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At least 19 recordsLinked to original sources

PubMed1978

Evidence that spo0A mutations are recessive in spo0A-/spo0A+ merodiploid strains of Bacillus subtilis.

The spo0A locus contains two types of closely linked mutations that block sporulation at stage 0: spo0A mutations (the most pleiotropic of the stage 0 markers) and spo0C mutations. It was previously thought that spo0A mutations were dominant in merodiploids of Bacillus subtilis, whereas spo0C mutations were recessive. We have shown that spo0A mutations were recessive when spo0A-/spo0A+ merodiploids were made in the genetic backgrounds of strains that were resistant to antibiotic produced by the wild-type strain. Reinvestigation of cultures of spo0A-/spo0A+ merodiploids constructed in the antibiotic-sensitive spo0A strain showed that they contained the spo0A allele at a low frequency, and they produced very few haploid Spo- segregants. These facts indicated that the cultures contained mostly homogenotic (spo0A+/spo0A+) cells. The reason for the poor survival of the spo0A-/spo0A+ merodiploids in the genetic background of the antibiotic-sensitive strain was not clear, but several possible explanations were given. It may have been related to the diploid state of other genes in the same merodiploid cells. The previous indication that spo0A mutations were dominant seems to have been based on the properties of a rare class of Spo- segregants that were probably selected in the presence of antibiotic.

Anti-Bacterial Agents↗

PubMed2025

Autoregulation of the Master Regulator Spo0A Controls Cell-Fate Decisions in Bacillus subtilis.

Spo0A in Bacillus subtilis is activated by phosphorylation (Spo0A~P) upon starvation and differentially controls a set of genes involved in biofilm formation and sporulation. The spo0A gene is transcribed by two distinct promoters, a σA-recognized upstream promoter Pv during growth, and a σH-recognized downstream promoter Ps during starvation, and appears to be autoregulated by four Spo0A~P binding sites (0A1-4 boxes) localized between two promoters. However, the autoregulatory mechanisms and their impact on differentiation remain elusive. Here, we determined the relative affinity of Spo0A~P for each 0A box and dissected each promoter in combination with the systematic 0A box mutations. The data revealed that (1) the Pv and Ps promoters are on and off, respectively, under nutrient-rich conditions without Spo0A~P, (2) the Ps promoter is activated by first 0A3 and then 0A1 during early starvation with low Spo0A~P, (3) during later starvation with high Spo0A~P, the Pv promoter is repressed by first 0A1 and then 0A2 and 0A4, and (4) during prolonged starvation, both promoters are silenced by all 0A boxes with very high Spo0A~P. Our results indicate that the autoregulation of spo0A is one of the key determinants to achieve a developmental increase in Spo0A~P, leading to a temporal window for entry into biofilm formation or sporulation.

Bacillus subtilis↗

PubMed1986

The effect of spo0 mutations on the expression of spo0A- and spo0F-lacZ fusions.

We have constructed spo0A-lacZ and spo0F-lacZ fusions with a temperate phage vector and have investigated how spo0 gene products are involved in the expression of each of these genes. The expression of spo0A-lacZ and spo0F-lacZ was stimulated at about the time of cessation of vegetative growth in Spo+ cells. This stimulation of spo0A-lacZ was impaired by mutations in the spo0B, D, E, F or H genes but was not affected by mutations in the spo0J or K genes. Similar results were obtained with the spo0F-lacZ fusion. The effect of the spo0A mutation on spo0A-lacZ expression was characteristic: the spo0A-directed beta-galactosidase activity found during vegetative growth was significantly enhanced in the spo0A mutant. This result suggests that spo0A gene expression is auto-regulated being repressed by its own gene product. Another remarkable observation was the effect of the sof-1 mutation, which is known to be a spo0A allele; it suppressed the sporulation deficiency of spo0B, spo0D and spo0F mutants. The spo0A-lacZ stimulation, which is impaired by any one of these spo0 mutations, was restored by the additional sof-1 mutation.

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↗

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↗

PubMed1991

Spo0A binds to a promoter used by sigma A RNA polymerase during sporulation in Bacillus subtilis.

Examination of the effects of 56 single-base-pair substitutions in the spoIIG promoter and studies of the interaction of the spo0A product (Spo0A) with this promoter in vitro demonstrated that Spo0A acts directly to enable this promoter to be used by sigma A-associated RNA polymerase (EC 2.7.7.6). The spoIIG operon from Bacillus subtilis is transcribed during sporulation by a form o RNA polymerase containing sigma A, the primary sigma factor in vegetative cells. The spoIIG promoter is unusual in that it contains sequences that are similar to those found at the -10 and -35 regions of promoters that are used by sigma A-associated RNA polymerase, but these sigma A-like recognition sequences are separated by 22 base pairs rather than the typical 17 or 18 base pairs. We found that single-base-pair substitutions in the around the -35-like sequence, and substitutions in a region upstream from this position, around position -87, reduced promoter activity. DNase I protection and electrophoretic gel mobility shift assays were used to demonstrate that Spo0A binds specifically to these regions in vitro. Evidently, the -35-like sequence is part of a Spo0A binding site and therefore is possibly not a sigma A-recognition sequence. These results support a model in which Spo0A activates the spoIIG promoter after the onset of endospore formation.

