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PubMed1998

DNA strand separation during activation of a developmental promoter by the Bacillus subtilis response regulator Spo0A.

Spo0A is the central regulator of commitment to sporulation in Bacillus subtilis. Spo0A is a member of the response regulator family of proteins and both represses and stimulates transcription from promoters when activated. In vivo Spo0A activation takes place by phosphorylation and in vitro activation can be accomplished by phosphorylation or removal of the N-terminal domain of the protein. We have examined the mechanism of Spo0A stimulation of transcription from the promoter of the spoIIG operon. This operon encodes one of the first compartment specific sigma factors whose appearance regulates sporulation development. When activated Spo0A was incubated with RNA polymerase and a DNA fragment containing the spoIIG promoter, bases between -13 and -3, relative to the start site of transcription, were denatured. Addition of activated Spo0A or RNA polymerase alone did not induce denaturation. Heteroduplex templates that contained the nontemplate sequence of the wild-type promoter on both strands between positions -3 and -13 were efficiently transcribed without activated Spo0A. These data suggest that DNA strand separation is a two-step process and that the activation of Spo0A creates a form that interacts with the polymerase to induce the first of the two steps.

Bacillus subtilis↗

PubMed2004

Dimer-induced signal propagation in Spo0A.

Spo0A, the response regulator protein controlling the initiation of sporulation in Bacillus, has two distinct domains, an N-terminal phosphoacceptor (or receiver) domain and a C-terminal DNA-binding (or effector) domain. The phosphoacceptor domain mediates dimerization of Spo0A on phosphorylation. A comparison of the crystal structures of phosphorylated and unphosphorylated response regulators suggests a mechanism of activation in which structural changes originating at the phosphorylatable aspartate extend to the alpha4beta5alpha5 surface of the protein. In particular, the data show an important role in downstream signalling for a conserved aromatic residue (Phe-105 in Spo0A), the conformation of which alters upon phosphorylation. In this study, we have prepared a Phe-105 to Ala mutant to probe the contribution of this residue to Spo0A function. We have also made an alanine substitution of the neighbouring residue Tyr-104 that is absolutely conserved in the Spo0As of spore-forming Bacilli. The spo0A(Y104A) and spo0A(F105A) alleles severely impair sporulation in vivo. In vitro phosphorylation of the purified proteins by phosphoramidate is unaffected, but dimerization and DNA binding are abolished by the mutations. We have identified intragenic suppressor mutations of spo0A(F105A) and shown that these second-site mutations in the purified proteins restore phosphorylation-dependent dimer formation. Our data support a model in which dimerization and signal transduction between the two domains of Spo0A are mediated principally by the alpha4beta5alpha5 signalling surface in the receiver domain.

Bacillus subtilis↗

PubMed1994

Expression in Bacillus subtilis of the Bacillus thuringiensis cryIIIA toxin gene is not dependent on a sporulation-specific sigma factor and is increased in a spo0A mutant.

Expression of the Bacillus thuringiensis cryIIIA gene encoding a Coleoptera-specific toxin is weak during vegetative growth and is activated at the onset of the stationary phase. cryIIIA'-'lacZ fusions and primer extension analysis show that the regulation of cryIIIA expression is similar in Bacillus subtilis and in B. thuringiensis. Activation of cryIIIA expression was not altered in B. subtilis mutant strains deficient for the sigma H and sigma E sporulation-specific sigma factors or for minor sigma factors such as sigma B, sigma D, or sigma L. This result and the nucleotide sequence of the -35 and -10 regions of the cryIIIA promoter suggest that cryIIIA expression might be directed by the E sigma A form of RNA polymerase. Expression of the cryIIIA'-'lacZ fusion is shut off after t2 (2 h after time zero) of sporulation in the B. subtilis wild-type strain grown on nutrient broth sporulation medium. However, no decrease in cryIIIA-directed beta-galactosidase activity occurred in sigma H, kinA, or spo0A mutant strains. Moreover, beta-galactosidase activity was higher and remained elevated after t2 in the spo0A mutant strain. beta-Galactosidase activity was weak in abrB and spo0A abrB mutant strains, suggesting that AbrB is responsible for the higher level of cryIIIA expression observed in a spo0A mutant. However, both in spo0A and spo0A abrB mutant strains, beta-galactosidase activity remained elevated after t2, suggesting that even in the absence of AbrB, cryIIIA expression is controlled through modulation of the phosphorylated form of Spo0A. When the cryIIIA gene is expressed in a B. subtilis spo0A mutant strain or in the 168 wild-type strain, large amounts of toxins are produced and accumulate to form a flat rectangular crystal characteristic of the coleopteran-specific B. thuringiensis strains.

