Mark A. Strauch

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.

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.

Postexponential Regulation of sin Operon Expression in Bacillus subtilis

The expression of many gene products required during the early stages of Bacillus subtilis sporulation is regulated by sinIR operon proteins. Transcription of sinIR from the P1 promoter is induced at the end of exponential growth. In vivo transcription studies suggest that P1 induction is repressed by the transition-state regulatory protein Hpr and is induced by the phosphorylated form of Spo0A. In vitro DNase I footprinting studies confirmed that Hpr, AbrB, and Spo0A are trans-acting transcriptional factors that bind to the P1 promoter region of sinIR. We have also determined that the P1 promoter is transcribed in vitro by the major vegetative sigma factor, final sigma(A), form of RNA polymerase.

Novel DNA binding domain and genetic regulation model of Bacillus subtilis transition state regulator AbrB

We have determined the high resolution NMR solution structure of the novel DNA binding domain of the Bacillus subtilis transition state regulator AbrB. Comparisons of the AbrB DNA binding domain with DNA binding proteins of known structure show that it is a member of a completely novel class of DNA recognition folds that employs a dimeric topology for cellular function. This new DNA binding conformation is referred to as the looped-hinge helix fold. Sequence homology investigations show that this DNA binding topology is found in other disparately related microbes. Structural analysis of the AbrB DNA binding domain together with bioanalytical and mutagenic data of full length AbrB allows us to construct a general model that describes the genetic regulation properties of AbrB.

Abh and AbrB Control of Bacillus subtilis Antimicrobial Gene Expression

The Bacillus subtilis abh gene encodes a protein whose N-terminal domain has 74% identity to the DNA-binding domain of the global regulatory protein AbrB. Strains with a mutation in abh showed alterations in the production of antimicrobial compounds directed against some other Bacillus species and gram-positive microbes. Relative to its wild-type parental strain, the abh mutant was found deficient, enhanced, or unaffected for the production of antimicrobial activity. Using lacZ fusions, we examined the effects of abh upon the expression of 10 promoters known to be regulated by AbrB, including five that transcribe well-characterized antimicrobial functions (SdpC, SkfA, TasA, sublancin, and subtilosin). For an otherwise wild-type background, the results show that Abh plays a negative regulatory role in the expression of four of the promoters, a positive role for the expression of three, and no apparent regulatory role in the expression of the other three promoters. Binding of AbrB and Abh to the promoter regions was examined using DNase I footprinting, and the results revealed significant differences. The transcription of abh is not autoregulated, but it is subject to a degree of AbrB-afforded negative regulation. The results indicate that Abh is part of the complex interconnected regulatory system that controls gene expression during the transition from active growth to stationary phase.

Complex Regulation of the Bacillus subtilis Aconitase Gene

The roles of the CcpC, CodY, and AbrB proteins in regulation of the Bacillus subtilis aconitase (citB) gene were found to be distinct and to vary with the conditions and phase of growth. CcpC, a citrate-inhibited repressor that is the primary factor regulating citB expression in minimal-glucose-glutamine medium, also contributed to repression of citB during exponential-phase growth in broth medium. A null mutation in codY had no effect on citB expression during growth in minimal medium even when combined with ccpC and abrB mutations. However, a codY mutation slightly relieved repression during exponential growth in broth medium and completely derepressed citB expression when combined with a ccpC mutation. An abrB mutation led to decreased expression of citB during stationary phase in both broth and minimal medium. All three proteins bound in vitro to specific and partially overlapping sites within the citB regulatory region. Interaction of CcpC and CodY with the citB promoter region was partially competitive.

A positive feedback loop controls transcription of the spo0F gene, a component of the sporulation phosphorelay In Bacillus subtilis

The spo0Fgene of Bacillus subtilisencodes 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 spo0Fexpression 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.

In vitro selection of optimal AbrB-binding sites: comparison to known in vivo sites indicates flexibility in AbrB binding and recognition of three-dimensional DNA structures

The AbrB protein of Bacillus subtilis regulates expression of numerous genes, primarily through specific binding interactions to DNA regions containing transcriptional promoters. Although over 15 target regions for AbrB binding to chromosomally located sequences have been analysed by DNase I footprinting, no obvious consensus sequence or motif has yet emerged from their examination. Using in vitro selection techniques, we have isolated optimal AbrB‐binding sites from oligonucleotides containing 22 or 44 random base pairs. The best of these sites have an apparent in vitro Kd which is fivefold lower than a similar‐sized DNA fragment containing the sequence corresponding to the AbrB‐binding site on the spoOE gene. We tested one of the sites in vivo and found that it confers AbrB‐mediated control upon a promoter not normally regulated by AbrB. In each of four separate trials, the selected sites possess motifs that converge to a simple consensus. It is argued that the nature and spacing of these motifs produce a type of three‐dimensional DNA structure recognizable by AbrB, and that known in vivo sites, which lack these motifs, possess an approximation of the optimal structural determinant.

Regulation of Bacillus subtilis Gene Expression during the Transition from Exponential Growth to Stationary Phase

Publisher Summary This chapter discusses the current understanding of B. subtilis regulatory proteins. A number of regulatory proteins function to activate or modulate expression levels of many transition-state genes. The main teleological difference between these regulators and the transition-state regulators just discussed is that, while the latter are active during vegetative growth to silence post-exponential specific genes, the former primarily make their presence felt only after the onset of the transition state. Genetic and biochemical aspects of B. subtilis post-exponential gene expression have been studied intensively for some time because of their association with the developmental program of endospore formation. Therefore, the transition state of B. subtilis can be viewed as a crossroads, where functions necessary for smooth entry into any of the alternate paths are present, but before any final commitment has been made. The proper regulation of gene expression during the transition state is obviously critical for optimizing survival. In fact, in its natural soil habitat, where nutrients are usually limiting, the transition state is probably the predominant metabolically active state of a Bacillus cell.

cis‐Unsaturated fatty acids specifically inhibit a signal‐transducing protein kinase required for initiation of sporulation in Bacillus subtilis

The initiation of sporutation 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.