Master Regulator Spo0A Research Articles

The Slowdown of Growth Rate Controls the Single-Cell Distribution of Biofilm Matrix Production via an SinI-SinR-SlrR Network.

In Bacillus subtilis, master regulator Spo0A controls several cell-differentiation pathways. Under moderate starvation, phosphorylated Spo0A (Spo0A~P) induces biofilm formation by indirectly activating genes controlling matrix production in a subpopulation of cells via an SinI-SinR-SlrR network. Under severe starvation, Spo0A~P induces sporulation by directly and indirectly regulating sporulation gene expression. However, what determines the heterogeneity of individual cell fates is not fully understood. In particular, it is still unclear why, despite being controlled by a single master regulator, biofilm matrix production and sporulation seem mutually exclusive on a single-cell level. In this work, with mathematical modeling, we showed that the fluctuations in the growth rate and the intrinsic noise amplified by the bistability in the SinI-SinR-SlrR network could explain the single-cell distribution of matrix production. Moreover, we predicted an incoherent feed-forward loop; the decrease in the cellular growth rate first activates matrix production by increasing in Spo0A phosphorylation level but then represses it via changing the relative concentrations of SinR and SlrR. Experimental data provide evidence to support model predictions. In particular, we demonstrate how the degree to which matrix production and sporulation appear mutually exclusive is affected by genetic perturbations. IMPORTANCE The mechanisms of cell-fate decisions are fundamental to our understanding of multicellular organisms and bacterial communities. However, even for the best-studied model systems we still lack a complete picture of how phenotypic heterogeneity of genetically identical cells is controlled. Here, using B. subtilis as a model system, we employ a combination of mathematical modeling and experiments to explain the population-level dynamics and single-cell level heterogeneity of matrix gene expression. The results demonstrate how the two cell fates, biofilm matrix production and sporulation, can appear mutually exclusive without explicitly inhibiting one another. Such a mechanism could be used in a wide range of other biological systems.

Response Regulator CD1688 Is a Negative Modulator of Sporulation in Clostridioides difficile.

ABSTRACTTwo-component signal transduction systems (TCSs), consisting of a sensor histidine kinase (HK) and a response regulator (RR), sense environmental stimuli and then modulate cellular responses, typically through changes in gene expression. Our previous work identified the DNA binding motif of CD1586, an RR implicated in Clostridioides difficile strain R20291 sporulation. To determine the role of this RR in the sporulation pathway in C. difficile, we generated a deletion strain of cd1688 in the historical 630 strain, the homolog of cd1586. The C. difficile Δcd1688 strain exhibited a hypersporulation phenotype, suggesting that CD1688 negatively regulates sporulation. Complementation of the C. difficile Δcd1688 strain restored sporulation. In contrast, a nonphosphorylatable copy of cd1688 did not restore sporulation to wild-type (WT) levels, indicating that CD1688 must be phosphorylated to properly modulate sporulation. Expression of the master regulator spo0A, the sporulation-specific sigma factors sigF, sigE, sigG, and sigK, and a signaling protein encoded by spoIIR was increased in the C. difficile Δcd1688 strain compared to WT. In line with the increased spoIIR expression, we detected an increase in mature SigE at an earlier time point, which arises from SpoIIR-mediated processing of pro-SigE. Taken together, our data suggest that CD1688 is a novel negative modulator of sporulation in C. difficile and contributes to mediating progression through the spore developmental pathway. These results add to our growing understanding of the complex regulatory events involved in C. difficile sporulation, insight that could be exploited for novel therapeutic development.IMPORTANCE Clostridioides difficile causes severe gastrointestinal illness and is a leading cause of nosocomial infections in the United States. This pathogen produces metabolically dormant spores that are the major vehicle of transmission between hosts. The sporulation pathway involves an intricate regulatory network that controls a succession of morphological changes necessary to produce spores. The environmental signals inducing the sporulation pathway are not well understood in C. difficile. This work identified a response regulator, CD1688, that, when deleted, led to a hypersporulation phenotype, indicating that it typically acts to repress sporulation. Improving our understanding of the regulatory mechanisms modulating sporulation in C. difficile could provide novel strategies to eliminate or reduce spore production, thus decreasing transmission and disease relapse.

The Transcription Factor CpcR Determines Cell Fate by Modulating the Initiation of Sporulation in Bacillus thuringiensis.

