The RicAFT (YmcA‐YlbF‐YaaT) complex carries two [4Fe‐4S]2+ clusters and may respond to redox changes

During times of environmental insult, Bacillus subtilis undergoes developmental changes leading to biofilm formation, sporulation and competence. Each of these states is regulated in part by the phosphorylated form of the master response regulator Spo0A (Spo0A～P). The phosphorylation state of Spo0A is controlled by a multi‐component phosphorelay. RicA, RicF and RicT (previously YmcA, YlbF and YaaT) have been shown to be important regulatory proteins for multiple developmental fates. These proteins directly interact and form a stable complex, which has been proposed to accelerate the phosphorelay. Indeed, this complex is sufficient to stimulate the rate of phosphotransfer amongst the phosphorelay proteins in vitro. In this study, we demonstrate that two [4Fe‐4S]²⁺ clusters can be assembled on the complex. As with other iron‐sulfur cluster‐binding proteins, the complex was also found to bind FAD, hinting that these cofactors may be involved in sensing the cellular redox state. This work provides the first comprehensive characterization of an iron‐sulfur protein complex that regulates Spo0A～P levels. Phylogenetic and genetic evidence suggests that the complex plays a broader role beyond stimulation of the phosphorelay.     

A revised model for the control of fatty acid synthesis by master regulator Spo0A in Bacillus subtilis

In starving Bacillus subtilis cells, the accDA operon encoding two subunits of the essential acetyl‐CoA carboxylase (ACC) has been proposed to be tightly regulated by direct binding of the master regulator Spo0A to a cis element (0A box) in the promoter region. When the 0A box is mutated, biofilm formation and sporulation have been reported to be impaired. Here, we present evidence that two 0A boxes, one previously known (0A‐1) and another newly discovered (0A‐2) in the accDA promoter region are positively and negatively regulated by Spo0A～P respectively. Cells with mutated 0A boxes experience slight delays in sporulation, but eventually sporulate with high efficiency. In contrast, cells harboring a single mutated 0A‐2 box are deficient for biofilm formation, while cells harboring either a mutated 0A‐1 box or both mutated 0A boxes form biofilms. We further show that the essential ACC enzyme localizes on or near the cell membrane by directly observing a functional GFP fusion to one of the enzyme's subunits. Collectively, we propose a revised model in which accDA is primarily transcribed by a major σ^A‐RNA polymerase, while Spo0A～P plays an additional role in the fine‐tuning of accDA expression upon starvation to support proper biofilm formation and sporulation.     

Structural Analysis of Divalent Metals Binding to the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Response Regulator Spo0F: The Possibility for <Emphasis Type="Italic">In Vitro</Emphasis> Metalloregulation in the Initiation of Sporulation

The presence of a divalent metal ion in a negatively charged aspartic acid pocket is essential for phosphorylation of response regulator proteins. Here, we present metal binding studies of the Bacillus subtilis response regulator Spo0F using NMR and μESI-MS. NMR studies show that the divalent metals Ca²⁺, Mg²⁺ and Mn²⁺ primarily bind, as expected, in the Asp pocket phosphorylation site. However, identical studies with Cu²⁺ show distinct binding effects in three specific locations: (i) the Asp pocket, (ii) a grouping of charged residues at a site opposite of the Asp pocket, and (iii) on the β4-α4 loop and the β5/α5 interface, particularly around and including H101. μESI-MS studies stoichiometrically confirm the NMR studies and demonstrate that most divalent metal ions bind to Spo0F primarily in a 1:1 ratio. Again, in the case of Cu²⁺, multiple metal-bound species are observed. Subsequent experiments reveal that Mg²⁺ supports phosphotransfer between KinA and Spo0F, while Cu²⁺ fails to support KinA phosphotransfer. Additionally, the presence of Cu²⁺ at non-lethal concentrations in sporulation media for B. subtilis and the related organism Pasteuria penetrans was found to inhibit spore formation while continuing to permit vegetative growth. Depending on the type of divalent metal ion present, in vitro phosphorylation of Spo0F by its cognate kinase KinA can be inhibited.     

