Relative levels and fractionation properties of Bacillus subtilis sigma(B) and its regulators during balanced growth and stress.

sigma B is a secondary sigma factor that controls the general stress response in Bacillus subtilis. sigma B-dependent genes are activated when sigma B is released from an inhibitory complex with an anti-sigma B protein (RsbW) and becomes free to associate with RNA polymerase. Two separate pathways, responding either to a drop in intracellular ATP levels or to environmental stress (e.g., heat, ethanol, or salt), cause the release of sigma B from RsbW. rsbR, rsbS, rsbT, and rsbU are four genes now recognized as the upstream half of an operon that includes sigB (sigma B) and its principal regulators. Using reporter gene assays, we find that none of these four genes are essential for stationary-phase (i.e., ATP-dependent) activation of sigma B, but rsbU and one or more of the genes contained within an rsbR,S,T deletion are needed for stress induction of sigma B. In other experiments, Western blot (immunoblot) analyses showed that the levels of RsbR, RsbS, Rsb, and RsbU, unlike those of the sigB operon's four downstream gene products (RsbV, RsbW, RsbX and sigma B), are not elevated during sigma B activation. Gel filtration and immunoprecipitation studies did not reveal the formation of complexes between any of the four upstream sigB operon products and the products of the downstream half of the operon. Much of the detectable RsbR, RsbS, RsbT, and RsbU did, however, fractionate as a large-molecular-mass (approximately 600-kDa) aggregate which was excluded from our gel filtration matrix. The downstream sigB operon products were not present in this excluded material. The unaggregated RsbR, RsbS, and RsbU, which were retarded by the gel matrix, elated from the column earlier than expected from their molecular weights. The RsbR and RsbS fractionation profile was consistent with homodimers (60 and 30 kDa, respectively), while the RsbU appeared larger, suggesting a protein complex of approximately 90 to 100 kDa.     

Obg, an essential GTP binding protein of Bacillus subtilis, is necessary for stress activation of transcription factor sigma(B).

sigma(B), the general stress response sigma factor of Bacillus subtilis, is activated when intracellular ATP levels fall or the bacterium experiences environmental stress. Stress activates sigma(B) by means of a collection of regulatory kinases and phosphatases (the Rsb proteins), which catalyze the release of sigma(B) from an anti-sigma factor inhibitor. By using the yeast dihybrid selection system to identify B. subtilis proteins that could interact with Rsb proteins and act as mediators of stress signaling, we isolated the GTP binding protein, Obg, as an interactor with several of these regulators (RsbT, RsbW, and RsbX). B. subtilis depleted of Obg no longer activated sigma(B) in response to environmental stress, but it retained the ability to activate sigma(B) by the ATP responsive pathway. Stress pathway components activated sigma(B) in the absence of Obg if the pathway's most upstream effector (RsbT) was synthesized in excess to the inhibitor (RsbS) from which it is normally released after stress. Thus, the Rsb proteins can function in the absence of Obg but fail to be triggered by stress. The data demonstrate that Obg, or a process under its control, is necessary to induce the stress-dependent activation of sigma(B) and suggest that Obg may directly communicate with one or more sigma(B) regulators.     

Mutational analysis of RsbT, an activator of the Bacillus subtilis stress response transcription factor, sigmaB

SigmaB, the stress-activated sigma factor of Bacillus subtilis, requires the RsbT protein as an essential positive regulator of its physical stress pathway. Stress triggers RsbT to both inactivate the principal negative regulator of the physical stress pathway (RsbS) by phosphorylation and activate a phosphatase (RsbU) required for sigmaB induction. Neither the regions of RsbT that are involved in responding to stress signaling nor those required for downstream events have been established. We used alanine scanning mutagenesis to examine the contributions of RsbT's charged amino acids to the protein's stability and activities. Eleven of eighteen rsbT mutations blocked sigmaB induction by stress. The carboxy terminus of RsbT proved to be particularly important for accumulation in Bacillus subtilis. Four of the five most carboxy-terminal mutations yielded rsbT alleles whose products were undetectable in B. subtilis extracts. Charged amino acids in the central region of RsbT were less critical, with four of the five substitutions in this region having no measurable effect on RsbT accumulation or activity. Only when the substitutions extended into a region of kinase homology was sigmaB induction affected. Six other RsbT variants, although present at levels adequate for activity, failed to activate sigmaB and displayed significant changes in their ability to interact with RsbT's normal binding partners in a yeast dihybrid assay. These changes either dramatically altered the proteins' tertiary structure without affecting their stability or defined regions of RsbT that are involved in multiple interactions.     