Bacillus subtilis↗

PubMed1998

Spo0A mutants of Bacillus subtilis with sigma factor-specific defects in transcription activation.

The transcription factor Spo0A of Bacillus subtilis has the unique ability to activate transcription from promoters that require different forms of RNA polymerase holoenzyme. One class of Spo0A-activated promoter, which includes spoIIEp, is recognized by RNA polymerase associated with the primary sigma factor, sigma A (sigmaA); the second, which includes spoIIAp, is recognized by RNA polymerase associated with an early-sporulation sigma factor, sigma H (sigmaH). Evidence suggests that Spo0A probably interacts directly with RNA polymerase to activate transcription from these promoters. To identify residues of Spo0A that may be involved in transcriptional activation, we used PCR mutagenesis of the entire spo0A gene and designed a screen using two distinguishable reporter fusions, spoIIE-gus and spoIIA-lacZ. Here we report the identification and characterization of five mutants of Spo0A that are specifically defective in activation of sigmaA-dependent promoters while maintaining activation of sigmaH-dependent promoters. These five mutants identify a 14-amino-acid segment of Spo0A, from residue 227 to residue 240, that is required for transcriptional activation of sigmaA-dependent promoters. This region may define a surface or domain of Spo0A that makes direct contacts with sigmaA-associated holoenzyme.

Alleles↗

PubMed2002

Northern, morphological, and fermentation analysis of spo0A inactivation and overexpression in Clostridium acetobutylicum ATCC 824.

The Clostridium acetobutylicum ATCC 824 spo0A gene was cloned, and two recombinant strains were generated, an spo0A inactivation strain (SKO1) and an spo0A overexpression strain [824(pMPSOA)]. SKO1 was developed by targeted gene inactivation with a replicative plasmid capable of double-crossover chromosomal integration--a technique never used before with solventogenic clostridia. SKO1 was severely deficient in solvent formation: it produced only 2 mM acetone and 13 mM butanol, compared to the 92 mM acetone and 172 mM butanol produced by the parental strain. After 72 h of growth on solid media, SKO1 formed long filaments of rod-shaped cells that failed to septate. SKO1 cells never achieved the swollen clostridial form typical of the parental strain and did not form endospores. No spo0A transcripts were detected in SKO1, while transcription of two solvent formation operons (aad-ctfA-ctfB and adc; both containing 0A boxes in their promoter regions) was limited. Strain 824(pMSPOA) produced higher butanol concentrations than the control strain [824(pIMP1)] and dramatically elevated spo0A transcript levels and displayed a bimodal pattern of spo0A transcription similar to that of B. subtilis. Microscopic studies indicated that sporulation was both enhanced and accelerated due to spo0A overexpression compared to that of both the 824(pIMP1) and parental strains. Consistent with that, expression of the key solvent formation genes (aad-ctfA-ctfB and adc) and three sporulation-specific genes (spoIIGA, sigE, and sigG) was observed earlier in strain 824(pMSPOA) than in the plasmid control. These data support the hypothesis that Spo0A is a transcriptional regulator that positively controls sporulation and solvent production. Its effect on solvent formation is a balancing act in regulating sporulation versus solvent gene expression: its overexpression apparently tips the balance in favor of accelerated and enhanced sporulation at the expense of overall solvent production.

Bacterial Proteins↗

PubMed1994

Subunit composition and domain structure of the Spo0A sporulation transcription factor of Bacillus subtilis.

The Spo0A transcription factor is responsible for the initiation of sporulation and is active in transcription only after phosphorylation by a specific signal transduction pathway, the phosphorelay. The effect of phosphorylation on the physical properties of Spo0A was determined. Spo0A and Spo0A approximately P both behaved as monomers during Sephacryl chromatography and gel electrophoresis, suggesting that phosphorylation did not modify the oligomerization state of the protein. Trypsin digested Spo0A at a single cleavage site between residues 142 and 143 within a hinge connecting two tightly folded domains. The amino domain retains ability to be phosphorylated by the phosphorelay. The carboxyl domain is active as a DNA-binding protein and retains the sequence specificity of the intact molecule for 0A boxes on the abrB promoter as revealed by footprinting studies. The carboxyl domain stimulated in vitro transcription from the spoIIG promoter 5-fold greater than an equal amount of Spo0A and about half as well as equivalent amounts of Spo0A approximately P. Thus, the unphosphorylated amino domain inhibits the transcription stimulation activity of the carboxyl domain. We suggest that phosphorylation activates transcription regulation functions of Spo0A by modifying the spatial relationships of the amino and carboxyl domains.

