Regulation of Bacillus subtilis gene expression during the transition from exponential growth to stationary phase.
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Bacillus subtilis↗
Biomedical subjects
M A Strauch
Publications and source records attributed to M A Strauch.
Transition-state regulators: sentinels of Bacillus subtilis post-exponential gene expression.
When Bacillus subtilis encounters a nutrient-depleted environment, it expresses a wide variety of genes that encode functions in alternative pathways of metabolism and energy production. Expression of these genes first occurs during the transition from active growth into stationary phase and is controlled by a class of proteins termed transition-state regulators. In several instances, a given gene is redundantly controlled by two or more of these regulators and many of these regulators control genes in numerous different pathways. The AbrB, Hpr and Sin proteins are the best-studied examples of these regulatory molecules. Their role is to prevent inappropriate and possibly detrimental functions from being expressed during exponential growth when they are not needed. They serve as elements integrating sporulation with ancillary stationary-phase phenomena and appear to participate in the timing of early sporulation events and in fine-tuning the magnitude of gene expression in response to specific environmental conditions.
Bacillus subtilis↗
A positive feedback loop controls transcription of the spoOF gene, a component of the sporulation phosphorelay in Bacillus subtilis.
The spo0F gene of Bacillus subtilis encodes a protein that functions as a secondary messenger in a phosphorelay system controlling the initiation of sporulation. Transcription of the spo0F gene was known to be dependent on an intact gene for the transcription regulator Spo0A. In vitro footprint analysis revealed that Spo0A protein bound to two locations in the spo0F promoter region. Deletion of a 40 bp region upstream of one of the promoters (P2) abolished the activation of spo0F expression that occurs at the onset of stationary phase and sporulation. This 40 bp region contains a Spo0A-binding site. These observations are consistent with a hypothesis that Spo0A binding to this region is responsible for activating spo0F transcription. Additionally, Spo0A binding at a downstream site could modulate the level of this activation. Since Spo0F protein is required for the formation of Spo0A-P (the form needed for transcriptional activation) a positive feedback loop controls transcription of spo0F.
Bacillus subtilis↗
Sporulation in prokaryotes and lower eukaryotes.
During the past year, highlights in sporulation research include the demonstration that phosphorylation of SpoOA is a critical factor in Bacillus subtilis development; the identification of C alpha proteins, adenylyl cyclase and protein kinase A genes in Dictyostelium; proof that an endogenous antisense RNA regulates gene expression in Dictyostelium; and characterization of a second type of differentiated cell in Myxococcus.
Animals↗
cis-unsaturated fatty acids specifically inhibit a signal-transducing protein kinase required for initiation of sporulation in Bacillus subtilis.
The initiation of sporulation in Bacillus subtilis is controlled by the Spo0A transcription factor which is activated by phosphorylation through a phosphorelay mechanism that is dependent upon the activity of one or more protein kinases. The enzymatic activity of one of these protein kinases, KinA, was found to be inhibited in vitro by certain fatty acids. The most potent inhibitors have at least one unsaturated double bond in the cis configuration and a chain length of 16-20 carbon atoms. Homologous isomers with a trans double bond are not inhibitory. Saturated straight- or branched-chain fatty acids are either much weaker inhibitors or have no effect. The inhibitors prevent autophosphorylation of KinA and are non-competitive with ATP. B. subtilis phospholipids were found to contain at least one as yet unidentified type of fatty acid that, when present in an unesterified form, inhibited KinA. The results suggest that the concentration of a specific unsaturated fatty acid may act as a signal linking the initiation of sporulation to the status of membrane synthesis and septation or some other specific membrane-associated activity.
Bacillus subtilis↗
Control of postexponential gene expression by transition state regulators.
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Bacillus subtilis↗
The transition state regulator Hpr of Bacillus subtilis is a DNA-binding protein.
