The growth-promoting and stress response activities of the Bacillus subtilis GTP binding protein Obg are separable by mutation.

Bacillus subtilis Obg is a ribosome-associating GTP binding protein that is needed for growth, sporulation, and induction of the bacterium's general stress regulon (GSR). It is unclear whether the roles of Obg in sporulation and stress responsiveness are direct or a secondary effect of its growth-promoting functions. The present work addresses this question by an analysis of two obg alleles whose phenotypes argue for direct roles for Obg in each process. The first allele [obg(G92D)] encodes a missense change in the protein's highly conserved "obg fold" region. This mutation impairs cell growth and the ability of Obg to associate with ribosomes but fails to block sporulation or the induction of the GSR. The second obg mutation [obg(Delta22)] replaces the 22-amino-acid carboxy-terminal sequence of Obg with an alternative 26-amino-acid sequence. This Obg variant cofractionates with ribosomes and allows normal growth but blocks sporulation and impairs the induction of the GSR. Additional experiments revealed that the block on sporulation occurs early, preventing the activation of the essential sporulation transcription factor Spo0A, while inhibition of the GSR appears to involve a failure of the protein cascade that normally activates the GSR to effectively catalyze the reactions needed to activate the GSR transcription factor (sigma(B)).

ClpP modulates the activity of the Bacillus subtilis stress response transcription factor, sigmaB.

The general stress regulon of Bacillus subtilis is controlled by the activity state of sigmaB, a transcription factor that is switched on following exposure to either physical or nutritional stress. ClpP is the proteolytic component of an ATP-dependent protease that is essential for the proper regulation of multiple adaptive responses in B. subtilis. Among the proteins whose abundance increases in ClpP- B. subtilis are several known to depend on sigmaB for their expression. In the current work we examine the relationship of ClpP to the activity of sigmaB. The data reveal that the loss of ClpP in otherwise wild-type B. subtilis results in a small increase in sigmaB activity during growth and a marked enhancement of sigmaB activity following its induction by either physical or nutritional stress. It appears to be the persistence of sigmaB's activity rather than its induction that is principally affected by the loss of ClpP. sigmaB-dependent reporter gene activity rose in parallel in ClpP+ and ClpP- B. subtilis strains but failed to display its normal transience in the ClpP- strain. The putative ClpP targets are likely to be stress generated and novel. Enhanced sigmaB activity in ClpP- B. subtilis was triggered by physical stress but not by the induced synthesis of the physical stress pathway's positive regulator (RsbT). In addition, Western blot analyses failed to detect differences in the levels of the principal known sigmaB regulators in ClpP+ and ClpP- B. subtilis strains. The data suggest a model in which ClpP facilitates the turnover of stress-generated factors, which persist in ClpP's absence to stimulate ongoing sigmaB activity.

Isolation and characterization of dominant mutations in the Bacillus subtilis stressosome components RsbR and RsbS.

The general stress response of Bacillus subtilis is controlled by the activity state of the sigma(B) transcription factor. Physical stress is communicated to sigma(B) via a large-molecular-mass (>10(6)-Da) structure (the stressosome) formed by one or more members of a family of homologous proteins (RsbR, YkoB, YojH, YqhA). The positive regulator (RsbT) of the sigma(B) stress induction pathway is incorporated into the complex bound to an inhibitor protein (RsbS). Exposure to stress empowers an RsbT-dependent phosphorylation of RsbR and RsbS, with the subsequent release of RsbT to activate downstream processes. The mechanism by which stress initiates these reactions is unknown. In an attempt to identify changes in stressosome components that could lead to sigma(B) activation, a DNA segment encoding these proteins was mutagenized and placed into B. subtilis to create a merodiploid strain for these genes. Eight mutations that allowed heightened sigma(B) activity in the presence of their wild-type counterparts were isolated. Two of the mutations are missense changes in rsbR, and six are amino acid changes in rsbS. Additional experiments suggested that both of the rsbR mutations and three of the rsbS mutations likely enhance sigma(B) activity by elevating the level of RsbS phosphorylation. All of the mutations were found to be dominant over wild-type alleles only when they are cotranscribed within an rsbR rsbS rsbT operon. The data suggest that changes in RsbR can initiate the downstream events that lead to sigma(B) activation and that RsbR, RsbS, and RsbT likely interact with each other concomitantly with their synthesis.

