Phosphorylation Of Spo0A Research Articles

Transcription Factor Spo0A Regulates the Biosynthesis of Difficidin in Bacillus amyloliquefaciens.

Bacillus amyloliquefaciens WH1 produces multiple antibiotics with antimicrobial activity and can control bacterial wilt disease caused by Ralstonia solanacearum. Antibacterial substances produced by WH1 and the regulation mechanism are unknown. In this study, it was found that difficidin, and to a minor extent bacillibactin, exhibited antibacterial activity against R. solanacearum. Lipopeptides, macrolactin, bacillaene, and bacilysin had no antibacterial activity. Ferric iron uptake transcriptional regulator Fur bound the promoter region of the dhb gene cluster of bacillibactin biosynthesis. Mutant Δfur showed a higher bacillibactin production and its antibacterial activity increased by 27% than wild-type WH1. Difficidin inhibited R. solanacearum growth and disrupted the integrity of the cells. Lack of transcription factor Spo0A abolished difficidin biosynthesis. Spo0A bound the promoter region of the dfn gene cluster of difficidin biosynthesis. Changing phosphorylation levels of Spo0A via deletion of phosphatase gene spo0E and histidine kinases genes kinA and kinD affected the biosynthesis of difficidin. Deletion of spo0E increased the phosphorylation level of Spo0A and consequently improved the difficidin production. The antibacterial activity of mutant Δspo0E and ΔkinA increased by 12% and 19%. The antibacterial activity of mutant ΔkinD decreased by 28%. Collectively, WH1 produced difficidin to disrupt the cell of R. solanacearum and secreted siderophore bacillibactin to compete for ferric iron. Spo0A regulated difficidin biosynthesis. Spo0A regulates quorum-sensing responses and controls the biosynthesis of secondary metabolites in B. amyloliquefaciens. This study has important findings in the regulation mechanism of antibiotic synthesis and helps to improve antibiotic yield in Bacillus. IMPORTANCE Pathogen R. solanacearum causes bacterial wilt disease in many crops. There is no chemical bactericide that can control bacterial wilt disease. It is vital to find antagonistic microorganisms and antibacterial substances that can efficiently control bacterial wilt disease. B. amyloliquefaciens WH1 could inhibit the growth of R. solanacearum. Via genetic mutation, it was found that difficidin and to a minor extent bacillibactin produced by WH1 acted efficiently against R. solanacearum. The transcription factor Spo0A regulated the synthesis of difficidin. Phosphorylation of Spo0A affected the production of difficidin. Increasing the phosphorylation level of Spo0A improved the difficidin production and antibacterial activity. In-depth analysis of the regulation mechanism of antibiotic difficidin is meaningful for enhancing the control efficiency of WH1. B. amyloliquefaciens WH1 and the antibacterial substances have vast application potential in controlling bacterial wilt disease.

Artificial activation of both σH and Spo0A in Bacillus subtilis enforced initiation of spore development at the vegetatively growing phase.

When Bacillus subtilis cells face environmental deterioration, such as exhaustion of nutrients and an increase in cell density, they form spores. It is known that phosphorylation of Spo0A and activation of σH are key events at the initiation of sporulation. However, the initiation of sporulation is an extremely complicated process, and the relationship between these two events remains to be elucidated. To determine the minimum requirements for triggering sporulation initiation, we attempted to induce cell sporulation at the log phase, regardless of nutrients and cell density. In rich media such as Luria-Bertani (LB) medium, the cells of B. subtilis do not sporulate efficiently, possibly because of excess nutrition. When the amount of xylose in the LB medium was limited, σH -dependent transcription of the strain, in which sigA was under the control of the xylose-inducible promoter, was induced, and the frequency of sporulation was elevated according to the decreased level of σA. We also employed a fusion of sad67, which codes for an active form of Spo0A, and the IPTG-inducible promoter. The combination of lowered σA expression and activated Spo0A allowed the cells in the log phase to stop growing and rush into spore development. This observation of enforced initiation of sporulation in the mutant strain was detected even in the presence of the wild-type strain, suggesting that only intracellular events initiate and fulfill spore development regardless of extracellular conditions. Under natural sporulation conditions, the amount of σA did not change drastically throughout growth. Mechanisms that sequester σA from the core RNA polymerase and help σH to become active exist, but this has not yet been elucidated.