Phosphorylation and RsbX-dependent dephosphorylation of RsbR in the RsbR-RsbS complex of Bacillus subtilis

In the pathway that controls sigmaB activity, the RsbR-RsbS complex plays an important role by trapping RsbT, a positive regulator of sigmaB of Bacillus subtilis. We have proposed that at the onset of stress, RsbR becomes phosphorylated, resulting in an enhanced activity of RsbT towards RsbS. RsbT is then free to interact with and activate RsbU, which in turn ultimately activates sigmaB. In this study with purified proteins, we used mutant RsbR proteins to analyze the role of its phosphorylatable threonine residues. The results show that the phosphorylation of either of the two RsbT-phosphorylatable threonine residues (T171 and T205) in RsbR enhanced the kinase activity of RsbT towards RsbS. However, it appeared that RsbT preferentially phosphorylates T171. We also present in vitro evidence that identifies RsbX as a potential phosphatase for RsbR T205.     

Serine kinase activity of a Bacillus subtilis switch protein is required to transduce environmental stress signals but not to activate its target PP2C phosphatase

The RsbT serine kinase has two known functions in the signal transduction pathway that activates the general stress factor σ^B of Bacillus subtilis . First, RsbT can phosphorylate and inactivate its specific antagonist protein, RsbS. Second, upon phosphorylation of RsbS, RsbT is released to stimulate RsbU, a PP2C phosphatase, thereby initiating a signalling cascade that ultimately activates σ^B. Here we describe a mutation that separates these two functions of RsbT. Although the mutant RsbT protein had essentially no kinase activity, it still retained the capacity to stimulate the RsbU phosphatase in vitro and to activate σ^B when overexpressed in vivo . These results support the hypothesis that phosphatase activation is accomplished via a long-lived interaction between RsbT and RsbU. In contrast, RsbT kinase activity was found to be integral for the transmission of external stimuli to σ^B. Thus, one route by which environmental stress signals could enter the σ^B network is by modulation of the RsbT kinase activity, thereby controlling the magnitude of the partner switch between the RsbS–RsbT complex and the RsbT–RsbU complex.     

The yeast two-hybrid system detects interactions between Bacillus subtilis sigmaB regulators.

SigmaB, the general stress response sigma factor of Bacillus subtilis, is regulated by the products of seven genes (rsbR, S, T, U, V, W, and X) with which it is cotranscribed. Biochemical techniques previously revealed physical associations among RsbW, RsbV, and sigmaB but failed to detect interactions of RsbR, S, T, U, or X with each other or RsbV, RsbW, or sigmaB. Using the yeast two-hybrid system, we have now obtained evidence for such interactions. The yeast reporter system was activated when RsbS was paired with either RsbR or RsbT, RsbR was paired with RsbT, and RsbV was paired with either RsbU or RsbW. In addition, RsbW2 and RsbR2 dimer formation was detected. RsbX failed to show interactions with itself or any of the other sigB operon products.     

A supramolecular complex in the environmental stress signalling pathway of Bacillus subtilis

SigmaB, an alternative sigma-factor of Bacillus subtilis, mediates the response of the cell to a variety of physical insults. Within the environmental stress signalling pathway RsbU, a protein phosphatase, is stimulated by its interaction with the protein kinase RsbT. In the absence of stress RsbT is expected to be trapped by an alternative binding partner, RsbS. Here, we have demonstrated that RsbS alone cannot act as an alternative partner for RsbT, but instead requires the presence of RsbR to create a high molecular mass RsbR:RsbS complex (approximately 1 MDa) able to capture RsbT. In this complex the phosphorylation state of RsbS, and not that of RsbR, controlled the binding to RsbT, whose kinase activity towards RsbS could be counterbalanced by the activity of RsbX, the phosphatase for RsbS-P. The RsbR:RsbS complex recruited RsbT from a mixture of RsbT and RsbU. The phosphorylated form of RsbR in the complex enhanced the kinase activity of RsbT towards RsbS. This supramolecular complex thus has the functional properties of an alternative partner for RsbT. Electron micrographs of this complex are presented, and the purification of the RsbR:RsbS complex from cellular extracts provides evidence for the existence of such a complex in vivo.     