In vivo random mutagenesis of Bacillus subtilis by use of TnYLB-1, a mariner-based transposon.

This report describes the construction and characterization of a mariner-based transposon system designed to be used in Bacillus subtilis, but potentially applicable to other gram-positive bacteria. Two pUC19-derived plasmids were created that contain the mariner-Himar1 transposase gene, modified for expression in B. subtilis, under the control of either sigmaA- or sigmaB-dependent promoters. Both plasmids also contain a transposable element (TnYLB-1) consisting of a Kan r cassette bracketed by the Himar1-recognized inverse terminal repeats, as well as the temperature-sensitive replicon and Erm r gene of pE194ts. TnYLB-1 transposes into the B. subtilis chromosome with high frequency (10(-2)) from either plasmid. Southern hybridization analyses of 15 transposants and sequence analyses of the insertion sites of 10 of these are consistent with random transposition, requiring only a "TA" dinucleotide as the essential target in the recipient DNA. Two hundred transposants screened for sporulation proficiency and auxotrophy yielded five Spo- clones, three with insertions in known sporulation genes (kinA, spoVT, and yqfD) and two in genes (ybaN and yubB) with unknown functions. Two auxotrophic mutants were identified among the 200 transposants, one with an insertion in lysA and another in a gene (yjzB) whose function is unknown.

Coexpression patterns of sigma(B) regulators in Bacillus subtilis affect sigma(B) inducibility.

RsbT is an essential component of the pathway that activates the Bacillus subtilis sigma(B) transcription factor in response to physical stress. rsbT is located within an operon that includes the genes for its principal negative regulator (RsbS) and the stress pathway component that it activates (RsbU), as immediate upstream and downstream neighbors. In the current work we demonstrate that RsbT's ability to function is strongly influenced by coexpression with these adjoining genes. When rsbT is expressed at a site displaced from rsbS and rsbU, RsbT accumulates but it is unable to activate sigma(B) following stress. RsbT activity is restored if rsbT is cotranscribed at the alternative site with the genes that normally abut it. Additionally, an rsbS allele whose product allows constitutively high RsbT-dependent sigma(B) activity displays this activity in rsbS merodiploid strains only when cotranscribed with rsbT and is recessive to a wild-type rsbS allele only if the wild-type rsbS gene is not cotranscribed with an rsbT gene of its own. The data suggest that RsbS and RsbT are synthesized in equivalent amounts and interact coincidently with their synthesis to form stable regulatory complexes that maintain RsbT in a state from which it can be stress activated.

Contributions of ATP, GTP, and redox state to nutritional stress activation of the Bacillus subtilis sigmaB transcription factor.

The general stress regulon of Bacillus subtilis is induced by activation of the sigma(B) transcription factor. sigma(B) activation occurs when one of two phosphatases responds to physical or nutritional stress to activate a positive sigma(B) regulator by dephosphorylation. The signal that triggers the nutritional stress phosphatase (RsbP) is unknown; however, RsbP activation occurs under culture conditions (glucose/phosphate starvation, azide or decoyinine treatment) that reduce the cell's levels of ATP and/or GTP. Variances in nucleotide levels in these instances may be coincidental rather than causal. RsbP carries a domain (PAS) that in some regulatory systems can respond directly to changes in electron transport, proton motive force, or redox potential, changes that typically precede shifts in high-energy nucleotide levels. The current work uses Bacillus subtilis with mutations in the oxidative phosphorylation and purine nucleotide biosynthetic pathways in conjunction with metabolic inhibitors to better define the inducing signal for RsbP activation. The data argue that a drop in ATP, rather than changes in GTP, proton motive force, or redox state, is the key to triggering sigma(B) activation.