Energy-efficient butanol production by Clostridium acetobutylicum with histidine kinase knockouts to improve strain tolerance and process robustness

Engineering histidine kinases in C. acetobutylicum enhanced cell viability and solventogenesis in ABE fermentation and enabled robust and energy-efficient butanol production.

Identification of an Important Orphan Histidine Kinase for the Initiation of Sporulation and Enterotoxin Production by Clostridium perfringens Type F Strain SM101.

Clostridium perfringens type F strains cause a common human foodborne illness and many cases of nonfoodborne human gastrointestinal diseases. Sporulation plays two critical roles during type F enteric disease. First, it produces broadly resistant spores that facilitate type F strain survival in the food and nosocomial environments. Second, production of C. perfringens enterotoxin (CPE), the toxin responsible for causing the enteric symptoms of type F diseases, is restricted to cells in the process of sporulation. While later steps in the regulation of C. perfringens sporulation have been discerned, the process leading to phosphorylation of Spo0A, the master early regulator of sporulation and consequent CPE production, has remained unknown. Using an insertional mutagenesis approach, the current study identified the orphan histidine kinase CPR0195 as an important factor regulating C. perfringens sporulation and CPE production. Specifically, a CPR0195 null mutant of type F strain SM101 made 103-fold fewer spores than its wild-type parent and produced no detectable CPE. In contrast, a null mutant of another putative C. perfringens orphan histidine kinase (CPR1055) did not significantly affect sporulation or CPE production. Studies using a spoIIA operon promoter-driven reporter plasmid indicated that CPR0195 functions early during sporulation, i.e., prior to production of sporulation-associated sigma factors. Furthermore, in vitro studies showed that the CPR0195 kinase domain can autophosphorylate and phosphorylate Spo0A. These results support the idea of CPR0195 as an important kinase that initiates C. perfringens sporulation by directly phosphorylating Spo0A. This kinase could represent a novel therapeutic target to block C. perfringens sporulation and CPE production during type F disease.IMPORTANCEClostridium perfringens type F enteric diseases, which include a very common form of food poisoning and many cases of antibiotic-associated diarrhea, develop when type F strains sporulate and produce C. perfringens enterotoxin (CPE) in the intestines. Spores are also important for transmission of type F disease. Despite the importance of sporulation for type F disease and the evidence that C. perfringens sporulation begins with phosphorylation of the Spo0A transcriptional regulator, the kinase phosphorylating Spo0A to initiate sporulation and CPE production had not been ascertained. In response, the current report now provides identification of an orphan histidine kinase named CPR0195 that can directly phosphorylate Spo0A. Results using a CPR0195 null mutant indicate that this kinase is very important for initiating C. perfringens sporulation and CPE production. Therefore, the CPR0195 kinase represents a potential target to block type F disease by interfering with intestinal C. perfringens sporulation and CPE production.

Immunoactive Clostridial Membrane Vesicle Production Is Regulated by a Sporulation Factor.

Recently, many Gram-positive bacteria as well as Gram-negative bacteria have been reported to produce membrane vesicles (MVs), but little is known regarding the regulators involved in MV formation. We found that a Gram-positive anaerobic pathogen, Clostridium perfringens, produces MVs predominantly containing membrane proteins and cell wall components. These MVs stimulated proinflammatory cytokine production in mouse macrophage-like cells. We suggested that MVs induced interleukin-6 production through the Toll-like receptor 2 (TLR2) signaling pathway. Thus, the MV could have a role in the bacterium-host interaction and bacterial infection pathogenesis. Moreover, we found that the sporulation master regulator gene spo0A was required for vesiculogenesis. A conserved, phosphorylated aspartate residue of Spo0A was indispensable for MV production, suggesting that the phosphorylation of Spo0A triggers MV production. Multiple orphan sensor kinases necessary for sporulation were also required to maximize MV production. These findings imply that C. perfringens actively produces immunoactive MVs in response to the environment changing, as recognized by membrane-spanning sensor kinases and by modulating the phosphorylation level of Spo0A.

A protein complex supports the production of Spo0A-P and plays additional roles for biofilms and the K-state in Bacillus subtilis.

