A DNA-binding protein tunes septum placement duringBacillus subtilissporulation

Abstract

AbstractBacillus subtilisis a soil bacterium capable of differentiating into a spore form resistant to desiccation, UV radiation, and heat. Early in spore development the cell possesses two copies of a circular chromosome, anchored to opposite cell poles via DNA proximal to the origin of replication (oriC). As sporulation progresses an FtsZ ring (Z-ring) assembles close to one pole and directs septation over one chromosome. The polar division generates two cell compartments with differing chromosomal contents. The smaller “forespore” compartment initially contains only 25–30% of one chromosome and this transient genetic asymmetry is required for differentiation. At the population level, the timely assembly of polar Z-rings and the precise capture of the chromosome in the forespore both require RefZ, a DNA-binding protein synthesized early in sporulation. To mediate precise capture of the chromosome RefZ must bind to specific DNA motifs (RBMs) that are localized near the poles around the time of septation, suggesting RefZ binds to theRBMsto affect positioning of the septum relative to the chromosome. RefZ’s mechanism of action is unknown, however, cells artificially induced to express RefZ during vegetative growth cannot assemble Z-rings or divide, leading to the hypothesis that RefZ-RBM complexes mediate precise chromosome capture by modulating FtsZ function. To investigate this possibility, we isolated 10 RefZ loss-of-function (rLOF) variants unable to inhibit cell division when expressed during vegetative growth, yet were still capable of bindingRBM-containing DNA. Sporulating cells expressing the rLOF variants in place of wild-type RefZ phenocopy a ΔrefZmutant, suggesting that RefZ mediates chromosome capture through an FtsZ-dependent mechanism. To better understand the molecular basis of RefZ’s activity, 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 theRBMsare critical determinants influencing RefZ’s ability to affect FtsZ dynamicsin vivo. 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.Author SummaryThe Gram-positive bacteriumB. subtiliscan differentiate into a dormant cell type called a spore. Early in sporulation the cell divides near one pole, generating two compartments: a larger mother cell and a smaller forespore (future spore). Only approximately 30 percent of one chromosome is initially captured in the forespore compartment at the time of division and this genetic asymmetry is critical for sporulation to progress. Precise chromosome capture requires RefZ, a sporulation protein that binds to specific DNA motifs (RBMs) positioned at the pole near the site of cell division. How RefZ functions at the molecular level is not fully understood. Here we show that RefZ-RBMcomplexes facilitate chromosome capture by acting through the major cell division protein FtsZ.