Bacillus subtilis↗

PubMed1989

Characterization of the gene for a protein kinase which phosphorylates the sporulation-regulatory proteins Spo0A and Spo0F of Bacillus subtilis.

The kinA (spoIIJ) locus contains a single gene which codes for a protein of 69,170 daltons showing strong homology to the transmitter kinases of two component regulatory systems. The purified kinase autophosphorylates in the presence of ATP and mediates the transfer of phosphate to the Spo0A and Spo0F sporulation regulatory proteins. Spo0F protein was a much better phosphoreceptor for this kinase than Spo0A protein in vitro. Mutants with deletion mutations in the kinA gene were delayed in their sporulation. They produced about a third as many spores as the wild type in 24 h, but after 72 h on solid medium, the level of spores approximated that found for the wild-type strain. Such mutations had no effect on the regulation of the abrB gene or on the timing of subtilisin expression and therefore did not impair the repression function of the Spo0A protein. Placement of the kinA locus on a multicopy vector suppressed the sporulation-defective phenotype of spo0B, spo0E, and spo0F mutations but not of spo0A mutations. The results suggest that the spo0B-, spo0E-, and spo0F-dependent pathway of activation (phosphorylation) of the Spo0A regulator may be by-passed through the kinA gene product if it is present at sufficiently high intracellular concentration. The results suggest that multiple kinases exist for the Spo0A protein.

Amino Acid Sequence↗

PubMedDate not supplied

Suppressors of a spo0A missense mutation and their effects on sporulation in Bacillus subtilis.

The spo0A gene product of Bacillus subtilis is a transcriptional regulator that is required for the initiation of sporulation. It has not been possible to isolate mutations that suppress the sporulation defect caused by spo0A null mutations. We describe the isolation and characterization of mutations that suppress the severe sporulation defect caused by a spo0A missense mutation (spo0A9V). Two suppressor mutations, spa2 and spa4, have been characterized in combination with, and separated from, the spo0A9V mutation. Both were located in the carboxyl half of Spo0A, in the putative DNA binding, transcriptional activation region. spa2 was in codon 174, causing a leucine to arginine change (spo0A174LR), and spa4 was in codon 162 (of 267), causing a histidine to arginine change (spo0A162HR). spa2 and spa4 significantly restored sporulation to the spo0A9V mutant, however, the appearance of heat resistant spores was delayed relative to wild-type. When separated from spo0A9V, that is, as single mutations in spo0A, spa4 caused a delay in sporulation, while spa2 allowed apparently normal sporulation. The spa mutations caused interesting phenotypes when combined with other early sporulation mutations. spa2 suppressed the sporulation defect caused by spo0E11. This was most easily seen in spo0E11 abrB double mutants, which had a much more severe sporulation defect than the spo0E11 single mutant. That is, spo0E11 and abrB mutations caused a synthetic (synergistic) sporulation phenotype. Both the spa2 spo0A9V and the spa4 spo0A9V alleles greatly enhanced the sporulation defect caused by mutations in spoIIJ, spo0J and spo0K. The significance of these synthetic sporulation defects is discussed.

Bacillus subtilis↗

PubMed1985

Deduced product of the stage 0 sporulation gene spo0F shares homology with the Spo0A, OmpR, and SfrA proteins.

The location of the stage 0 sporulation locus spo0F has been determined on a cloned fragment of Bacillus subtilis DNA. The spo0F gene and surrounding region was sequenced and was shown to code for a protein of Mr 14,286. The amino acid sequence of this deduced protein was 56% homologous to the amino-terminal domain of the spo0A gene product. The molecular weight of the Spo0F protein was approximately half that of the Spo0A protein, and its sequence was homologous to the amino-terminal half of the Spo0A protein. This same portion of the Spo0A protein showed ancestral relationship to the OmpR and SfrA regulatory proteins of Escherichia coli. Mutations in any of the genes encoding these proteins in either organism are highly pleiotropic and result in alterations in the regulation of membrane components, suggesting that they may have related roles in both organisms and that the stage 0 sporulation defect of spo0A and spo0F mutants is an indirect consequence of this regulatory system.

Amino Acid Sequence↗

PubMed1985

Characterization of the spo0A locus and its deduced product.

