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

Abstract

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.

Transcriptomic and Phenotypic Analysis of a spoIIE Mutant in Clostridium beijerinckii

“…An up-regulation of the genes involved in septum formation, fstZ, minD and refZ was also observed. These genes were proven to be crucial for the positioning of the septum formation and sporulation in B. subtilis ( Barák and Muchová, 2018 ; Brown et al, 2019 ). This result can be linked to the presence of several septa observed in the mutant ( Figure 5 and Supplementary Figures 2A,B ).…”

Bacteriophage SP01 Gene Product 56 Inhibits Bacillus subtilis Cell Division by Interacting with FtsL and Disrupting Pbp2B and FtsW Recruitment

“…Analogous temporary division blocks also occur within the B. subtilis host, such as during the aforementioned YneA-mediated SOS response to permit DNA repair (37), or during the transition of from vegetative growth to sporulation via RefZ activity on FtsZ (39,40). It also occurs during the DNA recombination events that accompany the developmental stress response of B. subtilis natural competence, where the peptide Maf is produced upon DNA uptake, and directly and transiently inhibits FtsZ assembly to permit uninterrupted genome maintenance (41).…”

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

“…We previously identified 10 RefZ loss-of-function variants (rLOFs) that retain the ability to bind DNA but are no longer able to perturb Z-ring assembly when artificially induced during vegetative growth (60). Cells harboring the rLOF alleles in place of wild-type refZ miscapture DNA in the forespore indistinguishably from the ∆refZ mutant, suggesting that RefZ-RBM complexes affect chromosome capture through direct or indirect effects on FtsZ (60). We hypothesized that RefZ's ability to modulate FtsZ activity would also be required to prevent aberrant divisions in the absence of noc.…”