Transcriptional regulation of adhesive properties of Bacillus subtilis to extracellular matrix proteins through the fibronectin-binding protein YloA.

Bacterial adherence to extracellular matrix proteins (ECMp) plays important roles during host-pathogen interaction, however its genetic regulation remains poorly understood. yloA of the model bacterium Bacillus subtilis shows high homology to genes encoding fibronectin-binding proteins of Gram-positive pathogens. Here, we characterized the regulatory network of YloA-dependent adhesive properties of the probiotic B. subtilis natto (Bsn). YloA-proficient, but not YloA-deficient, Bsn specifically bound to ECMp in a concentration-dependent manner and were proficient in biofilm formation. yloA expression showed a continuous increase in activity during the growth phase and decreased during the stationary phase. The transcription factors AbrB and DegU downregulated yloA expression during the logarithmic and stationary growth phases respectively. Analysis of the yloA promoter region revealed the presence of AT-rich direct and inverted repeats previously reported to function as DegU-recognized binding sites. In spo0A cells, yloA expression was completely turned off because of upregulation of AbrB throughout growth. Accordingly, DNase I footprinting analysis confirmed that AbrB bound to the promoter region of yloA. Interestingly, Bsn bound fibronectin with higher affinity, lower Kd, than several bacterial pathogens and competitively excluded them from binding to immobilized-fibronectin, a finding that might be important for the anti-infective properties of B. subtilis and its relatives.

Interactive regulation by the Bacillus subtilis global regulators CodY and ScoC.

CodY and ScoC are Bacillus subtilis transcriptional regulators that control the expression of dozens of genes and operons. Using scoC-lacZ fusions and DNA-binding experiments, we show here that scoC is directly repressed by CodY. This effect creates multiple forms of cascade regulation. For instance, expression of the dtpT gene, which is directly and negatively controlled by ScoC and encodes a putative oligopeptide permease, was activated indirectly by CodY due to CodY-mediated repression of scoC. The opp operon, which encodes an oligopeptide permease that is essential for sporulation and genetic competence development, proved to be a direct target of repression by both ScoC and CodY but was not significantly affected in codY or scoC single mutants. The combined actions of CodY and ScoC maintain opp repression when either one of the regulators loses activity but limit the level of repression to that provided by one of the regulators acting alone. Under conditions of nitrogen limitation, repression by ScoC of dtpT and opp was partly prevented by TnrA. Thus, the functioning of ScoC is determined by other transcription factors via modulation of its expression or DNA binding.

SigmaX is involved in controlling Bacillus subtilis biofilm architecture through the AbrB homologue Abh.

A characteristic feature of biofilm formation is the production of a protective extracellular polymeric matrix. In the gram-positive bacterium Bacillus subtilis, the biofilm matrix is synthesized by the products of the epsABCDEFGHIJKLMNO operon (hereafter called the eps operon) and yqxM-sipW-tasA loci. Transcription from these operons is repressed by two key regulators, AbrB and SinR. Relief of inhibition is necessary to allow biofilm formation to proceed. Here we present data indicating that Abh, a sequence and structural homologue of AbrB, regulates biofilm architecture by B. subtilis when colony morphology and pellicle formation are assessed. Data indicating that abh expression is dependent on the environmental signals that stimulate the activity of the extracytoplasmic function sigma-factor sigma(X) are shown. We demonstrate that expression of slrR, the proposed activator of yqxM transcription, is positively controlled by Abh. Furthermore, Abh is shown to activate transcription from the promoter of the eps operon through its control of SlrR. These findings add to the increasingly complex transcriptional network that controls biofilm formation by B. subtilis.

Insights into the nature of DNA binding of AbrB-like transcription factors.

Understanding the DNA recognition and binding by the AbrB-like family of transcriptional regulators is of significant interest since these proteins enable bacteria to elicit the appropriate response to diverse environmental stimuli. Although these "transition-state regulator" proteins have been well characterized at the genetic level, the general and specific mechanisms of DNA binding remain elusive. We present RDC-refined NMR solution structures and dynamic properties of the DNA-binding domains of three Bacillus subtilis transition-state regulators: AbrB, Abh, and SpoVT. We combined previously investigated DNase I footprinting, DNA methylation, gel-shift assays, and mutagenic and NMR studies to generate a structural model of the complex between AbrBN(55) and its cognate promoter, abrB8. These investigations have enabled us to generate a model for the specific nature of the transition-state regulator-DNA interaction, a structure that has remained elusive thus far.

Influence of the sigmaB stress factor and yxaB, the gene for a putative exopolysaccharide synthase under sigmaB Control, on biofilm formation.

