Transition State Regulator AbrB Research Articles

A Gene Encoding a Holin-Like Protein Involved in Spore Morphogenesis and Spore Germination inBacillus subtilis

We report here studies of expression and functional analysis of a Bacillus subtilis gene, ywcE, which codes for a product with features of a holin. Primer extension analysis of ywcE transcription revealed that a single transcript accumulated from the onset of sporulation onwards, produced from a sigma(A)-type promoter bearing the TG dinucleotide motif of "extended" -10 promoters. No primer extension product was detected in vivo during growth. However, specific runoff products were produced in vitro from the ywcE promoter by purified sigma(A)-containing RNA polymerase (Esigma(A)), and the in vivo and in vitro transcription start sites were identical. These results suggested that utilization of the ywcE promoter by Esigma(A) during growth was subjected to repression. Studies with a lacZ fusion revealed that the transition-state regulator AbrB repressed the transcription of ywcE during growth. This repression was reversed at the onset of sporulation in a Spo0A-dependent manner, but Spo0A did not appear to contribute otherwise to ywcE transcription. We found ywcE to be required for proper spore morphogenesis. Spores of the ywcE mutant showed a reduced outer coat which lacked the characteristic striated pattern, and the outer coat failed to attach to the underlying inner coat. The mutant spores also accumulated reduced levels of dipicolinic acid. ywcE was also found to be important for spore germination.

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

Nat. Struct. Biol. 7, 1139–1146 (2000). Based on a recent bioinformatics analysis, we have refined the dimeric interface of the N-terminal domain of the Bacillus subtilis transition state regulator AbrB. Comprehensive sets of ∼300 unambiguous distance constraints (NOEs) and hydrogen exchange data have been collected that now accurately define this interface.

Evaluation of the DNA Binding Tendencies of the Transition State Regulator AbrB

Global transition state regulator proteins represent one of the most diverse classes of prokaryotic transcription factors. One such transition state regulator, AbrB from Bacillus subtilis, is known to bind more than 60 gene targets yet displays specificity within this target set by binding each promoter with a different affinity. Microelectrospray ionization mass spectrometry (microESI-MS), circular dichroism, fluorescence, UV spectroscopy, and molecular modeling were used to elucidate differences among AbrB, DNA, and AbrB-DNA complexes. MicroESI-MS analysis of AbrB confirmed its stable macromolecular state as being tetrameric and verified the same stoichiometric state in complex with DNA targets. MicroESI-MS, circular dichroism, and fluorescence provided relative binding affinities for AbrB-DNA interactions in a qualitative manner. UV spectroscopy was used in a quantitative manner to determine solution phase dissociation constants for AbrB-DNA complexes. General DNA structural parameters for all known natural AbrB binding sequences were also studied and significant similarities in topological constraints (stretch, opening, and propeller twist) were observed. It is likely that these parameters contribute to the differential binding proclivities of AbrB. In addition to providing an improved understanding of transition state regulator-DNA binding properties and structural tendencies of target promoters, this comprehensive and corroborative spectroscopic study endorses the use of microESI-MS for rapidly ascertaining qualitative binding trends in noncovalent systems in a high-throughput manner.

Novel roles of the master transcription factors Spo0A and sigmaB for survival and sporulation of Bacillus subtilis at low growth temperature.

Spore development and stress resistance in Bacillus subtilis are governed by the master transcription factors Spo0A and sigma(B), respectively. Here we show that the coding genes for both regulatory proteins are dramatically induced, during logarithmic growth, after a temperature downshift from 37 to 20 degrees C. The loss of sigma(B) reduces the stationary-phase viability of cold-adapted cells 10- to 50-fold. Furthermore, we show that sigma(B) activity is required at a late stage of development for efficient sporulation at a low temperature. On the other hand, Spo0A loss dramatically reduces the stationary-phase viability of cold-adapted cells 10,000-fold. We show that the requirement of Spo0A for cellular survival during the cold is independent of the activity of the key transition state regulator AbrB and of the simple loss of sporulation ability. Furthermore, Spo0A, and not proficiency in sporulation, is required for the development of complete stress resistance of cold-adapted cells to heat shock (54 degrees C, 1 h), since a loss of Spo0A, but not a loss of the essential sporulation transcription factor sigma(F), reduced the cellular survival in response to heat by more than 1,000-fold. The overall results argue for new and important roles for Spo0A in the development of full stress resistance by nonsporulating cells and for sigma(B) in sporulation proficiency at a low temperature.