Associations between Bacillus subtilis sigmaB regulators in cell extracts.

The general stress regulon of Bacillus subtilis is induced by the activation of the sigma(B) transcription factor. Activation of sigma(B) occurs as a consequence of the dephosphorylation of its positive regulator RsbV by one of two phosphatases that respond to either physical or nutritional stress. The physical stress phosphatase (RsbU) requires a second protein (RsbT) for activity. Stress is thought to initiate a process that triggers the release of RsbT from a large inhibitory complex composed of multiple copies of two protein species, RsbR (and/or its paralogues) and RsbS. The stress-derived signal driving RsbT release is unknown, but it fails to develop in B. subtilis lacking either ribosome protein L11 or the ribosome-associated protein Obg. RsbR, RsbS, RsbT, Obg and ribosomes elute in common high-molecular-mass fractions during gel-filtration chromatography of crude B. subtilis extracts. This paper reports the investigation of the basis of this coelution by the examining of associations between these proteins in extracts prepared from wild-type and mutant B. subtilis, and Escherichia coli engineered to express RsbR, RsbS and RsbT. Large RsbR/RsbS complexes, distinct from ribosomes, were detected in extracts of both B. subtilis and E. coli. In E. coli, high-molecular-mass forms of RsbS were less abundant when RsbR was absent, but in B. subtilis, only when both RsbR and its principal paralogues were missing from the extract was this form less abundant. This finding is consistent with the notion that the RsbR paralogues, present in B. subtilis but not E. coli, can substitute for RsbR in such complexes. RsbT was not bound to RsbR/RsbS in any extract that was examined, including one prepared from a B. subtilis strain with an RsbS variant (RsbS59SA) that is believed to continuously associate with RsbT. The high-molecular-mass forms of RsbT were found to be Triton-sensitive and independent of any other B. subtilis protein for their formation. These probably represent RsbT aggregates. The data suggest that the contribution of ribosomes/Obg to sigma(B) activation does not involve formation of a stable association between these proteins and the Rsb complex. In addition, the binding of RsbT to RsbS/RsbR appears to be more labile than the binding between the previously analysed Rsb proteins which form inhibitory complexes. This, and the apparent proclivity of RsbT to aggregate, suggests an inherent instability in RsbT which may play a role in its regulation.

Guanine nucleotides stabilize the binding of Bacillus subtilis Obg to ribosomes.

Obg is a GTP-binding protein of Bacillus subtilis with essential, but undefined roles in the bacterium's growth, sporulation, and stress responses. Obg orthologs are widely conserved among both bacteria and eukaryotes. Gel filtration and affinity blot assays have suggested that Obg may be ribosome-associated. In the current work, we continue an examination of the putative Obg:ribosome interaction. Velocity centrifugation analyses of crude B. subtilis extracts or purified Obg:ribosome mixtures suggest that Obg is initially ribosome-bound, but can separate from ribosomes during sedimentation in the absence of added nucleotides. Addition of either GTP, GDP or ATP to the gradient prolonged the Obg:ribosome association, while inclusion of a nonhydrolyzable GTP analog (5-guanylyl-imidodiphosphate) preserved it. The data strengthen the notion that Obg is a ribosome-associated protein, demonstrate that Obg's association with ribosomes is stabilized by GTP, and indicate that the ribosome-bound Obg can likely hydrolyze GTP and be released as a consequence.

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.

Sporulation phenotype of a Bacillus subtilis mutant expressing an unprocessable but active sigmaE transcription factor.

