RefZ and Noc act synthetically to prevent aberrant divisions during Bacillus subtilis sporulation

Abstract

During sporulation, Bacillus subtilis undergoes an atypical cell division that requires overriding mechanisms which protect chromosomes from damage and ensure inheritance by daughter cells. Instead of assembling between segregated chromosomes at midcell, the FtsZ-ring (Z-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 wildtype 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 {Delta}refZ with deletions of genes previously implicated in FtsZ regulation and/or chromosome capture. Combining {Delta}refZ with deletions of ezrA, sepF, parA, or minD did not detectably affect sporulation. In contrast, a {Delta}refZ {Delta}noc mutant exhibited a sporulation defect, revealing a genetic interaction between RefZ and Noc. Using reporters of sporulation progression, we determined the {Delta}refZ {Delta}noc 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 {Delta}refZ {Delta}noc 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. IMPORTANCEThe 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 non-coding and non-regulatory 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.