The synthesis of a variety of proteins, including the well characterized degradative enzymes, which occurs during the transition state between vegetative growth and the onset of sporulation in Bacillus subtilis is controlled by a class of molecules known as transition state regulators. One of these regulators is the product of the hpr gene, first identified by mutations affecting the synthesis of extracellular proteases. We have purified the Hpr protein and found that it binds specifically to DNA fragments carrying the promoters and the upstream regions of the alkaline (aprE) and neutral (nprE) protease genes of B. subtilis. DNase I protection experiments revealed that the Hpr protein is able to bind at four and two regions of the aprE and nprE promoters, respectively. We have also located two Hpr binding sites in the promoter region of a gene of unknown function which is nevertheless known to be developmentally regulated during the transition state and which occurs in the same operon as the gene encoding another transition state regulator, Sin. The location of one of the Hpr binding sites on the aprE gene occurs adjacent to a region to which the Sin protein binds. However, in mixing competition experiments we have shown that Hpr and Sin binding occurred independently, and no visible alterations of protected regions were detected.
Bacillus subtilis↗
Transcriptional regulation of a Bacillus subtilis dipeptide transport operon.
The Bacillus subtilis dciA operon, which encodes a dipeptide transport system, was induced rapidly by several conditions that caused the cells to enter stationary phase and initiate sporulation. The in vivo start point of transcription was mapped precisely and shown to correspond to a site of transcription initiation in vitro by the major vegetative form of RNA polymerase. Post-exponential expression was prevented by a mutation in the spo0A gene (whose product is a known regulator of early sporulation genes) but was restored in a spo0A abrB double mutant. This implicated AbrB, another known regulator, as a repressor of dciA. In fact, purified AbrB protein bound to a portion of the dciA promoter region, protecting it against DNase I digestion. Expression of dciA in growing cells was also repressed independently by glucose and by a mixture of amino acids; neither of these effects was mediated by AbrB.
Adenosine↗
The transition state transcription regulator abrB of Bacillus subtilis is a DNA binding protein.
The product of the abrB gene of Bacillus subtilis is an ambiactive repressor and activator of the transcription of genes expressed during the transition state between vegetative growth and the onset of stationary phase and sporulation. Purified AbrB protein binds specifically in a highly co-operative fashion to fragments of DNA containing the promoters it affects. DNase I footprints of the binding regions in these promoters revealed large protected areas of 50-120 nucleotides or more depending on the promoter. Methylation protection experiments gave protected guanine residues on only one face of the DNA helix. A consensus sequence could be deduced around these guanine residues that was not found around non-protected guanine residues in the footprint region. The results suggested that stationary phase functions and sporulation are repressed during active growth by AbrB and other transition state regulators by binding to the affected promoters in a concentration-dependent manner.
Bacillus subtilis↗
The transition state transcription regulator AbrB of Bacillus subtilis is autoregulated during vegetative growth.
The DNA-binding AbrB protein of Bacillus subtilis is an ambiactive transcriptional regulator of genes expressed during the transition state between vegetative growth and the onset of stationary phase and sporulation. Studies on the transcriptional control of AbrB synthesis using abrB-lacZ fusions indicated that the abrB gene was autoregulated. This was consistent with the observation that purified AbrB protein bound specifically to the promoter region of its own gene in DNase I protection experiments. The structural gene mutation abrB4 abolished the autoregulation and purified AbrB4 protein did not have the promoter binding properties associated with the wild-type protein. Both AbrB and AbrB4 proteins were shown to be hexamers of 10,500 Dalton subunits and subunit exchange occurred between the proteins in vitro. However, the presence of only one or two mutant subunits dramaticaly altered the DNA-binding ability of the multimeric protein. The results support a model in which autoregulation of the abrB gene is an important factor in preventing sporulation-associated genes from being expressed during vegetative growth.
Bacillus subtilis↗
Sequence of the Bacillus subtilis glutamine synthetase gene region.