SigmaE, a sporulation-specific sigma factor of Bacillus subtilis, is formed from an inactive precursor (pro-sigmaE) by a developmentally regulated processing reaction that removes 27 amino acids from the proprotein's amino terminus. A sigE variant (sigE335) lacking 15 amino acids of the prosequence is not processed into mature sigmaE but is active without processing. In the present work, we investigated the sporulation defect in sigE335-expressing B. subtilis, asking whether it is the bypass of proprotein processing or a residual inhibition of sigmaE activity that is responsible. Fluorescence microscopy demonstrated that sigE335-expressing B. subtilis progresses further into sporulation (stage III) than do strains lacking sigmaE activity (stage II). Consistent with its stage III phenotype, and a defect in sigmaE activity rather than its timing, the sigE335 allele did not disturb early sporulation gene expression but did inhibit the expression of late sporulation genes (gerE and sspE). The Spo- phenotype of sigE335 was found to be recessive to wild-type sigE. In vivo assays of sigmaE activity in sigE, sigE335, and merodiploid strains indicate that the residual prosequence on sigmaE335, still impairs its activity to function as a transcription factor. The data suggest that the 11-amino-acid extension on sigmaE335 allows it to bind RNA polymerase and direct the resulting holoenzyme to sigmaE-dependent promoters but reduces the enzyme's ability to initiate transcription initiation and/or exit from the promoter.

RelA is a component of the nutritional stress activation pathway of the Bacillus subtilis transcription factor sigma B.

The general stress regulon of Bacillus subtilis is induced by the activation of the sigma(B) transcription factor. Activation of sigma(B) occurs when one of two phosphatases (RsbU and RsbP), each responding to a unique type of stress, actuates a positive regulator of sigma(B) by dephosphorylation. Nutritional stress triggers the RsbP phosphatase. The mechanism by which RsbP becomes active is unknown; however, its activation coincides with culture conditions that are likely to reduce the cell's levels of high-energy nucleotides. We now present evidence that RelA, a (p)ppGpp synthetase and the key enzyme of the stringent response, plays a role in nutritional stress activation of sigma(B). An insertion mutation that disrupts relA blocks the activation of sigma(B) in response to PO(4) or glucose limitation and inhibits the drop in ATP/GTP levels that normally accompanies sigma(B) induction under these conditions. In contrast, the activation of sigma(B) by physical stress (e.g., ethanol treatment) is not affected by the loss of RelA. RelA's role in sigma(B) activation appears to be distinct from its participation in the stringent response. Amino acid analogs which induce the stringent response and RelA-dependent (p)ppGpp synthesis do not trigger sigma(B) activity. In addition, neither a missense mutation in relA (relA240GE) nor a null mutation in rplK (rplK54), either of which is sufficient to inhibit the stringent response and RelA-dependent (p)ppGpp synthesis, fails to block sigma(B) activation by PO(4) or glucose limitation.

Tethering of the Bacillus subtilis sigma E proprotein to the cell membrane is necessary for its processing but insufficient for its stabilization.

sigma(E), a sporulation-specific transcription factor of Bacillus subtilis, is synthesized as an inactive proprotein with a 27-amino acid extension at its amino terminus. This "pro" sequence is removed by a developmentally regulated protease, but when present, it blocks sigma(E) activity, tethers sigma(E) to the bacterium's cytoplasmic membrane, and promotes sigma(E) stability. To investigate whether pro-sigma(E) processing and/or stabilization are tied to membrane sequestration, we used fluorescent protein fusions to examine the membrane binding of SigE variants. The results are consistent with membrane association as a prerequisite for pro-sigma(E) processing but not as a sufficient cause for the proprotein's stability.

HrcA is a negative regulator of the dnaK and groESL operons of Streptococcus pyogenes.

