A DNA-binding protein tunes septum placement duringBacillus subtilissporulation

Abstract

29Bacillus subtilis is a soil bacterium capable of differentiating into a spore form resistant 30 to desiccation, UV radiation, and heat. Early in spore development the cell possesses 31 two copies of a circular chromosome, anchored to opposite cell poles via DNA proximal 32 to the origin of replication (oriC). As sporulation progresses an FtsZ ring (Z-ring) 33 assembles close to one pole and directs septation over one chromosome. The polar 34 division generates two cell compartments with differing chromosomal contents. The 35 smaller "forespore" compartment initially contains only 25-30% of one chromosome and 36 this transient genetic asymmetry is required for differentiation. At the population level, 37 the timely assembly of polar Z-rings and the precise capture of the chromosome in the 38 forespore both require RefZ, a DNA-binding protein synthesized early in 39 sporulation. To mediate precise capture of the chromosome RefZ must bind to specific 40 DNA motifs (RBMs) that are localized near the poles around the time of septation, 41suggesting RefZ binds to the RBMs to affect positioning of the septum relative to the 42 chromosome. RefZ's mechanism of action is unknown, however, cells artificially 43 induced to express RefZ during vegetative growth cannot assemble Z-rings or divide, 44 leading to the hypothesis that RefZ-RBM complexes mediate precise chromosome 45 capture by modulating FtsZ function. To investigate this possibility, we isolated 10 RefZ 46 loss-of-function (rLOF) variants unable to inhibit cell division when expressed during 47 structure of RefZ was solved and wild-type RefZ and the rLOF variants were further 52 characterized. Our data suggest that RefZ's oligomerization state and specificity for the 53 RBMs are critical determinants influencing RefZ's ability to affect FtsZ dynamics in 54 vivo. We propose that RBM-bound RefZ complexes function as a developmentally 55 regulated nucleoid occlusion system for fine-tuning the position of the septum relative to 56 the chromosome during sporulation. 57 58 Author Summary 59The Gram-positive bacterium B. subtilis can differentiate into a dormant cell type called 60 a spore. Early in sporulation the cell divides near one pole, generating two 61 compartments: a larger mother cell and a smaller forespore (future spore). Only 62 approximately 30 percent of one chromosome is initially captured in the forespore 63 compartment at the time of division and this genetic asymmetry is critical for sporulation 64 to progress. Precise chromosome capture requires RefZ, a sporulation protein that 65 binds to specific DNA motifs (RBMs) positioned at the pole near the site of cell division. 66How RefZ functions at the molecular level is not fully understood. Here we show that 67RefZ-RBM complexes facilitate chromosome capture by acting through the major cell 68 division protein FtsZ. 69 70 71 72 73 at midcell, is carried out by a localized multi-subunit complex comprised of over 30 87 proteins called the "divisome" 8 . 88Bacteria also elicit subcellular heterogeneity by...