Bacillus subtilis YisK possesses oxaloacetate decarboxylase activity and exhibits Mbl-dependent localization.

YisK is an uncharacterized protein in Bacillus subtilis previously shown to interact genetically with the elongasome protein Mbl. YisK overexpression leads to cell widening and lysis, phenotypes that are dependent on mbl and suppressed by mbl mutations. In the present work, we characterize YisK's localization, structure, and enzymatic activity. We show that YisK localizes as puncta that depend on Mbl. YisK belongs to the fumarylacetoacetate hydrolase (FAH) superfamily, and crystal structures revealed close structural similarity to two oxaloacetate (OAA) decarboxylases: human mitochondrial FAHD1 and Corynebacterium glutamicum Cg1458. We demonstrate that YisK can also catalyze the decarboxylation of OAA (K m = 134 µM, K cat = 31 min-1). A catalytic dead variant (YisK E148A, E150A) retains wild-type localization and still widens cells following overexpression, indicating these activities are not dependent on YisK catalysis. Conversely, a non-localizing variant (YisK E30A) retains wild-type enzymatic activity in vitro but localizes diffusely and no longer widens cells following overexpression. Together, these results suggest that YisK may be subject to spatial regulation that depends on the cell envelope synthesis machinery. IMPORTANCE The elongasome is a multiprotein complex that guides lengthwise growth in some bacteria. We previously showed that, in B. subtilis, overexpression of an uncharacterized putative enzyme (YisK) perturbed function of the actin-like elongasome protein Mbl. Here, we show that YisK exhibits Mbl-dependent localization. Through biochemical and structural characterization, we demonstrate that, like its mitochondrial homolog FAHD1, YisK can catalyze the decarboxylation of the oxaloacetate to pyruvate and CO2. YisK is the first example of an enzyme implicated in central carbon metabolism with subcellular localization that depends on Mbl.

Magnesium Modulates Bacillus subtilis Cell Division Frequency.

By chance, we discovered a window of extracellular magnesium (Mg2+) availability that modulates the division frequency of Bacillus subtilis without affecting its growth rate. In this window, cells grown with excess Mg2+ produce shorter cells than do those grown in unsupplemented medium. The Mg2+-responsive adjustment in cell length occurs in both rich and minimal media as well as in domesticated and undomesticated strains. Of other divalent cations tested, manganese (Mn2+) and zinc (Zn2+) also resulted in cell shortening, but this occurred only at concentrations that affected growth. Cell length decreased proportionally with increasing Mg2+ from 0.2 mM to 4.0 mM, with little or no detectable change being observed in labile, intracellular Mg2+, based on a riboswitch reporter. Cells grown in excess Mg2+ had fewer nucleoids and possessed more FtsZ-rings per unit cell length, consistent with the increased division frequency. Remarkably, when shifting cells from unsupplemented to supplemented medium, more than half of the cell length decrease occurred in the first 10 min, consistent with rapid division onset. Relative to unsupplemented cells, cells growing at steady-state with excess Mg2+ showed an enhanced expression of a large number of SigB-regulated genes and the activation of the Fur, MntR, and Zur regulons. Thus, by manipulating the availability of one nutrient, we were able to uncouple the growth rate from the division frequency and identify transcriptional changes that suggest that cell division is accompanied by the general stress response and an enhanced demand to sequester and/or increase the uptake of iron, Mn2+, and Zn2+. IMPORTANCE The signals that cells use to trigger cell division are unknown. Although division is often considered intrinsic to the cell cycle, microorganisms can continue to grow and repeat rounds of DNA replication without dividing, indicating that cycles of division can be skipped. Here, we show that by manipulating a single nutrient, namely, Mg2+, cell division can be uncoupled from the growth rate. This finding can be applied to investigate the nature of the cell division signal(s).

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.

Metabolism Shapes the Cell.

More than 5 decades of work support the idea that cell envelope synthesis, including the inward growth of cell division, is tightly coordinated with DNA replication and protein synthesis through central metabolism. Remarkably, no unifying model exists to account for how these fundamentally disparate processes are functionally coupled. Recent studies demonstrate that proteins involved in carbohydrate and nitrogen metabolism can moonlight as direct regulators of cell division, coordinate cell division and DNA replication, and even suppress defects in DNA replication. In this minireview, we focus on studies illustrating the intimate link between metabolism and regulation of peptidoglycan (PG) synthesis during growth and division, and we identify the following three recurring themes. (i) Nutrient availability, not growth rate, is the primary determinant of cell size. (ii) The degree of gluconeogenic flux is likely to have a profound impact on the metabolites available for cell envelope synthesis, so growth medium selection is a critical consideration when designing and interpreting experiments related to morphogenesis. (iii) Perturbations in pathways relying on commonly shared and limiting metabolites, like undecaprenyl phosphate (Und-P), can lead to pleotropic phenotypes in unrelated pathways.