Bacillus thuringiensis is a bacterium capable of differentiating into a spore, a dormant and highly resistant cellular form. During the sporulation process, this bacterium produces insecticidal toxins in the form of a crystal inclusion, usually in the sporulating cell. We previously reported that the B. thuringiensis LM1212 strain can differentiate into two distinct subpopulations of sporeformers and crystal producers and that this division-of-labor phenotype provides the bacterium with a fitness advantage in competition with a typical B. thuringiensis strain. The transcription factor CpcR was characterized as the regulator responsible for this phenotype. Here, we examined how CpcR interacts with the sporulation network to control the cell differentiation. We found that the sporulation process was inhibited prior to polar septum formation and that Spo0A activity was impaired in the presence of cpcR in strain LM1212. Using bioinformatics and genetic tools, we identified a gene positively controlled by CpcR encoding a putative phosphatase of the Spo0E family known to specifically dephosphorylate phosphorylated Spo0A (Spo0A-P). We showed that this protein (called Spo0E1) is a negative regulator of sporulation and that variations in spo0E1 expression can modulate the production of spores. Using fluorescent reporters to follow gene expression at the single-cell level, we correlated expression of cpcR and sporulation genes to the formation of the two differentiated subpopulations. IMPORTANCE Formation of spores is a paradigm for study of cell differentiation in prokaryotes. Sporulation initiation is governed by a gradual increase in the level and activity of the master regulator Spo0A. Spo0A is usually indirectly phosphorylated by a multicomponent phosphorelay, and modulation of this phosphorelay system is a critical aspect of Bacillus physiology. Though we know that this phosphorelay system is usually affected by two negative regulatory mechanisms, i.e., rap genes and spo0E family genes, the regulatory mechanisms controlling the transcription of these genes are poorly understood. Here, we report that the transcription factor CpcR positively regulates a spo0E family gene and that variations in spo0E expression can modulate the production of spores in B. thuringiensis. This work emphasizes the diversity in modes of sporulation and illustrates the diversity in the strategies employed by bacteria to control this differentiation pathway and ensure their survival.

Investigating the Role of the Med Protein in Biofilm Formation

Bacillus subtilis is a soil‐dwelling bacterium that serves as a good model organism for the study of different adaptive responses to various environmental conditions. These adaptive responses, including spore formation, cannibalism, and biofilm formation, are controlled by the master regulator Spo0A whose phosphorylation is under the control of five kinases. Initial interest in the protein Med came from its role in the transcription of operons related to cannibalism, and studies show the effect of Med is dependent on one of the five kinases, KinD. These same studies suggest Med may play a role in biofilm formation in a KinD‐dependent manner. The goal of our study of the Med protein is to determine whether it plays a direct role in biofilm formation by directly detecting environmental signals or indirectly through interactions with KinD. To determine whether Med is involved in detecting environmental signals, Δmed and ΔkinD bacteria were compared to wildtype B. subtilis using luciferase reporter assays that quantify the effect of different small molecules on the transcription of biofilm genes. Luciferase assays were compared to biofilm assays under the same conditions to visualize the effect on the biofilm phenotypes. We find that Δmed mutants had a defect in biofilm formation and reduced transcription of biofilm genes compared with wildtype bacteria. However, Δmed mutants were the same as wildtype bacteria in terms of their pattern of stimulation by small molecules. These results indicate that Med is not directly sensing environmental signals and instead is likely to be involved in the biofilm formation pathway indirectly through KinD, which has been shown to bind small molecules through its extracellular CACHE domain. We therefore shifted our focus to investigating interactions between Med and KinD and how Med may affect KinD’s ability to sense small molecules. Med and KinD were purified by tagging them with GST and His tags respectively. Additionally, in the case of KinD we purified constructs of the extracellular and intracellular domains to more precisely determine where the Med protein may be interacting with KinD. Presence of the proteins was determined by SDS‐PAGE and interactions between them was determined by Western blot analysis.Support or Funding InformationResearch was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R15GM135861.

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 σA -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.

Phosphorylation of the Bacillus subtilis Replication Controller YabA Plays a Role in Regulation of Sporulation and Biofilm Formation.

