Transition State Regulator Research Articles

The signal‐transduction network for Pho regulation in Bacillus subtilis

Depletion of nutrients, including phosphate, is a stress often encountered by a bacterial cell, and results in slowed growth, marking the cessation of exponential growth. Genes that are transcriptionally activated during phosphate starvation have been used to examine the signal-transduction mechanisms governing the Pho regulon in Bacillus subtilis. Alkaline phosphatase, the traditional reporter protein for Pho regulation in prokaryotes, is encoded by a multigene family in B. subtilis. Characterization of the alkaline phosphatase family was a breakthrough in the study of regulation of the Pho regulon, especially the discovery of promoter elements exclusively responsive to phosphate-starvation regulation. Current data suggest that at least three two-component signal-transduction systems interact, forming a regulatory network that controls the phosphate-deficiency response in B. subtilis. The interconnected pathways involve the PhoP-PhoR system, whose primary role is to mediate the phosphate-deficiency response; the SpoO phosphorelay required for the initiation of sporulation; and a newly discovered signal-transduction system, ResD-ResE, which also has a role in respiratory regulation during late growth. Parallel pathways positively regulate the Pho response via PhoP-PhoR. One pathway includes the ResD-ResE system, while the other involves a transition-state regulator, AbrB. The SpoO system represses the Pho response by negatively regulating both pathways. This review will discuss how the characterization of the APase multigene family made possible studies which show that the Pho regulon in B. subtilis is regulated by the integrated action of the Res, Pho and Spo signal-transduction systems.

The citrulline biosynthetic operon, argC-F, and a ribose transport operon, rbs, from Bacillus subtilis are negatively regulated by Spo0A.

A method is described here that can be used to identify operons whose expression is controlled by any particular regulator protein. This method was used to identify operons whose expression is negatively regulated by Spo0A in Bacillus subtilis. Twenty-eight strains were identified, each of which contains an operon-lacZ transcriptional fusion, negatively regulated, either directly or indirectly, by Spo0A. In one of these strains (CSA8), the lacZ gene is fused to the argC-F operon positioned at 100 degrees on the B. subtilis chromosome. The regulated expression of this operon by Spo0A-P is mediated indirectly through the transition state regulator AbrB and is manifest only during growth on solid medium. In a second strain (CSA15), the lacZ gene is fused to an operon encoding a transport system which displays features characteristic of the ABC group of transporters, and which has a very high level of identity to the ribose transport system from Escherichia coli. Expression of the ribose transport operon is directed by a single SigA-type promoter. Transcription from this promoter is repressed by the phosphorylated form of Spo0A during the late-exponential/transition phase of the growth cycle and this control is not mediated through the transition-state regulator, AbrB.

A complex array of Hpr consensus DNA recognition sequences proximal to the enterotoxin gene in Clostridium perfringens type A

Enterotoxin production in Clostridium perfringens is both strain dependent and sporulation associated. Underlying these phenotypic observations must lie a genetic and molecular explanation and the principal keys will be held within the DNA sequence both upstream and downstream of the structural gene cpe. In accordance with the above we have sequenced 4.1 kbp of DNA upstream of cpe in the type strain NCTC 8239. A region of DNA extending up to 1.5 kb 5' to cpe is conserved in all enterotoxin-positive strains. This region contains a putative ORF with substantial homology to an ORF in the Salmonella typhimurium IS200 insertion element and, in addition, contains multiple perfect consensus DNA-binding sequences for the Bacillus subtilis transition state regulator Hpr. The detailed structural elements revealed by the sequence analysis are presented and used to develop a new perspective on the molecular basis of enterotoxin production in this important food-poisoning bacterium.

Regulation of peptide antibiotic production in Bacillus

In Bacillus species, starvation leads to the activation of a number of processes that affect the ability to survive during periods of nutritional stress. Activities that are induced include the development of genetic competence, sporulation, the synthesis of degradative enzymes, motility, and antibiotic production. The genes that function in these processes are activated during the transition from exponential to stationary phase and are controlled by mechanisms that operate primarily at the level of transcription initiation. One class of genes functions in the synthesis of special metabolites such as the peptide antibiotics tyrocidine and gramicidin S as well as the cyclic lipopeptide surfactin. These genes include the grs and tyc operons in Bacillus brevis, which encode gramicidin S synthetase and tyrocidine synthetase, respectively, and the srfA operon of Bacillus subtilis which encodes the enzymes of the surfactin synthetase complex. Peptide antibiotic biosynthesis genes are regulated by factors as diverse as the early sporulation gene product Spo0A, the transition-state regulator AbrB, and gene products (ComA, ComP, and ComQ) required for the initiation of the competence developmental pathway.

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.

Negative regulation of Bacillus subtilis sporulation by the spo0E gene product

Transcription of the Bacillus subtilis spo0E gene is controlled by the AbrB transition state regulator. In AbrB+ strains, a single transcript, P1, was observed for the spo0E gene. In an abrB4 mutant strain, a second transcription start site 3 bases upstream from P1 was found to be used for the predominant transcript. P1 transcription was insensitive to the state of the abrB gene. Mutants carrying deletion or antibiotic cassette insertion mutations in the spo0E gene were Spo+. Multiple copies of the spo0E gene, not just the promoter region, were found to render strains incapable of sporulation. Spo+ strains that arose spontaneously from such Spo- strains were found to have deletions in the spo0E coding sequence on the plasmid. Strains carrying a deletion of the spo0E gene segregated Spo- colonies. These colonies were found to have secondary mutations in or near the spo0A, spo0B, or spo0F gene, suggesting that deletion of the spo0E gene results in increased pressure to sporulate that is compensated for by inactivation of one or more of the components of the signal transduction system leading to the initiation of sporulation. spo0E deletions were suppressors of the spo0F221 missense mutation but had no effect on the regulation of the spo0F, kinA, spo0A, or spo0B genes. The results suggest that the spo0E gene product is a negative regulator of the signal transduction pathway leading to sporulation.

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.