The Bacillus subtilis transition state regulator AbrB binds to the -35 promoter region of comK.

Genetic competence is a differentiation process initiated by Bacillus subtilis as a result of nutritional deprivation, and is controlled by a complex signal transduction cascade. The promoter of comK, encoding the competence transcription factor, is regulated by at least four different transcription factors: Rok, CodY, DegU and ComK itself. Genetic data have shown that comK expression is influenced by the transition state regulator AbrB as well. In this paper we show that AbrB binds specifically to the comK promoter and covers the RNA polymerase binding site, making it the fifth transcription factor regulating the activity of the comK promoter.

A plasmid-borne Rap-Phr system of Bacillus subtilis can mediate cell-density controlled production of extracellular proteases.

Bacillus subtilis uses two-component signal transduction systems to sense intra- and extracellular stimuli to adapt to fluctuating environmental situations. Regulator aspartate phosphatases (Raps) have important roles in these processes, as they can dephosphorylate certain response-regulators, and are themselves subject to cell-density-controlled inhibition by secreted Phr (phosphate regulator) peptides. Eleven chromosomal genes encode this family of phosphatases, but in addition, certain strains contain endogenous plasmids with genes for homologous Rap-Phr systems. Plasmid pTA1060 encodes Rap60 and its antagonistic signalling molecule Phr60. Strikingly, expression of Rap60 in B. subtilis 168 strongly repressed the production of proteolytic enzymes. In fact, the transcription of the aprE gene, encoding a major extracellular protease, was shown to be decreased upon Rap60 expression, whereas this effect could be antagonized by the extracellular addition of synthetic Phr60 pentapeptide. Finally, transcription studies suggest that Rap60 dephosphorylates a component of the phosphorelay and is coupled to aprE transcription by the transition-state regulator AbrB. In conclusion, these data show that endogenous plasmids contain functional Rap-Phr systems and for the first time, that Rap-Phr systems can mediate cell-density controlled production of secreted proteases. This quorum-sensing mechanism might enable B. subtilis to suppress protease production under conditions of low cell densities when nutrients are still available in sufficient amounts.

The Bacillus subtilis transition state regulator AbrB binds to the −35 promoter region of comK

Genetic competence is a differentiation process initiated by Bacillus subtilis as a result of nutritional deprivation, and is controlled by a complex signal transduction cascade. The promoter of comK, encoding the competence transcription factor, is regulated by at least four different transcription factors: Rok, CodY, DegU and ComK itself. Genetic data have shown that comK expression is influenced by the transition state regulator AbrB as well. In this paper we show that AbrB binds specifically to the comK promoter and covers the RNA polymerase binding site, making it the fifth transcription factor regulating the activity of the comK promoter.

Antibiotics that inhibit cell wall biosynthesis induce expression of the Bacillus subtilis sigma(W) and sigma(M) regulons.

Bacillus subtilis encodes seven extracytoplasmic function (ECF) sigma factors. The sigma(W) regulon includes functions involved in detoxification and protection against antimicrobials, whereas sigma(M) is essential for growth at high salt concentrations. We now report that antibiotics that inhibit cell wall biosynthesis induce both sigma(W) and sigma(M) regulons as monitored using DNA microarrays. Induction of selected sigma(W)-dependent genes was confirmed using lacZ reporter fusions and Northern blot analysis. The ability of vancomycin to induce the sigma(W) regulon is dependent on both sigma(W) and the cognate anti-sigma, RsiW, but is independent of the transition state regulator AbrB. These results suggest that the membrane-localized RsiW anti-sigma(W) factor mediates the transcriptional response to cell wall stress. Our findings are consistent with the idea that one function of ECF sigma factors is to coordinate antibiosis stress responses and cell envelope homeostasis.