Bacillus subtilis cells can adopt different life-styles in response to various environmental cues, including planktonic cells during vegetative growth, sessile cells during biofilm formation and sporulation. While switching life-styles, bacteria must coordinate the progression of their cell cycle with their physiological status. Our current understanding of the regulatory pathways controlling the decision-making processes and triggering developmental switches highlights a key role of protein phosphorylation. The regulatory mechanisms that integrate the bacterial chromosome replication status with sporulation involve checkpoint proteins that target the replication initiator DnaA or the kinase phosphorelay controlling the master regulator Spo0A. B. subtilis YabA is known to interact with DnaA to prevent over-initiation of replication during vegetative growth. Here, we report that YabA is phosphorylated by YabT, a Ser/Thr kinase expressed during sporulation and biofilm formation. The phosphorylation of YabA has no effect on replication initiation control but hyper-phosphorylation of YabA leads to an increase in sporulation efficiency and a strong inhibition of biofilm formation. We also provide evidence that YabA phosphorylation affects the level of Spo0A-P in cells. These results indicate that YabA is a multifunctional protein with a dual role in regulating replication initiation and life-style switching, thereby providing a potential mechanism for cross-talk and coordination of cellular processes during adaptation to environmental change.

Lysinibacillus fusiformis M5 Induces Increased Complexity in Bacillus subtilis 168 Colony Biofilms via Hypoxanthine.

In recent years, biofilms have become a central subject of research in the fields of microbiology, medicine, agriculture, and systems biology, among others. The sociomicrobiology of multispecies biofilms, however, is still poorly understood. Here, we report a screening system that allowed us to identify soil bacteria which induce architectural changes in biofilm colonies when cocultured with Bacillus subtilis We identified the soil bacterium Lysinibacillus fusiformis M5 as an inducer of wrinkle formation in B. subtilis colonies mediated by a diffusible signaling molecule. This compound was isolated by bioassay-guided chromatographic fractionation. The elicitor was identified to be the purine hypoxanthine using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. We show that the induction of wrinkle formation by hypoxanthine is not dependent on signal recognition by the histidine kinases KinA, KinB, KinC, and KinD, which are generally involved in phosphorylation of the master regulator Spo0A. Likewise, we show that hypoxanthine signaling does not induce the expression of biofilm matrix-related operons epsABCDEFGHIJKLMNO and tasA-sipW-tapA Finally, we demonstrate that the purine permease PbuO, but not PbuG, is necessary for hypoxanthine to induce an increase in wrinkle formation of B. subtilis biofilm colonies. Our results suggest that hypoxanthine-stimulated wrinkle development is not due to a direct induction of biofilm-related gene expression but rather is caused by the excess of hypoxanthine within B. subtilis cells, which may lead to cell stress and death.IMPORTANCE Biofilms are a bacterial lifestyle with high relevance regarding diverse human activities. Biofilms can be beneficial, for instance, in crop protection. In nature, biofilms are commonly found as multispecies communities displaying complex social behaviors and characteristics. The study of interspecies interactions will thus lead to a better understanding and use of biofilms as they occur outside laboratory conditions. Here, we present a screening method suitable for the identification of multispecies interactions and showcase L. fusiformis as a soil bacterium that is able to live alongside B. subtilis and modify the architecture of its biofilms.

Termination factor Rho: From the control of pervasive transcription to cell fate determination in Bacillus subtilis

In eukaryotes, RNA species originating from pervasive transcription are regulators of various cellular processes, from the expression of individual genes to the control of cellular development and oncogenesis. In prokaryotes, the function of pervasive transcription and its output on cell physiology is still unknown. Most bacteria possess termination factor Rho, which represses pervasive, mostly antisense, transcription. Here, we investigate the biological significance of Rho-controlled transcription in the Gram-positive model bacterium Bacillus subtilis. Rho inactivation strongly affected gene expression in B. subtilis, as assessed by transcriptome and proteome analysis of a rho–null mutant during exponential growth in rich medium. Subsequent physiological analyses demonstrated that a considerable part of Rho-controlled transcription is connected to balanced regulation of three mutually exclusive differentiation programs: cell motility, biofilm formation, and sporulation. In the absence of Rho, several up-regulated sense and antisense transcripts affect key structural and regulatory elements of these differentiation programs, thereby suppressing motility and biofilm formation and stimulating sporulation. We dissected how Rho is involved in the activity of the cell fate decision-making network, centered on the master regulator Spo0A. We also revealed a novel regulatory mechanism of Spo0A activation through Rho-dependent intragenic transcription termination of the protein kinase kinB gene. Altogether, our findings indicate that distinct Rho-controlled transcripts are functional and constitute a previously unknown built-in module for the control of cell differentiation in B. subtilis. In a broader context, our results highlight the recruitment of the termination factor Rho, for which the conserved biological role is probably to repress pervasive transcription, in highly integrated, bacterium-specific, regulatory networks.

