits nucleoid organization by associating with polarly localized RBMs to modulate the positioning of FtsZ relative to the chromosome during sporulation.

A DNA-binding protein defines the precise region of chromosome capture during Bacillus sporulation.

During sporulation, Bacillus subtilis divides around the nucleoid near one cell pole, initially capturing approximately one quarter of one chromosome in the newly formed forespore compartment. While it is known that a specific region of the nucleoid is reproducibly captured in the forespore, the mechanism underlying the precision of capture is unknown. Here we describe a role for RefZ, a DNA-binding protein that regulates FtsZ, and its cognate binding motifs (RBMs) in defining the specific region of chromosome initially captured in the forespore. RefZ is conserved across the Bacillus genus and remains functional as an inhibitor of cell division in a species-swapping experiment. The RBMs are also conserved in their positioning relative to oriC across 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. refZ and RBM mutants capture chromosomal regions normally excluded from the forespore, suggesting that RefZ-RBM complexes play a role in regulating the position of cell division relative to the chromosome during sporulation.