Bacillus subtilis can enter three developmental pathways to form spores, biofilms or K-state cells. The K-state confers competence for transformation and antibiotic tolerance. Transition into each of these states requires a stable protein complex formed by YlbF, YmcA and YaaT. We have reported that this complex acts in sporulation by accelerating the phosphorylation of the response regulator Spo0A. Phosphorelay acceleration was also predicted to explain their involvement in biofilm formation and the K-state. This view has been challenged in the case of biofilms, by the suggestion that the three proteins act in association with the mRNA degradation protein RNaseY (Rny) to destabilize the sinR transcript. Here, we reaffirm the roles of the three proteins in supporting the phosphorylation of Spo0A for all three developmental pathways and show that in their absence sinR mRNA is not stabilized. We demonstrate that the three proteins also play unknown Spo0A-P-independent roles in the expression of biofilm matrix and in the production of ComK, the master transcription factor for competence. Finally, we show that domesticated strains of B. subtilis carry a mutation in sigH, which influences the expression kinetics of the early spore gene spoIIG, thereby increasing the penetrance of the ylbF, ymcA and yaaT sporulation phenotypes.

Transcriptional Profile during Deoxycholate-Induced Sporulation in a Clostridium perfringens Isolate Causing Foodborne Illness.

Clostridium perfringens type A is a common source of foodborne illness (FBI) in humans. Vegetative cells sporulate in the small intestinal tract and produce the major pathogenic factor C. perfringens enterotoxin. Although sporulation plays a critical role in the pathogenesis of FBI, the mechanisms inducing sporulation remain unclear. Bile salts were shown previously to induce sporulation, and we confirmed deoxycholate (DCA)-induced sporulation in C. perfringens strain NCTC8239 cocultured with human intestinal epithelial Caco-2 cells. In the present study, we performed transcriptome analyses of strain NCTC8239 in order to elucidate the mechanism underlying DCA-induced sporulation. Of the 2,761 genes analyzed, 333 were up- or downregulated during DCA-induced sporulation and included genes for cell division, nutrient metabolism, signal transduction, and defense mechanisms. In contrast, the virulence-associated transcriptional regulators (the VirR/VirS system, the agr system, codY, and abrB) were not activated by DCA. DCA markedly increased the expression of signaling molecules controlled by Spo0A, the master regulator of the sporulation process, whereas the expression of spo0A itself was not altered in the presence or absence of DCA. The phosphorylation of Spo0A was enhanced in the presence of DCA. Collectively, these results demonstrated that DCA induced sporulation, at least partially, by facilitating the phosphorylation of Spo0A and activating Spo0A-regulated genes in strain NCTC8239 while altering the expression of various genes. Disease caused by Clostridium perfringens type A consistently ranks among the most common bacterial foodborne illnesses in humans in developed countries. The sporulation of C. perfringens in the small intestinal tract is a key event for its pathogenesis, but the factors and underlying mechanisms by which C. perfringens sporulates in vivo currently remain unclear. Bile salts, major components of bile, which is secreted from the liver for the emulsification of lipids, were shown to induce sporulation. However, the mechanisms underlying bile salt-induced sporulation have not yet been clarified. In the present study, we demonstrate that deoxycholate (one of the bile salts) induces sporulation by facilitating the phosphorylation of Spo0A and activating Spo0A-regulated genes using a transcriptome analysis. Thus, this study enhances our understanding of the mechanisms underlying sporulation, particularly that of bile salt-induced sporulation, in C. perfringens.

Sporulation during growth in a gut isolate of Bacillus subtilis.

Sporulation by Bacillus subtilis is a cell density-dependent response to nutrient deprivation. Central to the decision of entering sporulation is a phosphorelay, through which sensor kinases promote phosphorylation of Spo0A. The phosphorelay integrates both positive and negative signals, ensuring that sporulation, a time- and energy-consuming process that may bring an ecological cost, is only triggered should other adaptations fail. Here we report that a gastrointestinal isolate of B. subtilis sporulates with high efficiency during growth, bypassing the cell density, nutritional, and other signals that normally make sporulation a post-exponential-phase response. Sporulation during growth occurs because Spo0A is more active per cell and in a higher fraction of the population than in a laboratory strain. This in turn, is primarily caused by the absence from the gut strain of the genes rapE and rapK, coding for two aspartyl phosphatases that negatively modulate the flow of phosphoryl groups to Spo0A. We show, in line with recent results, that activation of Spo0A through the phosphorelay is the limiting step for sporulation initiation in the gut strain. Our results further suggest that the phosphorelay is tuned to favor sporulation during growth in gastrointestinal B. subtilis isolates, presumably as a form of survival and/or propagation in the gut environment.