The PAS domains of the major sporulation kinase in Bacillus subtilis play a role in tetramer formation that is essential for theautokinase activity.

Sporulation in Bacillus subtilis is induced upon starvation. In a widely accepted model, an N‐terminal “sensor” domain of the major sporulation kinase KinA recognizes a hypothetical starvation signal(s) and autophosphorylates a histidine residue to activate the master regulator Spo0A via a multicomponent phosphorelay. However, to date no confirmed signal has been found. Here, we demonstrated that PAS‐A, the most N‐terminal of the three PAS domains (PAS‐ABC), is dispensable for the activity, contrary to a previous report. Our data indicated that the autokinase activity is dependent on the formation of a functional tetramer, which is mediated by, at least, PAS‐B and PAS‐C. Additionally, we ruled out the previously proposed notion that NAD +/NADH ratio controls KinA activity through the PAS‐A domain by demonstrating that the cofactors show no effects on the kinase activity in vitro. In support of these data, we found that the cofactors exist in approximately 1000‐fold excess of KinA in the cell and the cofactors’ ratio does not change significantly during growth and sporulation, suggesting that changes in the cofactor ratio might not play a role in controlling KinA activity. These data may refute the widely‐held belief that the activity of KinA is regulated in response to an unknown starvation signal(s).

Slowdown of growth controls cellulardifferentiation.

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.

Impact of Serine/Threonine Protein Kinases on the Regulation of Sporulation in Bacillus subtilis.

Bacteria possess many kinases that catalyze phosphorylation of proteins on diverse amino acids including arginine, cysteine, histidine, aspartate, serine, threonine, and tyrosine. These protein kinases regulate different physiological processes in response to environmental modifications. For example, in response to nutritional stresses, the Gram-positive bacterium Bacillus subtilis can differentiate into an endospore; the initiation of sporulation is controlled by the master regulator Spo0A, which is activated by phosphorylation. Spo0A phosphorylation is carried out by a multi-component phosphorelay system. These phosphorylation events on histidine and aspartate residues are labile, highly dynamic and permit a temporal control of the sporulation initiation decision. More recently, another kind of phosphorylation, more stable yet still dynamic, on serine or threonine residues, was proposed to play a role in spore maintenance and spore revival. Kinases that perform these phosphorylation events mainly belong to the Hanks family and could regulate spore dormancy and spore germination. The aim of this mini review is to focus on the regulation of sporulation in B. subtilis by these serine and threonine phosphorylation events and the kinases catalyzing them.

Coordination of cell decisions and promotion of phenotypic diversity in B. subtilis via pulsed behavior of the phosphorelay.

The phosphorelay of Bacillus subtilis, a kinase cascade that activates master regulator Spo0A ~ P in response to starvation signals, is the core of a large network controlling the cell's decision to differentiate into sporulation and other phenotypes. This article reviews recent advances in understanding the origins and purposes of the complex dynamical behavior of the phosphorelay, which pulses with peaks of activity coordinated with the cell cycle. The transient imbalance in the expression of two critical genes caused by their strategic placement at opposing ends of the chromosome proved to be the key for this pulsed behavior. Feedback control loops in the phosphorelay use these pulses to implement a timer mechanism, which creates several windows of opportunity for phenotypic transitions over multiple generations. This strategy allows the cell to coordinate multiple differentiation programs in a decision process that fosters phenotypic diversity and adapts to current conditions.

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.

Chromosomal Arrangement of Phosphorelay Genes Couples Sporulation and DNA Replication

Genes encoding proteins in a common regulatory network are frequently located close to one another on the chromosome to facilitate co-regulation or couple gene expression to growth rate. Contrasting with these observations, here, we demonstrate a functional role for the arrangement of Bacillus subtilis sporulation network genes on opposite sides of the chromosome. We show that the arrangement of two sporulation network genes, one located close to the origin and the other close to the terminus, leads to a transient gene dosage imbalance during chromosome replication. This imbalance is detected by the sporulation network to produce cell-cycle coordinated pulses of the sporulation master regulator Spo0A∼P. This pulsed response allows cells to decide between sporulation and continued vegetative growth during each cell cycle spent in starvation. The simplicity of this coordination mechanism suggests that it may be widely applicable in a variety of gene regulatory and stress-response settings. VIDEO ABSTRACT.