The DnaA inhibitor SirA acts in the same pathway as Soj (ParA) to facilitate oriC segregation during Bacillus subtilis sporulation.

DNA replication and chromosome segregation must be carefully regulated to ensure reproductive success. During Bacillus subtilis sporulation, chromosome copy number is reduced to two, and cells divide asymmetrically to produce the future spore (forespore) compartment. For successful sporulation, oriC must be captured in the forespore. New rounds of DNA replication are prevented in part by SirA, a protein that utilizes residues in its N-terminus to directly target Domain I of the bacterial initiator, DnaA. Using a quantitative forespore chromosome organization assay, we show that SirA also acts in the same pathway as another DnaA regulator, Soj, to promote oriC capture in the forespore. By analyzing loss-of-function variants of both SirA and DnaA, we observe that SirA's ability to inhibit DNA replication can be genetically separated from its role in oriC capture. In addition, we identify substitutions near the C-terminus of SirA and in DnaA Domain III that enhance interaction between the two proteins. One such variant, SirAP141T , remained functional in regard to inhibiting replication, but was unable to support oriC capture. Collectively, our results support a model in which SirA targets DnaA Domain I to inhibit DNA replication, and DnaA Domain III to facilitate Soj-dependent oriC capture in the forespore.

YodL and YisK Possess Shape-Modifying Activities That Are Suppressed by Mutations in Bacillus subtilis mreB and mbl.

UNLABELLED: Many bacteria utilize actin-like proteins to direct peptidoglycan (PG) synthesis. MreB and MreB-like proteins are thought to act as scaffolds, guiding the localization and activity of key PG-synthesizing proteins during cell elongation. Despite their critical role in viability and cell shape maintenance, very little is known about how the activity of MreB family proteins is regulated. Using a Bacillus subtilis misexpression screen, we identified two genes, yodL and yisK, that when misexpressed lead to loss of cell width control and cell lysis. Expression analysis suggested that yodL and yisK are previously uncharacterized Spo0A-regulated genes, and consistent with these observations, a ΔyodL ΔyisK mutant exhibited reduced sporulation efficiency. Suppressors resistant to YodL's killing activity occurred primarily in mreB mutants and resulted in amino acid substitutions at the interface between MreB and the highly conserved morphogenic protein RodZ, whereas suppressors resistant to YisK occurred primarily in mbl mutants and mapped to Mbl's predicted ATP-binding pocket. YodL's shape-altering activity appears to require MreB, as a ΔmreB mutant was resistant to the effects of YodL but not YisK. Similarly, YisK appears to require Mbl, as a Δmbl mutant was resistant to the cell-widening effects of YisK but not of YodL. Collectively, our results suggest that YodL and YisK likely modulate MreB and Mbl activity, possibly during the early stages of sporulation. IMPORTANCE: The peptidoglycan (PG) component of the cell envelope confers structural rigidity to bacteria and protects them from osmotic pressure. MreB and MreB-like proteins are thought to act as scaffolds for PG synthesis and are essential in bacteria exhibiting nonpolar growth. Despite the critical role of MreB-like proteins, we lack mechanistic insight into how their activities are regulated. Here, we describe the discovery of two B. subtilis proteins, YodL and YisK, which modulate MreB and Mbl activities. Our data suggest that YodL specifically targets MreB, whereas YisK targets Mbl. The apparent specificities with which YodL and YisK are able to differentially target MreB and Mbl make them potentially powerful tools for probing the mechanics of cytoskeletal function in bacteria.

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.

A Secreted Factor Coordinates Environmental Quality with Bacillus Development.

Entry into sporulation is governed by the master regulator Spo0A. Spo0A accumulates in its active form, Spo0A-P, as cells enter stationary phase. Prior reports have shown that the acute induction of constitutively active Spo0A during exponential growth does not result in sporulation. However, a subsequent study also found that a gradual increase in Spo0A-P, mediated through artificial expression of the kinase, KinA, during exponential growth, is sufficient to trigger sporulation. We report here that sporulation via KinA induction depends on the presence of an extracellular factor or factors (FacX) that only accumulates to active levels during post-exponential growth. FacX is retained by dialysis with a cutoff smaller than 500 Dalton, can be concentrated, and is susceptible to proteinase K digestion, similar to described quorum-sensing peptides shown to be involved in promoting sporulation. However, unlike previously characterized peptides, FacX activity does not require the Opp or App oligopeptide transporter systems. In addition, FacX activity does not depend on SigH, Spo0A, or ComX. Importantly, we find that in the presence of FacX, B. subtilis can be induced to sporulate following the artificial induction of constitutively active Spo0A. These results indicate that there is no formal requirement for gradual Spo0A-P accumulation and instead support the idea that sporulation requires both sufficient levels of active Spo0A and at least one other signal or condition.