Macromolecular Assembly of the Transition State Regulator AbrB in Its Unbound and Complexed States Probed by Microelectrospray Ionization Mass Spectrometry

The Bacillus subtilis global transition-state regulator AbrB specifically recognizes over 60 different DNA regulatory regions of genes expressed during cellular response to suboptimal environments. Most interestingly the DNA regions recognized by AbrB share no obvious consensus base sequence. To more clearly understand the functional aspects of AbrB activity, microelectrospray ionization mass spectrometry has been employed to resolve the macromolecular assembly of unbound and DNA-bound AbrB. Analysis of the N-terminal DNA binding domain of AbrB (AbrBN53, residues 1–53) demonstrates that AbrBN53 is a stable dimer, showing no apparent exchange with a monomeric form as a function of pH, ionic strength, solvent, or protein concentration. AbrBN53 demonstrates a capacity for DNA binding, underscoring the role of the N-terminal domain in both DNA recognition and dimerization. Full-length AbrB is shown to exist as a homotetramer. An investigation of the binding of AbrBN53 and AbrB to the natural DNA target element sinIR shows that AbrBN53 binds as a dimer and AbrB binds as a tetramer. This study represents the first detailed characterization of the stoichiometry of a transition-state regulator binding to one of its target promoters.

Stoichiometries of protein-protein/DNA binding and conformational changes for the transition-state regulator AbrB measured by pseudo cell-size exclusion chromatography-mass spectrometry.

We have developed on-line pseudo cell-size exclusion chromatography-mass spectrometry (PsC-SEC-MS) for the rapid, real time analyses of noncovalently bound protein complexes. The methodology can be used to determine constituent components of such complexes, as well as exact stoichiometries. Furthermore, it enables the efficient determination of gross conformational changes upon complexation. The power of the new approach is demonstrated in the analysis of the global transition-state regulator AbrB and its complex with a target DNA sequence from the promoter sinIR. Using PsC-SEC-MS, we confirm that AbrB is assembled as a homotetramer and not as a homohexamer as previously suggested. Additionally, we show that AbrB binds to the sinIR DNA target element as a homotetramer, affording a 4:1 protein:DNA stoichiometry. Finally, we demonstrate that when the complex binds to sinIR, the hydrodynamic volume (size) of the complex is notably reduced compared to that of the apoprotein, indicating a protein conformational change.

Dual control of subtilin biosynthesis and immunity in Bacillus subtilis.

The production of the peptide antibiotic (lantibiotic) subtilin in Bacillus subtilis ATCC 6633 is highly regulated. Transcriptional organization and regulation of the subtilin gene cluster encompassing 11 genes was characterized. Two polycistronic mRNAs encoding transcript spaBTC (6.8 kb) and encoding transcript spaIFEG (3.5 kb) as well as the monocistronic spaS (0.3 kb) mRNA were shown by Northern hybridization. Primer extension experiments and beta-galactosidase fusions confirmed three independent promoter sites preceding genes spaB, spaS and spaI. beta-Galactosidase expression of spaB, spaS and spaI promoter lacZ fusions initiated in mid-exponential growth. Maximal activities were reached at the transition to stationary growth and were collinear with subtilin production. The lacZ activity was dependent on co-expression with the two-component regulatory system spaRK. The presence of subtilin was needed for efficient expression of all three promoter lacZ fusions. This suggests a transcriptional autoregulation according to a quorum-sensing mechanism with subtilin as autoinducer and signal transduction via SpaRK. Additionally, spaR expression was found to be under positive control of the alternative sigma factor H. Deletion of sigma H strongly decreased subtilin production. Full subtilin production could be restored after in-trans complementation of spaR. Deletion of the major B. subtilis transition state regulator AbrB strongly increased subtilin production. These results show that the spaRK two-component regulatory system, and hence subtilin biosynthesis and immunity, is under dual control of two independent regulatory systems: autoinduction via subtilin and transcriptional regulation via sigma factor H.

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The DNA-binding domain in the Bacillus subtilis transition-state regulator AbrB employs significant motion for promiscuous DNA recognition

AbrB is a Bacillus subtilis protein responsible for regulating a diverse array of unrelated genes during periods of sub-optimal growth conditions. DNA binding by AbrB is unique in that sequence recognition is specific, yet no obvious consensus sequence of bound promoter regions is apparent. The N-terminal domain is a recently characterized representative of a novel class of DNA-binding proteins that possess a looped-hinge helix DNA-binding topology. Although the structural characterization of this DNA-binding topology contributed to an understanding of the architectural basis for recognition of DNA target sequences, specific mechanisms responsible for promiscuity in DNA sequence recognition still were not apparent. Analysis of 15N backbone relaxation parameters shows that dynamic motion of regions directly linked to DNA binding show concerted motion on the microsecond-millisecond timescale. Furthermore, dynamic motion of the hinge region suggests that the DNA-binding region is capable of conformational orientations that allow it to accommodate DNA sequence variability in the cognate binding sites.