Bacillus subtilis↗

PubMed1996

The Bacillus subtilis response regulator Spo0A stimulates transcription of the spoIIG operon through modification of RNA polymerase promoter complexes.

Sporulation in Bacillus subtilis is 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 vivo transcription from the promoter for the spoIIG operon, 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 spoIIG operon promoter using a single round transcription assay and the kinetics of formation of spoIIG promoter-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.

Bacillus subtilis↗

PubMedDate not supplied

Spo0A activates and represses its own synthesis by binding at its dual promoters.

The Spo0A protein of Bacillus subtilis controls the onset of sporulation by regulating transcription of various genes in both positive and negative manners depending on the promoters affected. The expression of the spo0A gene occurs from two promoters (Pv,Ps), separated by 148 bp, and transcription switches from Pv to Ps early in the sporulation program. DNase I footprint analysis of the spo0A promoter region revealed three distinct sites of Spo0A binding: -4 to +19 relative to Pv, -17 to +1 relative to Ps, and a region between Pv and Ps. The Pv region and the region between the two promoters was sufficient for repression of Pv. Induction of Ps also required these regions which are upstream of -52 relative to Ps. Mutant Spo0A proteins containing asp----asn mutations at asp10 and asp56 were inactive in repression of the abrB promoter in vivo yet still retained DNA-binding activity. The results presented are consistent with a model in which the phosphorylated form of Spo0A acts directly at its promoters to achieve induction of Ps and repression of Pv. These effects at the spo0A promoter were independent of the presence of the major kinase, KinA.

Bacillus subtilis↗

PubMed2004

The Bacillus subtilis response regulator Spo0A stimulates sigmaA-dependent transcription prior to the major energetic barrier.

At the spoIIG promoter phosphorylated Spo0A (Spo0A approximately P) binds 0A boxes overlapping the -35 element, interacting with RNA polymerase to facilitate open complex formation. We have compared in vitro transcription from a series of heteroduplex templates containing denatured regions within the promoters. Transcription from heteroduplex templates with 12, 8, or 6 base pairs denatured was independent of Spo0A approximately P, but heteroduplexes with 4 or 2 base pairs denatured required Spo0A approximately P for maximal levels of transcription. Investigation of the thermal dependence of transcription suggested that strand separation was the primary thermodynamic barrier to transcription initiation but indicated that Spo0A approximately P does not reduce this energetic barrier. Kinetic assays revealed that Spo0A approximately P stimulated both the rate of formation of initiated complexes as well as increasing the number of complexes capable of initiating transcription. These results imply that Spo0A approximately P stimulates transcription at least in part by stabilizing the RNA polymerase-spoIIG complex until contacts between RNA polymerase and the -10 element induce strand separation.

Bacillus subtilis↗

PubMed1987

Role of AbrB in Spo0A- and Spo0B-dependent utilization of a sporulation promoter in Bacillus subtilis.

Transcription of the Bacillus subtilis gene spoVG is induced at the onset of sporulation and is dependent on the products of the stage-0 regulatory genes spo0A, spo0B, and spo0H. We show here that the dependence of spoVG transcription on Spo0A and Spo0B (but not Spo0H) can be bypassed by a mutation at abrB, a previously identified locus at which mutations that suppress some of the phenotypes of spo0A are often located, or by a cis-acting mutation within the spoVG promoter. To explain the epistatis of abrB to spo0A and spo0B mutations, we propose that AbrB acts, directly or indirectly, to block transcription of spoVG and that Spo0A and Spo0B cause inactivation of the abrB gene product(s). Spo0A-Spo0B-dependent inactivation of AbrB could be a general explanation for the pleiotropic effects of spo0A and spo0B mutations on B. subtilis gene expression.

Bacillus subtilis↗

PubMed1992

Binding of Spo0A stimulates spoIIG promoter activity in Bacillus subtilis.