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.

Phosphorylation of Spo0A by the Histidine Kinase KinD Requires the Lipoprotein Med in Bacillus subtilis

The response regulatory protein Spo0A of Bacillus subtilis is activated by phosphorylation by multiple histidine kinases via a multicomponent phosphorelay. Here we present evidence that the activity of one of the kinases, KinD, depends on the lipoprotein Med, a mutant of which has been known to cause a cannibalism phenotype. We show that the absence of Med impaired and the overproduction of Med stimulated the transcription of two operons (sdp and skf) involved in cannibalism whose transcription is known to depend on Spo0A in its phosphorylated state (Spo0A∼P). Further, these effects of Med were dependent on KinD but not on kinases KinA, KinB, and KinC. Additionally, we show that deletion or overproduction of Med impaired or enhanced, respectively, biofilm formation and that these effects, too, depended specifically on KinD. Finally, we report that overproduction of Med bypassed the dominant negative effect on transcription of sdp of a truncated KinD retaining the transmembrane segments but lacking the kinase domain. We propose that Med directly or indirectly interacts with KinD in the cytoplasmic membrane and that this interaction is required for KinD-dependent phosphorylation of Spo0A.

MecA dampens transitions to spore, biofilm exopolysaccharide and competence expression by two different mechanisms

The adapter protein MecA targets the transcription factor ComK for degradation by the ClpC/ClpP proteolytic complex, thereby negatively regulating competence in Bacillus subtilis. Here we show that MecA also decreases the frequency of transitions to the sporulation pathway as well as the expression of eps, which encodes synthesis of the biofilm matrix exopolysaccharide. We present genetic and biophysical evidence that MecA downregulates eps expression and spore formation by directly interacting with Spo0A. MecA does not target Spo0A for degradation, and apparently does not prevent the phosphorylation of Spo0A. We propose that it inhibits the transcriptional activity of Spo0A∼P by direct binding. Thus, in its interaction with Spo0A, MecA differs from its role in the regulation of competence where it targets ComK for degradation. MecA acts as a general buffering protein for development, acting by two distinct mechanisms to regulate inappropriate transitions to energy-intensive pathways.

Ancestral sporulation initiation

Members of the classes Bacilli and Clostridia are able to form endospores, with clostridia representing the ancestral phylogenetic line. In contrast to Bacillus subtilis, the process of sporulation initiation at the molecular level is less well understood in Clostridium acetobutylicum, the model organism of the clostridia. Especially the question of how the master regulator of sporulation, Spo0A, becomes phosphorylated, remained unsolved so far. Steiner et al. now provide compelling genetic and biochemical evidence for two different pathways of direct phosphorylation of Spo0A in C. acetobutylicum by three orphan sensor kinases. Cac0903 and Cac3319 act together, while the other pathway is only dependent on Cac0323. Abortion of sporulation initiation can be achieved by a kinase-like protein, Cac0437.

Multiple orphan histidine kinases interact directly with Spo0A to control the initiation of endospore formation in Clostridium acetobutylicum

The phosphorylated Spo0A transcription factor controls the initiation of endospore formation in Clostridium acetobutylicum, but genes encoding key phosphorelay components, Spo0F and Spo0B, are missing in the genome. We hypothesized that the five orphan histidine kinases of C. acetobutylicum interact directly with Spo0A to control its phosphorylation state. Sequential targeted gene disruption and gene expression profiling provided evidence for two pathways for Spo0A activation, one dependent on a histidine kinase encoded by cac0323, the other on both histidine kinases encoded by cac0903 and cac3319. Purified Cac0903 and Cac3319 kinases autophosphorylated and transferred phosphoryl groups to Spo0A in vitro, confirming their role in Spo0A activation in vivo. A cac0437 mutant hyper-sporulated, suggesting that Cac0437 is a modulator that prevents sporulation and maintains cellular Spo0A∼P homeostasis during growth. Accordingly, Cac0437 has apparently lost the ability to autophosphorylate in vitro; instead it catalyses the ATP-dependent dephosphorylation of Spo0A∼P releasing inorganic phosphate. Direct phosphorylation of Spo0A by histidine kinases and dephosphorylation by kinase-like proteins may be a common feature of the clostridia that may represent the ancestral state before the great oxygen event some 2.4 billion years ago, after which additional phosphorelay proteins were recruited in the evolutionary lineage that led to the bacilli.