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.

Molecular characterization of the transition state regulator AbrB from Bacillus stearothermophilus

The Bacillus subtilis transition state regulator AbrB su is a DNA-binding protein that acts on several genes either as activator, repressor, or preventer. However, among genes under its control, neither common binding sites could be identified nor could the structural features of this broad and specific interaction be elucidated. Attempts to elucidate these interesting features by crystallizing AbrB su have failed so far. Therefore, to solve this problem, we focused in this work on identifying an AbrB su homologue from Bacillus stearothermophilus. Using a novel method, the entire abrB st gene of B. stearothermophilus was cloned and sequenced. The gene encodes a 95 amino acid protein that shows 77% identity and 85% similarity to the mesophilic B. subtilis protein. A calmodulin binding peptide-tagged fusion of the thermophilic gene was constructed for overexpression and efficient affinity column purification of the AbrB st protein. The purified protein showed, after removal of the tag, an oligomerization behavior through hexamer formation that is essential for its DNA binding activity.

Dual Control of sbo-alb Operon Expression by the Spo0 and ResDE Systems of Signal Transduction under Anaerobic Conditions in Bacillus subtilis

The Bacillus subtilis sbo-alb operon contains sboA, the structural gene for the bacteriocin subtilosin, and the alb genes required for subtilosin production. Transcription from the sbo-alb promoter is highly induced by oxygen limitation. The transcriptional regulation of the sbo-alb operon is under dual control involving the transition state regulator AbrB and the two-component regulatory proteins ResD and ResE.

Genes of the sbo-alb locus of Bacillus subtilis are required for production of the antilisterial bacteriocin subtilosin.

Bacillus subtilis JH642 and a wild strain of B. subtilis called 22a both produce an antilisterial peptide that can be purified by anion-exchange and gel filtration chromatography. Amino acid analysis confirmed that the substance was the cyclic bacteriocin subtilosin. A mutant defective in production of the substance was isolated from a plasmid gene disruption library. The plasmid insertion conferring the antilisterial-peptide-negative phenotype was located in a seven-gene operon (alb, for antilisterial bacteriocin) residing immediately downstream from the sbo gene, which encodes the precursor of subtilosin. An insertion mutation in the sbo gene also conferred loss of antilisterial activity. Comparison of the presubtilosin and mature subtilosin sequences suggested that certain residues undergo unusual posttranslational modifications unlike those occurring during the synthesis of class I (lantibiotic) or some class II bacteriocins. The putative products of the genes of the operon identified show similarities to peptidases and transport proteins that may function in processing and export. Two alb gene products resemble proteins that function in pyrroloquinoline quinone biosynthesis. The use of lacZ-alb and lacZ-sbo gene fusions, along with primer extension analysis, revealed that the sbo-alb genes are transcribed from a major promoter, residing upstream of sbo, that is very likely utilized by the sigma(A) form of RNA polymerase. The sbo and alb genes are negatively regulated by the global transition state regulator AbrB and are also under positive autoregulation that is not mediated by the subtilosin peptide but instead requires one or more of the alb gene products.

Expression of AbrB, a transition state regulator from Bacillus subtilis, is growth phase dependent in a manner resembling that of Fis, the nucleoid binding protein from Escherichia coli.

The transition state regulator AbrB functions as an activator, a repressor, and a preventer of gene expression in Bacillus subtilis. In this paper, we show that expression of abrB is growth phase dependent. Accumulation of abrB transcript is restricted to a short period spanning the transition between the lag and exponential phases of the growth cycle. The level of abrB transcript then falls sharply, and transcript cannot be detected at the mid-exponential period of the growth cycle. The level of AbrB protein is also maximal during early exponential growth but decreases gradually throughout the remainder of the growth cycle. The abrupt reduction of abrB transcript level during the early period of the growth cycle is effected by the phosphorylated form of the response regulator Spo0p3and to a lesser extent by negative autoregulation. The growth cycle-dependent expression of abrB is very similar to that observed for fis in Escherichia coli and in Salmonella typhimurium. Although AbrB and Fis are not homologous proteins, they display extensive similarity in terms of size, DNA binding characteristics, growth cycle-dependent patterns of expression, and their control over the expression of a varied group of operons. We hypothesize therefore that AbrB, like Fis, is a nucleoid binding protein.

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.

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.