The genome of Streptococcus pyogenes, an important human pathogen, encodes homologs of the principal bacterial heat shock proteins DnaK and GroES, -EL, as well as HrcA, a negative regulator of dnaK and groESL expression in other Gram-positive bacteria. Using nuclease protection assays to measure dnaK/groESL mRNA abundance and a "non-polar" insertion to disrupt hrcA, we demonstrate that heat shock triggers a 4- to 8-fold increase in dnaK and groESL-specific mRNAs within 5 min of the temperature shift and that HrcA is a negative regulator of S. pyogenes dnaK/groESL mRNA abundance in unstressed S. pyogenes. Although the loss of HrcA elevated dnaK and groESL mRNA levels under non-heat shock conditions, the relative abundance of these RNAs increased further in heat shocked S. pyogenes, suggesting an additional element contributing to their synthesis or stability.

Loss of ribosomal protein L11 blocks stress activation of the Bacillus subtilis transcription factor sigma(B).

sigma(B), the general stress response sigma factor of Bacillus subtilis, is activated when the cell's energy levels decline or the bacterium is exposed to environmental stress (e.g., heat shock, ethanol). Physical stress activates sigma(B) through a collection of regulatory kinases and phosphatases (the Rsb proteins) which catalyze the release of sigma(B) from an anti-sigma(B) factor inhibitor. The means by which diverse stresses communicate with the Rsb proteins is unknown; however, a role for the ribosome in this process was suggested when several of the upstream members of the sigma(B) stress activation cascade (RsbR, -S, and -T) were found to cofractionate with ribosomes in crude B. subtilis extracts. We now present evidence for the involvement of a ribosome-mediated process in the stress activation of sigma(B). B. subtilis strains resistant to the antibiotic thiostrepton, due to the loss of ribosomal protein L11 (RplK), were found to be blocked in the stress activation of sigma(B). Neither the energy-responsive activation of sigma(B) nor stress-dependent chaperone gene induction (a sigma(B)-independent stress response) was inhibited by the loss of L11. The Rsb proteins required for stress activation of sigma(B) are shown to be active in the RplK(-) strain but fail to be triggered by stress. The data demonstrate that the B. subtilis ribosomes provide an essential input for the stress activation of sigma(B) and suggest that the ribosomes may themselves be the sensors for stress in this system.

The Bacillus subtilis GTP binding protein obg and regulators of the sigma(B) stress response transcription factor cofractionate with ribosomes.

Obg, an essential GTP binding protein of Bacillus subtilis, is necessary for stress activation of the sigma(B) transcription factor. We investigated Obg's cellular associations by differential centrifugation of crude B. subtilis extracts, using an anti-Obg antibody as a probe to monitor Obg during the fractionation, and by fluorescent microscopy of a B. subtilis strain in which Obg was fused to green fluorescent protein. The results indicated that Obg is part of a large cytoplasmic complex. In subsequent analyses, Obg coeluted with ribosomal subunits during gel filtration of B. subtilis lysates on Sephacryl S-400 and specifically bound to ribosomal protein L13 in an affinity blot assay. Probing the gel filtration fractions with antibodies specific for sigma(B) and its coexpressed regulators (Rsb proteins) revealed coincident elution of the upstream components of the sigma(B) stress activation pathway (RsbR, -S, and -T) with Obg and the ribosomal subunits. The data implicate ribosome function as a possible mediator of the activity of Obg and the stress induction of sigma(B).

Stress triggers a process that limits activation of the Bacillus subtilis stress transcription factor sigma(B).

Stress-induced activation of the Bacillus subtilis transcription factor sigma(B) is transitory. To determine whether the process that limits sigma(B) activation is itself triggered by stress, B. subtilis strains in which the stress pathway was artificially activated by the induced expression of a positive regulatory protein (RsbT) were exposed to ethanol stress and were monitored for the persistence of sigma(B) activity. Without ethanol treatment, the induced cultures displayed continuously high sigma(B) activity. Ethanol treatment restricted ongoing sigma(B) activity, but only in strains with intact rsbX and -S genes. The loss of other gene products (RsbR and Obg) known to participate in the stress activation pathway had little influence in blocking the ethanol effect. The data argue that stress upregulates the activity of the RsbX-S regulatory pair to restrict sigma(B) induction following stress.