Bacillus subtilis forms structured communities of biofilms encased in an exopolysaccharide matrix on solid surfaces and at the air-liquid interface. It is postulated that nonoptimal growth conditions induce this multicellular behavior. We showed that under laboratory conditions a strain deleted for sigB was unable to form a floating pellicle on the surface of a liquid medium. However, overexpression of yxaB, encoding a putative exopolysaccharide synthase, from a p(Spac) promoter in a sigB-deleted strain resulted in partial recovery of the wild-type phenotype, indicating the participation of the YxaB protein in this multicellular process. We present data concerning the regulation of transcription of genes yxaB and yxaA, encoding a putative glycerate kinase. Both genes are cotranscribed as a single transcription unit from a sigma(A)-dependent promoter during vegetative growth of a liquid bacterial culture. The promoter driving transcription of the yxaAB operon is regulated by AbrB. In addition, the second gene in the operon, yxaB, possesses its own promoter, which is recognized by RNA polymerase containing the sigma(B) subunit. This transcription start site is used under general stress conditions, resulting in increased expression.

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.

LiaRS-dependent gene expression is embedded in transition state regulation in Bacillus subtilis.

Maintaining envelope integrity is crucial for the survival of any bacterial cell, especially those living in a complex and ever-changing habitat such as the soil ecosystem. The LiaRS two-component system is part of the regulatory network orchestrating the cell-envelope stress response in Bacillus subtilis. It responds to perturbations of the cell envelope, especially the presence of antibiotics that interfere with the lipid II cycle, such as bacitracin or vancomycin. LiaRS-dependent regulation is strictly repressed by the membrane protein LiaF in the absence of inducing conditions. Here, it is shown that the LiaR-dependent liaI promoter is induced at the onset of stationary phase without addition of exogenous stresses. Its activity is embedded in the complex regulatory cascade governing adaptation at the onset of stationary phase. The liaI promoter is directly repressed by the transition state regulator AbrB and responds indirectly to the activity of Spo0A, the master regulator of sporulation. The activity of the liaI promoter is therefore tightly regulated by at least five regulators to ensure an appropriate level of liaIH expression.

NMR structure of AbhN and comparison with AbrBN: FIRST insights into the DNA binding promiscuity and specificity of AbrB-like transition state regulator proteins.

Understanding the molecular mechanisms of transition state regulator proteins is critical, since they play a pivotal role in the ability of bacteria to cope with changing environments. Although much effort has focused on their genetic characterization, little is known about their structural and functional conservation. Here we present the high resolution NMR solution structure of the N-terminal domain of the Bacillus subtilis transition state regulator Abh (AbhN), only the second such structure to date. We then compare AbhN to the N-terminal DNA-binding domain of B. subtilis AbrB (AbrBN). This is the first such comparison between two AbrB-like transition state regulators. AbhN and AbrBN are very similar, suggesting a common structural basis for their DNA binding. However, we also note subtle variances between the AbhN and AbrBN structures, which may play important roles in DNA target specificity. The results of accompanying in vitro DNA-binding studies serve to highlight binding differences between the two proteins.

Independent and interchangeable multimerization domains of the AbrB, Abh, and SpoVT global regulatory proteins.

The global regulators AbrB, Abh, and SpoVT are paralogous proteins showing their most extensive sequence homologies in the DNA-binding amino-terminal regions (about 50 residues). The carboxyl-terminal portion of AbrB has been hypothesized to be a multimerization domain with little if any role in DNA-binding recognition or specificity. To investigate the multimerization potentials of the carboxyl-terminal portions of AbrB, Abh, and SpoVT we utilized an in vivo multimerization assay system based upon fusion of the domains to the DNA binding domain of the lambda cI repressor protein. The results indicate that the N and C domains of all three paralogues are independent dimerization modules and that the intact Abh and SpoVT proteins are most probably tetramers. Chimeric proteins consisting of the AbrB N-terminal DNA-binding domain fused to the C domain of either Abh or SpoVT are indistinguishable from wild-type AbrB in their ability to regulate an AbrB target promoter in vivo.

The DNA-binding specificity of the Bacillus anthracis AbrB protein.

The Bacillus subtilis AbrB protein is a DNA-binding global regulator of a plethora of functions that are expressed during the transition from exponential growth to stationary phase and under suboptimal growth conditions. AbrB orthologues have been identified in a variety of prokaryotic organisms, notably in all species of Bacillus, Clostridium and Listeria that have been examined. Based on amino acid sequence identity in the N-terminal domains of the orthologues from B. subtilis and Bacillus anthracis, it was predicted that the proteins might display identical DNA-binding specificities. The binding of purified B. anthracis AbrB (AbrB(BA)) and purified B. subtilis AbrB (AbrB(BS)) at DNA targets of B. subtilis, B. anthracis and a synthetic origin was compared. In all cases examined, DNA-binding specificity was identical as judged by DNase I footprinting. In B. subtilis cells, the B. anthracis promoters from the atxA and abrB genes were regulated by AbrB(BS), and the B. subtilis promoter from the yxbB operon was regulated by AbrB(BA).

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.

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.

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.

AbrB is a regulator of the sigma(W) regulon in Bacillus subtilis.

The Bacillus subtilis global regulator AbrB was found to negatively control expression of sigW and genes of the sigma(W) regulon. AbrB bound to DNA regions in the autoregulatory sigW promoter and to some, but not all, of the other sigma(W)-dependent promoters in B. subtilis. Defects in antibiotic resistance properties caused by spo0A mutations are at least partially correlated with AbrB repression of the sigma(W) regulon.

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.