In vivo functional characterization of the transmembrane histidine kinase KinC in Bacillus subtilis.

In response to starvation, Bacillus subtilis cells differentiate into different subsets, undergoing cannibalism, biofilm formation or sporulation. These processes require a multiple component phosphorelay, wherein the master regulator Spo0A is activated upon phosphorylation by one or a combination of five histidine kinases (KinA-KinE) via two intermediate phosphotransferases, Spo0F and Spo0B. In this study, we focused on KinC, which was originally identified as a sporulation kinase and was later shown to regulate cannibalism and biofilm formation. First, genetic experiments using both the domesticated and undomesticated (biofilm forming) strains revealed that KinC activity and the membrane localization are independent of both the lipid raft marker proteins FloTA and cytoplasmic potassium concentration, which were previously shown to be required for the kinase activity. Next, we demonstrated that KinC controls cannibalism and biofilm formation in a manner dependent on phosphorelay. For further detailed characterization of KinC, we established an IPTG-inducible expression system in the domesticated strain, in which biofilm formation is defective, for simplicity of study. Using this system, we found that the N-terminal transmembrane domain is dispensable but the PAS domain is needed for the kinase activity. An in vivo chemical cross-linking experiment demonstrated that the soluble and functional KinC (KinC(ΔTM1+2)) forms a tetramer. Based on these results, we propose a revised model in which KinC becomes active by forming a homotetramer via the N-terminal PAS domain, but its activity is independent of both the lipid raft and the potassium leakage, which was previously suggested to be induced by surfactin.

Computational modelling and analysis of the molecular network regulating sporulation initiation in Bacillus subtilis

BackgroundBacterial spores are important contaminants in food, and the spore forming bacteria are often implicated in food safety and food quality considerations. Spore formation is a complex developmental process involving the expression of more than 500 genes over the course of 6 to 8 hrs. The process culminates in the formation of resting cells capable of resisting environmental extremes and remaining dormant for long periods of time, germinating when conditions promote further vegetative growth. Experimental observations of sporulation and germination are problematic and time consuming so that reliable models are an invaluable asset in terms of prediction and risk assessment. In this report we develop a model which assists in the interpretation of sporulation dynamics.ResultsThis paper defines and analyses a mathematical model for the network regulating Bacillus subtilis sporulation initiation, from sensing of sporulation signals down to the activation of the early genes under control of the master regulator Spo0A. Our model summarises and extends other published modelling studies, by allowing the user to execute sporulation initiation in a scenario where Isopropyl β-D-1-thiogalactopyranoside (IPTG) is used as an artificial sporulation initiator as well as in modelling the induction of sporulation in wild-type cells. The analysis of the model results and the comparison with experimental data indicate that the model is good at predicting inducible responses to sporulation signals. However, the model is unable to reproduce experimentally observed accumulation of phosphorelay sporulation proteins in wild type B. subtilis. This model also highlights that the phosphorelay sub-component, which relays the signals detected by the sensor kinases to the master regulator Spo0A, is crucial in determining the response dynamics of the system.ConclusionWe show that there is a complex connectivity between the phosphorelay features and the master regulatory Spo0A. Additional we discovered that the experimentally observed regulation of the phosphotransferase Spo0B for wild-type B. subtilis may be playing an important role in the network which suggests that modelling of sporulation initiation may require additional experimental support.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-014-0119-x) contains supplementary material, which is available to authorized users.

Physiology and Sporulation in Clostridium.

Clostridia are Gram-positive, anaerobic, endospore-forming bacteria, incapable of dissimilatory sulfate reduction. Comprising approximately 180 species, the genus Clostridium is one of the largest bacterial genera. Physiology is mostly devoted to acid production. Numerous pathways are known, such as the homoacetate fermentation by acetogens, the propionate fermentation by Clostridium propionicum, and the butyrate/butanol fermentation by C. acetobutylicum, a well-known solvent producer. Clostridia degrade sugars, alcohols, amino acids, purines, pyrimidines, and polymers such as starch and cellulose. Energy conservation can be performed by substrate-level phosphorylation as well as by the generation of ion gradients. Endospore formation resembles the mechanism elucidated in Bacillus. Morphology, contents, and properties of spores are very similar to bacilli endospores. Sporulating clostridia usually form swollen mother cells and accumulate the storage substance granulose. However, clostridial sporulation differs by not employing the so-called phosphorelay. Initiation starts by direct phosphorylation of the master regulator Spo0A. The cascade of sporulation-specific sigma factors is again identical to what is known from Bacillus. The onset of sporulation is coupled in some species to either solvent (acetone, butanol) or toxin (e.g., C. perfringens enterotoxin) formation. The germination of spores is often induced by various amino acids, often in combination with phosphate and sodium ions. In medical applications, C. butyricum spores are used as a C. difficile prophylaxis and as treatment against diarrhea. Recombinant spores are currently under investigation and testing as antitumor agents, because they germinate only in hypoxic tissues (i.e., tumor tissue), allowing precise targeting and direct killing of tumor cells.