The nucleotide sequence of the glutamine synthetase (GS) region of Bacillus subtilis has been determined and found to contain several unique features. An open reading frame (ORF) upstream of the GS structural gene is part of the same operon as GS and is involved in regulation. Two downstream ORFs are separated from glnA by an apparent Rho-independent termination site. One of the downstream ORFs encodes a very hydrophobic polypeptide and contains its own potential RNA polymerase and ribosome-binding sites. The derived amino acid (aa) sequence of B. subtilis GS is similar to that of several other prokaryotes, especially to the GS of Clostridium acetobutylicum. The B. subtilis and C. acetobutylicum enzymes differ from the others in the lack of a stretch of about 25 aa as well as the presence of extra cysteine residues in a region known to contain regulatory as well as catalytic mutations. The region around the tyrosine residue that is adenylylated in GS from many species is fairly similar in the B. subtilis GS despite its lack of adenylylation.
Amino Acid Sequence↗
Characterization of the glutamyl-tRNA(Gln)-to-glutaminyl-tRNA(Gln) amidotransferase reaction of Bacillus subtilis.
In Bacillus subtilis, the formation of glutaminyl-tRNA is accomplished by first charging tRNA(Gln) with glutamate, which is then amidated. Glutamine was preferred over asparagine and ammonia as the amide donor in vitro. There is a functional analogy of this reaction to that catalyzed by glutamine synthetase. Homogeneous glutamine synthetase, from either B. subtilis or Escherichia coli, catalyzed the amidotransferase reaction but only about 3 to 5% as well as a partially purified preparation from B. subtilis. Several classes of glutamine synthetase mutants of B. subtilis, however, were unaltered in the amidotransferase reaction. In addition, there was no inhibition by inhibitors of either glutamine synthetase or other amidotransferases. A unique, rather labile activity seems to be required for this reaction.
Ammonia↗
Identification and characterization of mutations in Escherichia coli that selectively influence the growth of hybrid lambda bacteriophages carrying the immunity region of bacteriophage P22.
Mutations in two Escherichia coli genes, sipA and sipB, result in a specific inhibition of the growth of certain hybrid lambdoid bacteriophages, lambda immP22, that have the early regulatory regions and adjacent genes from bacteriophage P22. The sipB391 mutation maps near minute 56 and exerts the strongest inhibitory effect on the growth of the hybrid phages. The sipA1 mutation maps near minute 72 and plays an auxiliary role: enhancing the action of sipB391. Such a role is not limited to sipA1, since there is a similar enhancement by the nusA1 and nusE71 mutations. The Sip-imposed restriction on the growth of lambda immP22 phages is not observed if the phage carries a mutation in the c1 gene. Perhaps this reflects the fact that the c1 product regulates phage DNA replication and is a major determinant in the decision governing whether the phage takes the lytic or lysogenic pathway. Consistent with this idea is the observation that lambda immP22 DNA replication is severely inhibited in bacteria carrying the sipB391 mutation. It is suggested that sip mutations exaggerate the normal role of c1 in limiting lytic growth. This causes a failure in the expression of sufficient amounts of some or all of the lytic gene products required for phage growth.
Bacteriophage lambda↗
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↗
The effect of supercoiling on the in vitro transcription of the spoIIA operon from Bacillus subtilis.
The spoIIA operon codes for an alternative sigma factor which appears in the early stages of a sigma factor expression cascade during sporulation in Bacillus subtilis. We have used a single round in vitro transcription assay to probe requirements for transcription initiation at the spoIIA promoter. Core RNA polymerase or holoenzyme containing sigma A was reconstituted with sigma H protein and used to transcribe the spoIIA promoter. Formation of heparin resistant transcription initiation complexes required that the spoIIA template be supercoiled. Topoisomers of the spoIIA template were created and transcribed at various temperatures. Changes in the superhelicity of template DNA had a significant influence on the amount of transcription complexes formed at the spoIIA promoter.
Bacillus subtilis↗