The Biofilm Regulatory Network from Bacillus subtilis: A Structure-Function Analysis

Bacterial biofilms are notorious for their ability to protect bacteria from environmental challenges, most importantly the action of antibiotics. Bacillus subtilis is an extensively studied model organism used to understand the process of biofilm formation. A complex network of principal regulatory proteins including Spo0A, AbrB, AbbA, Abh, SinR, SinI, SlrR, and RemA, work in concert to transition B. subtilis from the free-swimming planktonic state to the biofilm state. In this review, we explore, connect, and summarize decades worth of structural and biochemical studies that have elucidated this protein signaling network. Since structure dictates function, unraveling aspects of protein molecular mechanisms will allow us to devise ways to exploit critical features of the biofilm regulatory pathway, such as possible therapeutic intervention. This review pools our current knowledge base of B. subtilis biofilm regulatory proteins and highlights potential therapeutic intervention points.     

Spo0A positively regulates epr expression by negating the repressive effect of co-repressors, SinR and ScoC, in Bacillus subtilis

Bacillus subtilis under nutritional deprivation exhibits several physiological responses such as synthesis of degradative enzymes, motility, competence, sporulation, etc. At the onset of post-exponential phase the global response regulator, Spo0A, directly or indirectly activates the expression of genes involved in the above processes. These genes are repressed during the exponential phase by a group of proteins called transition state regulators, e.g. AbrB, ScoC and SinR. One such post-exponentially expressed gene is epr, which encodes a minor extracellular serine protease and is involved in the swarming motility of B. subtilis. Deletion studies of the upstream region of epr promoter revealed that epr is co-repressed by transition state regulators, SinR and ScoC. Our study shows that Spo0A positively regulates epr expression by nullifying the repressive effect of co-repressors, SinR and ScoC. We demonstrate via in vitro mobility shift assays that Spo0A binds to the upstream region of epr promoter and in turn occludes the binding site of one of the co-repressor, SinR. This explains the mechanism behind the positive regulatory effect of Spo0A on epr expression.     

Slowdown of growth controls cellular differentiation

How can changes in growth rate affect the regulatory networks behavior and the outcomes of cellular differentiation? We address this question by focusing on starvation response in sporulating Bacillus subtilis. We show that the activity of sporulation master regulator Spo0A increases with decreasing cellular growth rate. Using a mathematical model of the phosphorelay—the network controlling Spo0A—we predict that this increase in Spo0A activity can be explained by the phosphorelay protein accumulation and lengthening of the period between chromosomal replication events caused by growth slowdown. As a result, only cells growing slower than a certain rate reach threshold Spo0A activity necessary for sporulation. This growth threshold model accurately predicts cell fates and explains the distribution of sporulation deferral times. We confirm our predictions experimentally and show that the concentration rather than activity of phosphorelay proteins is affected by the growth slowdown. We conclude that sensing the growth rates enables cells to indirectly detect starvation without the need for evaluating specific stress signals.     

Spatiotemporally regulated proteolysis to dissect the role of vegetative proteins during Bacillus subtilis sporulation: cell‐specific requirement of σH and σA

Sporulation in Bacillus subtilis is a paradigm of bacterial development, which involves the interaction between a larger mother cell and a smaller forespore. The mother cell and the forespore activate different genetic programs, leading to the production of sporulation‐specific proteins. A critical gap in our understanding of sporulation is how vegetative proteins, made before sporulation initiation, contribute to spore formation. Here we present a system, spatiotemporally regulated proteolysis (STRP), which enables the rapid, developmentally regulated degradation of target proteins, thereby providing a suitable method to dissect the cell‐ and developmental stage‐specific role of vegetative proteins. STRP has been used to dissect the role of two major vegetative sigma factors, σ^H and σ^A, during sporulation. The results suggest that σ^H is only required in predivisional cells, where it is essential for sporulation initiation, but that it is dispensable during subsequent steps of spore formation. However, evidence has been provided that σ^A plays different roles in the mother cell, where it replenishes housekeeping functions, and in the forespore, where it plays an unexpected role in promoting spore germination and outgrowth. Altogether, the results demonstrate that STRP has the potential to provide a comprehensive molecular dissection of every stage of sporulation, germination and outgrowth.