Triggering sporulation in Bacillus subtilis with artificial two‐component systems reveals the importance of proper Spo0A activation dynamics

Sporulation initiation in Bacillus subtilis is controlled by the phosphorylated form of the master regulator Spo0A which controls transcription of a multitude of sporulation genes. In this study, we investigated the importance of temporal dynamics of phosphorylated Spo0A (Spo0A∼P) accumulation by rewiring the network controlling its phosphorylation. We showed that simultaneous induction of KinC, a kinase that can directly phosphorylate Spo0A, and Spo0A itself from separately controlled inducible promoters can efficiently trigger sporulation even under nutrient rich conditions. However, the sporulation efficiency in this artificial two-component system was significantly impaired when KinC and/or Spo0A induction was too high. Using mathematical modelling, we showed that gradual accumulation of Spo0A∼P is essential for the proper temporal order of the Spo0A regulon expression, and that reduction in sporulation efficiency results from the reversal of that order. These insights led us to identify premature repression of DivIVA as one possible explanation for the adverse effects of accelerated accumulation of Spo0A∼P on sporulation. Moreover, we found that positive feedback resulting from autoregulation of the native spo0A promoter leads to robust control of Spo0A∼P accumulation kinetics. Thus we propose that a major function of the conserved architecture of the sporulation network is controlling Spo0A activation dynamics.

Bacillus subtilisbiofilm induction by plant polysaccharides

Bacillus subtilis is a plant-beneficial Gram-positive bacterium widely used as a biofertilizer. However, relatively little is known regarding the molecular processes underlying this bacterium's ability to colonize roots. In contrast, much is known about how this bacterium forms matrix-enclosed multicellular communities (biofilms) in vitro. Here, we show that, when B. subtilis colonizes Arabidopsis thaliana roots it forms biofilms that depend on the same matrix genes required in vitro. B. subtilis biofilm formation was triggered by certain plant polysaccharides. These polysaccharides served as a signal for biofilm formation transduced via the kinases controlling the phosphorylation state of the master regulator Spo0A. In addition, plant polysaccharides are used as a source of sugars for the synthesis of the matrix exopolysaccharide. The bacterium's response to plant polysaccharides was observed across several different strains of the species, some of which are known to have beneficial effects on plants. These observations provide evidence that biofilm genes are crucial for Arabidopsis root colonization by B. subtilis and provide insights into how matrix synthesis may be triggered by this plant.

A complex of YlbF, YmcA and YaaT regulates sporulation, competence and biofilm formation by accelerating the phosphorylation of Spo0A.

Bacillus subtilis has adopted a bet-hedging strategy to ensure survival in changing environments. From a clonal population, numerous sub-populations can emerge, expressing different sets of genes that govern the developmental processes of sporulation, competence and biofilm formation. The master transcriptional regulator Spo0A controls the entry into all three fates and the production of the phosphorylated active form of Spo0A is precisely regulated via a phosphorelay, involving at least four proteins. Two proteins, YmcA and YlbF were previously shown to play an unidentified role in the regulation of biofilm formation, and in addition, YlbF was shown to regulate competence and sporulation. Using an unbiased proteomics screen, we demonstrate that YmcA and YlbF interact with a third protein, YaaT to form a tripartite complex. We show that all three proteins are required for proper establishment of the three above-mentioned developmental states. We show that the complex regulates the activity of Spo0A in vivo and, using in vitro reconstitution experiments, determine that they stimulate the phosphorelay, probably by interacting with Spo0F and Spo0B. We propose that the YmcA-YlbF-YaaT ternary complex is required to increase Spo0A~P levels above the thresholds needed to induce development.