The highly pleiotropic stage 0 sporulation locus of Bacillus subtilis, spo0A, has been cloned in bacteriophage lambda, subcloned in plasmids, and sequenced. The locus was found to code for a protein of 29,691 Da. Analysis of the in vivo transcripts from this region by nuclease S1 protection experiments located the start and stop of transcription of the locus. The transcription start site was preceded by a promoter resembling sigma 37-dependent promoters. Two mutations originally assigned to a second locus, spo0C, in this region because of their weakly pleiotropic phenotypes were cloned and sequenced. The mutations were found to be different missense alterations in the same base of the 10th codon preceding the carboxyl end of the Spo0A protein. These results, along with the finding that mutations in the spo0A gene product [Hoch, J. A., Trach, K., Kawamura, F. & Saito, H. (1985) J. Bacteriol. 161, 552-555] suppress the requirement for spo0B, spo0E, and spo0F gene products in transcription from sigma 28-dependent promoters, suggest that the Spo0A protein interacts directly with the transcription machinery to effect the initiation of sporulation. The deduced amino acid sequence of the Spo0A protein was highly related to that of the OmpR regulatory protein of Escherichia coli.

Amino Acid Sequence↗

PubMed1988

Structure of the gene for the transition state regulator, abrB: regulator synthesis is controlled by the spo0A sporulation gene in Bacillus subtilis.

Sporulation begins coincidentally with the expression of several stationary-phase-associated gene products during the transition state of a culture from exponential to stationary phase. Mutations in the stage 0 sporulation genes prevent the expression of these gene products in addition to blocking sporulation. Suppressor mutations in the abrB gene, in a spo0 background, restore stationary-phase-associated gene expression but not sporulation. The nature of the abrB gene product was investigated by isolating and sequencing the abrB gene. The abrB gene coded for a 96-amino-acid protein (molecular weight 10773) and contained a helix-turn-helix structure common to DNA binding proteins. Analysis of expression of the abrB gene using lacZ transcription fusions and direct measurement of mRNA content by hybridization showed that the spo0A gene repressed transcription of the abrB gene. Primer extension analysis of abrB gene mRNA revealed two initiation sites. The downstream site was dramatically repressed in spo0A+ strains, while the upstream site appeared not to be regulated by spo0A. Five abrB mutant alleles were cloned and sequenced. One mutation, abrB4, resided within the structural gene and continued to overexpress abrB messenger RNA from both promoters. A promoter mutation, abrB15, reduced transcription from the downstream promoter but not the upstream promoter. Thus, the phenotype of abrB mutations results from inactivation of the abrB gene product or by prevention of its overexpression. The results suggest that the abrB gene codes for a regulator which controls several genes whose products are normally produced during the transition phase between active growth and sporulation. The level of this regulator is, in turn, controlled by the spo0A gene. The pleiotropic phenotypes of spo0A mutants result from uncontrolled overexpression of the abrB regulator.

Amino Acid Sequence↗

PubMed1991

Mutational dissociation of the positive and negative regulatory properties of the Spo0A sporulation transcription factor of Bacillus subtilis.

The Spo0A regulatory protein controls the onset of stationary phase and sporulation by controlling transcription in both a negative and a positive manner depending on the promoter affected. Missense mutations, e.g., spo0A9V, which result in alterations in the eleventh amino acid preceding the C terminus of the Spo0A protein, give rise to a protein active as a negative regulator of the abrB gene but unable to activate transcription of the spoIIA gene. Second-site suppressors of spo0A9V occurred within the spo0A gene at codons 162 and 174. These suppressors did not suppress a spo0F mutation, indicating that the suppressed protein still requires phosphorylation for activity. The results suggest that the C terminus of Spo0A interacts with the transcription complex to activate transcription.

Amino Acid Sequence↗

PubMed1992

The spo0A gene is implicated in the maintenance of non-complementing diploids in Bacillus subtilis.

Bacillus subtilis can exist in a diploid state in which two genetically distinct chromosomes co-exist in the same cell and yet only one of them is expressed, thereby determining the phenotype. Such cells are called non-complementing diploids (Ncds). In this study, two types of experiments are reported which indicate that a previously known pleiotropic gene, spo0A, plays a role in the maintaining the diploid state, as follows. (i) When protoplasts of two Spo0A mutant strains were fused, the resulting products continued to segregate cells of both parental phenotypes for many more divisions than had been reported previously. (ii) When a stable Ncd (an Ncd in which the unexpressed markers are not spontaneously activated at a detectable level) harbouring a chloramphenicol acetyltransferase gene on the silent chromosome was transformed with spo0A null alleles the transformants often expressed chloramphenicol acetyltransferase activity. Together these results indicate that the spo0A gene is involved in maintenance of the diploid state in both unstable and stable Ncds.