Broadly heterogeneous activation of the master regulator for sporulation in Bacillus subtilis

A model system for investigating how developmental regulatory networks determine cell fate is spore formation in Bacillus subtilis. The master regulator for sporulation is Spo0A, which is activated by phosphorylation via a phosphorelay that is subject to three positive feedback loops. The ultimate decision to sporulate is, however, stochastic in that only a portion of the population sporulates even under optimal conditions. It was previously assumed that activation of Spo0A and hence entry into sporulation is subject to a bistable switch mediated by one or more feedback loops. Here we reinvestigate the basis for bimodality in sporulation. We show that none of the feedback loops is rate limiting for the synthesis and phosphorylation of Spo0A. Instead, the loops ensure a just-in-time supply of relay components for rising levels of phosphorylated Spo0A, with phosphate flux through the relay being limiting for Spo0A activation and sporulation. In addition, genes under Spo0A control did not exhibit a bimodal pattern of expression as expected for a bistable switch. In contrast, we observed a highly heterogeneous pattern of Spo0A activation that increased in a nonlinear manner with time. We present a computational model for the nonlinear increase and propose that the phosphorelay is a noise generator and that only cells that attain a threshold level of phosphorylated Spo0A sporulate.

The Spo0E phosphatase of Bacillus subtilis is a substrate of the FtsH metalloprotease

In the absence of the ATP-dependent metalloprotease FtsH, the sporulation frequency of Bacillus subtilis cells is reduced by several orders of magnitude. This indicates that FtsH has to degrade or to regulate the steady-state level of one or more proteins that interfere with successful sporulation. Here, we show that the amount of the master regulator protein Spo0A is reduced in an ftsH knockout and the small amounts of Spo0A protein present are inactive. Phosphorylation of Spo0A occurs through a phosphorelay. Four negative regulators have been identified here which directly interfere with the phosphorelay through ftsH, namely the phosphatases RapA, RapB, RapE and Spo0E. If a null allele in any one of them was combined with an ftsH knockout, the sporulation frequency was increased by two to three orders of magnitude, but remained below 1 %. When purified Spo0E was incubated with FtsH, partial degradation of the phosphatase was observed. In contrast, two mutant versions of Spo0E with truncated C-termini remained stable. Transfer of the C-terminal 25 aa of Spo0E to a shorter homologue of Spo0E, YnzD, which is not a substrate of FtsH, conferred instability. When a mutant Spo0A was produced that was active in the absence of phosphorylation, spores were formed at a normal rate in an ftsH knockout, indicating that ftsH is needed only during phase 0.

DisA, a Busy Bee That Monitors Chromosome Integrity

When nutrients are limited many prokaryotic and some eukaryotic cells tuck their chromosomes safely away in resistant spores. However, before starting the sporulation process, the prokaryote Bacillus subtilis wisely ensures that its chromosome is intact. In this issue of Cell, Bejerano-Sagie et al. (2006) describe a protein, DisA, that is responsible for the surveillance of chromosome integrity during sporulation.

Control of sporulation gene expression in Bacillus subtilis by the chromosome partitioning proteins Soj (ParA) and Spo0J (ParB).

Two chromosome partitioning proteins, Soj (ParA) and Spo0J (ParB), regulate the initiation of sporulation in Bacillus subtilis. In a spo0J null mutant, sporulation is inhibited by the action of Soj. Soj negatively regulates expression of several sporulation genes by binding to the promoter regions and inhibiting transcription. All of the genes known to be inhibited by Soj are also activated by the phosphorylated form of the transcription factor Spo0A (Spo0A approximately P). We found that, in a spo0J null mutant, Soj affected sporulation, in part, by decreasing the level of Spo0A protein. Soj negatively regulated transcription of spo0A and associated with the spo0A promoter region in vivo. Expression of spo0A from a heterologous promoter in a spo0J null mutant restored Spo0A levels and partly bypassed the sporulation and gene expression defects. Soj did not appear to significantly affect phosphorylation of Spo0A. Thus, in the absence of Spo0J, Soj inhibits sporulation and sporulation gene expression by inhibiting accumulation of the activator protein Spo0A and by acting downstream of Spo0A to inhibit gene expression directly.