The spoIIG promoter is used by RNA polymerase containing sigma A (E sigma A), the primary form of RNA polymerase found in vegetative cells in Bacillus subtilis. However, the spoIIG promoter is active only after the onset of sporulation. Activation of the spoIIG promoter requires the product of the spo0A gene (Spo0A). Spo0A is a sequence-specific DNA-binding protein which binds to two sites in the spoIIG promoter that are essential for promoter activity. We found that single-base-pair substitutions in these two regions that reduced promoter activity in vivo caused reduced binding of Spo0A in vitro, and one substitution that increased promoter activity in vivo increased the affinity of Spo0A for this DNA in vitro. Furthermore, Spo0A stimulated transcription from the spoIIG promoter by E sigma A in vitro. These results support the model that binding of Spo0A activates E sigma A-dependent transcription from the spoIIG promoter after the onset of sporulation.

Bacillus subtilis↗

PubMed1993

Bacillus subtilis transcription regulator, Spo0A, decreases alkaline phosphatase levels induced by phosphate starvation.

Alkaline phosphatase (APase) is induced as a culture enters stationary phase because of limiting phosphate. The results presented here show that expression of APase is regulated both negatively and positively. PhoP, a homolog of a family of bacterial transcription factors, and PhoR, a homolog of bacterial histidine protein kinases, are required for induction of APases when phosphate becomes limiting. The induction period lasts 2 to 3 h, after which the rate of APase accumulation is decreased. Mutant strains defective in the Spo0A transcription factor failed to decrease APase production. The consequent hyperinduction of APase in a spo0A strain was dependent on phoP and phoR. spo0B and spo0F strains also overexpressed APase, suggesting that phosphorylated Spo0A is required for repression of APase. An abrB mutant allele in the presence of the mutant spo0A allele in these strains did not significantly change the APase hyperinduction phenotype, demonstrating that Spo0A repression of abrB expression is not the mechanism by which Spo0A-P regulates APase expression. Our previous report that spo0A mutants do not express APases is in conflict with the present data. We show here that the previously used mutants and a number of commonly used spo0 strains, all of which have an APase deficiency phenotype, contain a previously unrecognized mutation in phoR.

Alkaline Phosphatase↗

PubMed2000

Mutational analysis of conserved residues in the putative DNA-binding domain of the response regulator Spo0A of Bacillus subtilis.

The Spo0A protein of Bacillus subtilis is a DNA-binding protein that is required for the expression of genes involved in the initiation of sporulation. Spo0A binds directly to and both activates and represses transcription from the promoters of several genes required during the onset of endospore formation. The C-terminal 113 residues are known to contain the DNA-binding activity of Spo0A. Previous studies identified a region of the C-terminal half of Spo0A that is highly conserved among species of endospore-forming Bacillus and Clostridium and which encodes a putative helix-turn-helix DNA-binding domain. To test the functional significance of this region and determine if this motif is involved in DNA binding, we changed three conserved residues, S210, E213, and R214, to Gly and/or Ala by site-directed mutagenesis. We then isolated and analyzed the five substitution-containing Spo0A proteins for DNA binding and sporulation-specific gene activation. The S210A Spo0A mutant exhibited no change from wild-type binding, although it was defective in spoIIA and spoIIE promoter activation. In contrast, both the E213G and E213A Spo0A variants showed decreased binding and completely abolished transcriptional activation of spoIIA and spoIIE, while the R214G and R214A variants completely abolished both DNA binding and transcriptional activation. These data suggest that these conserved residues are important for transcriptional activation and that the E213 residue is involved in DNA binding.

Bacillus subtilis↗

PubMed1995

Dimer form of phosphorylated Spo0A, a transcriptional regulator, stimulates the spo0F transcription at the initiation of sporulation in Bacillus subtilis.

The Spo0A protein of Bacillus subtilis is a transcriptional regulator that shows extensive homology to the regulator proteins in bacterial two-component regulatory systems. Phosphorylation of Spo0A is absolutely necessary for the initiation of sporulation. We now show that phospho-Spo0A is a dimer, binds specifically to the spo0F promoter region, and stimulates the transcription from the P2 promoter recognized by sigma H-RNA polymerase. Biochemical and biological analyses suggest that phospho-Spo0A interacts directly with the "0A-like box" sequence (TGTCGTA) located in the spo0F promoter region. Phosphorylation of Spo0A enhanced its affinity to the 0A-like box. Evidence is also presented that the spo0F promoter region contains a static bend having two sets of oligo(dA-dT) tracts. It was demonstrated that the bending region overlaps with the recognition site for the phospho-Spo0A.