The RsbRST stress module in bacteria: a signalling system that may interact with different output modules

In the Gram-positive bacterium Bacillus subtilis, the activity of the alternative sigma factor sigma(B) is triggered upon exposure of the bacteria to environmental stress conditions or to nutrient limitation. sigma(B) activity is controlled by protein-phosphorylation-dependent interactions of anti-sigma with anti-anti-sigma factors. Under stress conditions, the phosphatase RsbU triggers release of sigma(B) and thus induces the expression of stress genes. RsbU activity is controlled by three proteins, RsbR, RsbS and RsbT which form a supramolecular complex called the stressosome. Here we review the occurrence of the genes encoding the stressosome proteins (called the RsbRST module) in a wide variety of bacteria. While this module is linked to the gene encoding sigma(B) and its direct regulators in B. subtilis and its close relatives, genes encoding two-component regulatory systems and more complex phosphorelays are clustered with the RsbRST module in bacteria as diverse as cyanobacteria, bacteroidetes, proteobacteria, and deinococci. The conservation of the RsbRST module and its clustering with different types of regulatory systems suggest that the stressosome proteins form a signal sensing and transduction unit that relays information to very different output modules.     

Stress activation of Bacillus subtilis sigma B can occur in the absence of the sigma B negative regulator RsbX.

Environmental stress activates sigma B, the general stress response sigma factor of Bacillus subtilis, by a pathway that is negatively controlled by the RsbX protein. To determine whether stress activation of sigma B occurs by a direct effect of stress on RsbX, we constructed B. subtilis strains which synthesized various amounts of RsbX or lacked RsbX entirely and subjected these strains to ethanol stress. Based on the induction of a sigma B-dependent promoter, stress activation of sigma B can occur in the absence of RsbX. Higher levels of RsbX failed to detectably influence stress induction, but reduced levels of RsbX resulted in greater and longer-lived sigma B activation. The data suggest that RsbX is not a direct participant in the sigma B stress induction process but rather serves as a device to limit the magnitude of the stress response.     

Phenotypes of Bacillus subtilis mutants altered in the precursor-specific region of sigma E.

sigma E is a sporulation-specific sigma factor of Bacillus subtilis that is synthesized from an inactive precursor protein (P31). The structural gene (sigE) for P31 was reengineered by oligonucleotide-directed mutagenesis to encode sigma E directly. The sequence specifying the first amino acid of sigma E (GGC) was placed immediately downstream of the initiating codon (ATG) of P31. The resulting sigE allele (sigE delta 84) encodes a sigma E-like protein which differs from the "processed product" by a single Met residue at its amino terminus. B. subtilis strains which carried this allele were Spo- and contained no detectable sigma E. The sigE delta 84 allele generated a product in Escherichia coli which, by quantitative Western immunoblot analysis, was present at 10 to 20% of the level of product (P31) obtained from a wild-type allele. A sigma E-like product was also not detected in two B. subtilis strains with missense mutations in the sequence encoding the processed region of P31. These results suggest that sigma E is a highly labile protein that is stabilized during its synthesis by an element of the precursor sequence. A mutant allele (sigE delta 48) which made an active sigma E-like protein in B. subtilis was isolated. This gene specified a product in which five amino acids, not derived from the P31 processed region, were joined to P31 at a position eight amino acids upstream of the processing site. The sigE delta 48 product was not processed, but it activated the sigma E -dependent spoIID promoter in vivo. The sigE delta 48 product therefore lost both an essential target for processing and a region which inhibited sigma sigma E activity. Cells which carried sig E delta 48 were Spo-. The basis of the sigE delta 48-dependent defect in sporulation is unknown, but the sigma E delta 48 activity appeared to persist beyond the time in development (4 h after onset sporulation) when wild-type sigma E activity declines. Thus, it may interfere with the proper regulation of late sporulation genes.