Bacillus subtilis↗

PubMed1985

Identification of the transcriptional suppressor sof-1 as an alteration in the spo0A protein.

The mutation sof-1 suppresses the sporulation defect of mutations in either the spo0B, spo0E, or spo0F stage 0 sporulation genes. Through the use of integrative plasmids carrying the portion of the chromosome including the spo0A locus and flanking regions, the sof-1 mutation was localized near the spo0A locus. A plasmid carrying a fragment of DNA with sof genetic activity was constructed. Nucleic acid sequence analysis of this fragment revealed a single base change that resulted in a substitution of lysine for asparagine in the 12th codon of the spo0A gene. The results indicate that certain missense mutations in the spo0A gene bypass the necessity for the spo0B, spo0E, and spo0F gene products in sporulation. Several models for the interaction of these gene products may be imagined.

Bacillus subtilis↗

PubMed1990

Structural alterations in the Bacillus subtilis Spo0A regulatory protein which suppress mutations at several spo0 loci.

Secondary site mutations that restore sporulation to sporulation-defective spo0F or spo0B deletion mutants were found to reside in the spo0A gene. Sequence analysis of 23 such sof mutants showed that the sof mutations fell into six classes of missense codon changes, primarily in the conserved amino-terminal domain of the response regulator Spo0A protein. Changes were observed in codons 12, 14, 60, 92, and 121. The residues affected were predominantly located in the potential turn regions at one end of the amino-terminal conserved domain on the same topological face as the active site aspartate residues. The ability of sof mutations to suppress deficiencies in the transmitter kinases, KinA and KinB, of two-component regulatory systems was tested. All of the sof mutations suppressed the sporulation deficiency of kinA mutants but only two classes among five tested suppressed kinB mutations. sof mutants segregated Spo- colonies at high frequency. Five of these Spo- mutants were found to result from mutations in the spo0A locus that reversed the effect of the sof mutatation. One of these was sequenced and found to have the original sof mutation and a new mutation, sos, at codon 105. The accumulation of sos mutations in sof strains suggested that the sof mutations have a subtle, yet deleterious, effect on the growth of the cell. The results suggested that the sof mutations increase the avidity for or reactivity with transmitter kinases in an allele-specific manner, although in some cases it is possible that the sof mutations obviate the need for phosphorylation to activate the Spo0A protein. An alternative hypothesis is presented in which the sof mutations play the role of bypass mutations for kinases.

Amino Acid Sequence↗

PubMed1992

Spo0A controls the sigma A-dependent activation of Bacillus subtilis sporulation-specific transcription unit spoIIE.

The spoIIE operon is a developmentally regulated transcription unit activated in the second hour of sporulation in Bacillus subtilis. Its promoter has an unusual structure, containing sequences which conform perfectly to the consensus for vegetative promoters recognized by sigma A-associated RNA polymerase (E sigma A), but with a spacing of 21 bp between the apparent -10 and -35 elements instead of the 17- or 18-bp spacing typical of promoters utilized by E sigma A. Mutations introduced into the apparent -10 element affected transcription in a manner consistent with its functioning as a polymerase recognition sequence. The deleterious effect of one -10 mutation was also suppressed in an allele-specific manner by a mutation in sigA known to suppress analogous -10 mutations in conventional vegetative promoters recognized by E sigma A. Similar suppression experiments failed to provide evidence for a direct interaction between E sigma A and the "-35-like" element, however, and DNase I protection experiments suggested instead that the Spo0A protein binds to a site overlapping this -35-like hexamer. Moreover, the effects of mutations within the -35-like hexamer on the binding of Spo0A in vitro paralleled their effects on transcription in vivo. We suggest that spoIIE belongs to a class of early-intermediate sporulation genes whose transcription by E sigma A is activated by the Spo0A protein.

Bacillus subtilis↗

PubMed1992

Bacillus subtilis early sporulation genes kinA, spo0F, and spo0A are transcribed by the RNA polymerase containing sigma H.

The Bacillus subtilis genes kinA (spoIIJ), spo0F, and spo0A encode components of the sporulation signal transduction pathway. Recent work has suggested that these genes are transcribed by a minor form of RNA polymerase, E sigma H (sigma H is the product of spo0H, another early sporulation gene). We directly tested this hypothesis by performing in vitro transcription assays with reconstituted E sigma H and a set of plasmids containing the kinA, spo0F, and spo0A promoter regions. We were able to obtain distinct transcripts of the expected sizes with all three genes by using linearized or supercoiled templates. Furthermore, primer extension experiments indicate that the transcription start sites for the three genes in vitro and in vivo are the same. In addition, we measured steady-state levels of kinA, spo0F, and spo0A mRNAs during growth in sporulation medium; all of them were increased at or near the beginning of the stationary phase.

Bacillus subtilis↗