The "pro" sequence of the sporulation-specific sigma transcription factor sigma(E) directs it to the mother cell side of the sporulation septum.

sigma(E), a mother cell-specific transcription factor of sporulating Bacillus subtilis, is derived from an inactive precursor protein (pro-sigma(E)). Activation of sigma(E) occurs when a sporulation-specific protease (SpoIIGA) cleaves 27 amino acids from the pro-sigma(E) amino terminus. This reaction is believed to take place at the mother cell-forespore septum. Using a chimera of pro-sigma(E) and green fluorescent protein (GFP) to visualize the intracellular location of pro-sigma(E) by fluorescence microscopy, and lysozyme treatment to separate the mother cell and forespore compartments, we determined that the pro-sigma(E)::GFP signal, localized to the forespore septum prior to lysozyme treatment, is restricted to the mother cell compartment after treatment. Thus, pro-sigma(E)::GFP had been sequestered to the mother cell side of the septum. This segregation of pro-sigma(E)::GFP, and presumably pro-sigma(E), to the mother cell is likely to be the reason why sigma(E) activity is restricted to that compartment.

Sigma factor displacement from RNA polymerase during Bacillus subtilis sporulation.

As Bacillus subtilis proceeds through sporulation, the principal vegetative cell sigma subunit (sigma(A)) persists in the cell but is replaced in the extractable RNA polymerase (RNAP) by sporulation-specific sigma factors. To explore how this holoenzyme changeover might occur, velocity centrifugation techniques were used in conjunction with Western blot analyses to monitor the associations of RNAP with sigma(A) and two mother cell sigma factors, sigma(E) and sigma(K), which successively replace sigma(A) on RNAP. Although the relative abundance of sigma(A) with respect to RNAP remained virtually unchanged during sporulation, the percentage of the detectable sigma(A) which cosedimented with RNAP fell from approximately 50% at the onset of sporulation (T(0)) to 2 to 8% by 3 h into the process (T(3)). In a strain that failed to synthesize sigma(E), the first of the mother cell-specific sigma factors, approximately 40% of the sigma(A) remained associated with RNAP at T(3). The level of sigma(A)-RNAP cosedimentation dropped to less than 10% in a strain which synthesized a sigma(E) variant (sigma(ECR119)) that could bind to RNAP but was unable to direct sigma(E)-dependent transcription. The E-sigma(E)-to-E-sigma(K) changeover was characterized by both the displacement of sigma(E) from RNAP and the disappearance of sigma(E) from the cell. Analyses of extracts from wild-type and mutant B. subtilis showed that the sigma(K) protein is required for the displacement of sigma(E) from RNAP and also confirmed that sigma(K) is needed for the loss of the sigma(E) protein. The results indicate that the successive appearance of mother cell sigma factors, but not necessarily their activities, is an important element in the displacement of preexisting sigma factors from RNAP. It suggests that competition for RNAP by consecutive sporulation sigma factors may be an important feature of the holoenzyme changeovers that occur during sporulation.

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.

A Bacillus-specific factor is needed to trigger the stress-activated phosphatase/kinase cascade of sigmaB induction.

The general stress regulon of Bacillus subtilis is controlled by the transcription factor sigmaB. Environmental stress activates sigmaB via a phosphatase/kinase cascade that triggers sigmaB's release from an anti sigma factor complex. To determine if the members of the phosphatase/kinase cascade are sufficient to detect environmental stress and activate sigmaB, we expressed sigmaB and its regulators in E. coli. In E. coli, as in B. subtilis, the intact collection of regulators silenced sigmaB, while allowing sigmaB to be active if the cascade's most upstream negative regulator was deleted. The regulators could not, however, activate sigmaB in response to ethanol treatment or heat shock. In other experiments, the GroEL and DnaK chaperones, known to be important in controlling stress sigma factors in E. coli, were found to be unimportant for sigmaB activity in B. subtilis. The findings argue that stress induction of sigmaB requires novel factors that are B. subtilis specific.