Bacillus subtilis↗

PubMed2002

Dimer formation and transcription activation in the sporulation response regulator Spo0A.

The response regulator Spo0A is the master control element in the initiation of sporulation in Bacillus subtilis. Like many other multi-domain response regulators, the latent activity of the effector, C-terminal domain is stimulated by phosphorylation on a conserved aspartic acid residue in the regulatory, N-terminal domain. If a threshold concentration of phosphorylated Spo0A is achieved, the transcription of genes required for sporulation is activated, whereas the genes encoding stationary phase sentinels are repressed, and sporulation proceeds. Despite detailed genetic, biochemical and structural characterisation, it is not understood how the phosphorylation signal in the receiver domain is transduced into DNA binding and transcription activation in the distal effector domain. An obstacle to our understanding of Spo0A function is the uncertainty concerning changes in quaternary structure that accompany phosphorylation. Here we have revisited this question and shown unequivocally that Spo0A forms dimers upon phosphorylation and that the subunit interactions in the dimer are mediated principally by the receiver domain. Purified dimers of two mutants of Spo0A, in which the phosphorylatable aspartic acid residue has been substituted, activate transcription from the spoIIG promoter in vitro, whereas monomers do not. This suggests that dimers represent the activated form of Spo0A.

Bacillus subtilis↗

PubMed2005

Proteome analysis and comparison of Clostridium acetobutylicum ATCC 824 and Spo0A strain variants.

The proteomic profiles of several Clostridium acetobutylicum strains were compared by two-dimensional gel electrophoresis and mass spectroscopy. The proteomic profile of C. acetobutylicum wild type strain ATCC 824 with and without a commonly used control plasmid and with a spo0A overexpression plasmid pMSPOA was compared. A total of 2,081 protein spots were analyzed; 23 proteins were chosen to be identified of which 18 were unique and 5 were proteins located in more than one location. The proteins identified were classified into heat shock stress response, acid and solvent formation, and transcription and translation proteins. Spo0A was identified and its protein expression was confirmed to be absent in the spo0A knockout SKO1 strain as expected, as was the protein Adc, which is known to be regulated by Spo0A. The expression of six proteins was not detected in strain SKO1 indicating these proteins require Spo0A. Spo0A overexpression affected the abundance of proteins involved in glycolysis, translation, heat shock stress response, and energy production. Two features were identified: five of the 23 proteins identified were located in more than one position and clusters of protein spots resembled fingers of a straightened hand. Normally a protein localizes to only one spot on the gel; localization of a protein to more than one spot is indicative of post-translational modifications, suggesting that such modification of proteins may be a more prevalent mechanism in C. acetobutylicum than previously thought. The clusters of protein spots resembling fingers of a straightened hand were in the acidic high molecular weight areas. Two such protein spots were identified as variants of the same protein, GroEL.

Bacterial Proteins↗

PubMed2004

Disruption of the gene (spo0A) encoding sporulation transcription factor blocks endospore formation and enterotoxin production in enterotoxigenic Clostridium perfringens type A.

This study identified a functional spo0A ORF in enterotoxigenic Clostridium perfringens type A. To evaluate the function of spo0A, an isogenic spo0A knock-out mutant was constructed. The spo0A mutant was unable to form endospores and produce enterotoxin, however, these defects could be restored by complementing the mutant with a recombinant plasmid carrying the wild-type spo0A gene. These results provide evidence that spo0A expression is essential for sporulation and enterotoxin production in C. perfringens.

Amino Acid Sequence↗

PubMed2002

Two-vector assay as a tool for examining Spo0A gene transcription regulation.

We have modified an assay using two compatible vectors that coexist in Escherichia coli cells and applied it in the investigation of the transcriptional activity of Spo0A, a key regulator of sporulation in Bacillus subtilis. We have chosen the promoters of the Spo0A dependent genes, spoIIE and spoIIA, involved in sporulation, in order to study the transcription activity solely of the DNA binding domain of Spo0A. We have prepared the two-vector system so that one vector contained the cloned C-Spo0A under the control of an inducible promoter, and the second vector (the promoter probe vector), was composed of the Spo0A dependent spoIIE and spoIIA promoters. Using this two-vector system in E. coli, we proved that C-Spo0A is able to interact with the E. coli transcription apparatus, recognizes both promoters and activates transcription from these promoters.

Bacillus subtilis↗