SigmaK can negatively regulate sigE expression by two different mechanisms during sporulation of Bacillus subtilis.

Temporal and spatial gene regulation during Bacillus subtilis sporulation involves the activation and inactivation of multiple sigma subunits of RNA polymerase in a cascade. In the mother cell compartment of sporulating cells, expression of the sigE gene, encoding the earlier-acting sigma factor, sigmaE, is negatively regulated by the later-acting sigma factor, sigmaK. Here, it is shown that the negative feedback loop does not require SinR, an inhibitor of sigE transcription. Production of sigmaK about 1 h earlier than normal does affect Spo0A, which when phosphorylated is an activator of sigE transcription. A mutation in the spo0A gene, which bypasses the phosphorelay leading to the phosphorylation of Spo0A, diminished the negative effect of early sigmaK production on sigE expression early in sporulation. Also, early production of sigmaK reduced expression of other Spo0A-dependent genes but not expression of the Spo0A-independent ald gene. In contrast, both sigE and ald were overexpressed late in development of cells that fail to make sigmaK. The ald promoter, like the sigE promoter, is believed to be recognized by sigmaA RNA polymerase, suggesting that sigmaK may inhibit sigmaA activity late in sporulation. To exert this negative effect, sigmaK must be transcriptionally active. A mutant form of sigmaK that associates with core RNA polymerase, but does not direct transcription of a sigmaK-dependent gene, failed to negatively regulate expression of sigE or ald late in development. On the other hand, the negative effect of early sigmaK production on sigE expression early in sporulation did not require transcriptional activity of sigmaK RNA polymerase. These results demonstrate that sigmaK can negatively regulate sigE expression by two different mechanisms, one observed when sigmaK is produced earlier than normal, which does not require sigmaK to be transcriptionally active and affects Spo0A, and the other observed when sigmaK is produced at the normal time, which requires sigmaK RNA polymerase transcriptional activity. The latter mechanism facilitates the switch from sigmaE to sigmaK in the cascade controlling mother cell gene expression.

The Spo0A sof mutations reveal regions of the regulatory domain that interact with a sensor kinase and RNA polymerase.

Spo0A is a two-domain response regulator required for the initiation of sporulation in Bacillus subtilis. Spo0A is activated by phosphorylation of its regulatory domain by a multicomponent phosphorelay. To define the role of the regulatory domain in the activation of Spo0A, we have characterized four of the sof mutations in vitro. The sof mutations were identified previously as suppressors of the sporulation-negative phenotype resulting from a deletion of the gene for one of the phosphorelay components, spo0F. Like wild-type Spo0A, the transcription stimulation properties of all of the Sof proteins were dependent upon phosphorylation. Sof mutants from two classes were improved substrates for direct phosphorylation by the KinA sensor kinase, providing an explanation for their suppression properties. Two other Sof proteins showed a phosphorylation-dependent enhancement of the stability of the Sof approximately P-RNA polymerase-DNA complex. One of these mutants, Sof114, increased the stability of the Sof114 approximately P-RNAP-DNA complex without increasing its own affinity for the spoIIG promoter. A comparison of the location of the sof mutations with mutations in CheY suggests that phosphorylation of Spo0A results in the exposure of a region in the regulatory domain that interacts with RNA polymerase, thereby contributing to the signal transduction mechanism.

The Bacillus subtilisResponse Regulator Spo0A Stimulates Transcription of the spoIIGOperon through Modification of RNA Polymerase Promoter Complexes

Sporulation in Bacillus subtilisis dependent on the response regulator Spo0A, which both represses and activates transcription in vitro. The activity of Spo0A is increased by phosphorylation. We previously demonstrated that the phosphorylation increased the ability of Spo0A to stimulate in vivotranscription from the promoter for the spoIIGoperon, one of the operons known to be regulated by Spo0A in vivo. In the work reported here we have examined the kinetics of transcription initiation at the spoIIGoperon promoter using a single round transcription assay and the kinetics of formation of spoIIGpromoter – RNA polymerase complexes using DNase I footprinting. Both the kinetic assays and the footprint assays indicated that the initial binding of the polymerase to the template was not dependent on the presence of Spo0A. The phosphorylated form of Spo0A stimulated the rate of initiation by affecting a step that occurred after the initial interaction of the polymerase with the template. Phosphorylation of Spo0A may stimulate transcription by modifying preinitiation complexes containing the polymerase and the promoter.