CodY Regulates SigD Levels and Activity by Binding to Three Sites in the fla/che Operon.

UNLABELLED: Exponentially growing cultures of Bacillus subtilis (PY79) are composed primarily of nonmotile, chained cells. The alternative sigma factor, SigD, promotes the phenotypic switch from nonmotile, chained cells to unchained, motile cells. In the present work, we investigated the role of the GTP-sensing protein CodY in the regulation of SigD. Deletion of codY resulted in a significant increase in SigD accumulation and activity and shifted the proportion of unchained cells up from ∼15% to ∼75%, suggesting that CodY is an important regulator of SigD. CodY was previously shown to bind to the PD3 and Pfla/che promoters located upstream of the first gene in the sigD-containing fla/che operon. Using electrophoretic mobility shift assays, we found that CodY also binds to two other previously uncharacterized sites within the fla/che operon. Mutations in any one of the three binding sites resulted in SigD levels similar to those seen with the ΔcodY mutant, suggesting that each site is sufficient to tip cells toward a maximal level of CodY-dependent SigD accumulation. However, mutations in all three sites were required to phenocopy the ΔcodY mutant's reduced level of cell chaining, consistent with the idea that CodY binding in the fla/che operon is also important for posttranslational control of SigD activity. IMPORTANCE: One way that bacteria adapt quickly and efficiently to changes in environmental quality is to employ global transcriptional regulators capable of responding allosterically to key cellular metabolites. In this study, we found that the conserved GTP-sensing protein CodY directly regulates cell motility and chaining in B. subtilis by controlling expression and activity of SigD. Our results suggest that B. subtilis becomes poised for cell dispersal as intracellular GTP levels are depleted.

RelA inhibits Bacillus subtilis motility and chaining.

The nucleotide second messengers pppGpp and ppGpp [(p)ppGpp] are responsible for the global downregulation of transcription, translation, DNA replication, and growth rate that occurs during the stringent response. More recent studies suggest that (p)ppGpp is also an important effector in many nonstringent processes, including virulence, persister cell formation, and biofilm production. In Bacillus subtilis, (p)ppGpp production is primarily determined by the net activity of RelA, a bifunctional (p)ppGpp synthetase/hydrolase, and two monofunctional (p)ppGpp synthetases, YwaC and YjbM. We observe that in B. subtilis, a relA mutant grows exclusively as unchained, motile cells, phenotypes regulated by the alternative sigma factor SigD. Our data indicate that the relA mutant is trapped in a SigD "on" state during exponential growth, implicating RelA and (p)ppGpp levels in the regulation of cell chaining and motility in B. subtilis. Our results also suggest that minor variations in basal (p)ppGpp levels can significantly skew developmental decision-making outcomes.

RefZ facilitates the switch from medial to polar division during spore formation in Bacillus subtilis.

During sporulation, Bacillus subtilis redeploys the division protein FtsZ from midcell to the cell poles, ultimately generating an asymmetric septum. Here, we describe a sporulation-induced protein, RefZ, that facilitates the switch from a medial to a polar FtsZ ring placement. The artificial expression of RefZ during vegetative growth converts FtsZ rings into FtsZ spirals, arcs, and foci, leading to filamentation and lysis. Mutations in FtsZ specifically suppress RefZ-dependent division inhibition, suggesting that RefZ may target FtsZ. During sporulation, cells lacking RefZ are delayed in polar FtsZ ring formation, spending more time in the medial and transition stages of FtsZ ring assembly. A RefZ-green fluorescent protein (GFP) fusion localizes in weak polar foci at the onset of sporulation and as a brighter midcell focus at the time of polar division. RefZ has a TetR DNA binding motif, and point mutations in the putative recognition helix disrupt focus formation and abrogate cell division inhibition. Finally, chromatin immunoprecipitation assays identified sites of RefZ enrichment in the origin region and near the terminus. Collectively, these data support a model in which RefZ helps promote the switch from medial to polar division and is guided by the organization of the chromosome. Models in which RefZ acts as an activator of FtsZ ring assembly near the cell poles or as an inhibitor of the transient medial ring at midcell are discussed.