Transition State Regulator AbrB Research Articles

Regulation of heterologous subtilin production in Bacillus subtilis W168

BackgroundSubtilin is a peptide antibiotic (lantibiotic) natively produced by Bacillus subtilis ATCC6633. It is encoded in a gene cluster spaBTCSIFEGRK (spa-locus) consisting of four transcriptional units: spaS (subtilin pre-peptide), spaBTC (modification and export), spaIFEG (immunity) and spaRK (regulation). Despite the pioneer understanding on subtilin biosynthesis, a robust platform to facilitate subtilin research and improve subtilin production is still a poorly explored spot.ResultsIn this work, the intact spa-locus was successfully integrated into the chromosome of Bacillus subtilis W168, which is the by far best-characterized Gram-positive model organism with powerful genetics and many advantages in industrial use. Through systematic analysis of spa-promoter activities in B. subtilis W168 wild type and mutant strains, our work demonstrates that subtilin is basally expressed in B. subtilis W168, and the transition state regulator AbrB strongly represses subtilin biosynthesis in a growth phase-dependent manner. The deletion of AbrB remarkably enhanced subtilin gene expression, resulting in comparable yield of bioactive subtilin production as for B. subtilis ATCC6633. However, while in B. subtilis ATCC6633 AbrB regulates subtilin gene expression via SigH, which in turn activates spaRK, AbrB of B. subtilis W168 controls subtilin gene expression in SigH-independent manner, except for the regulation of spaBTC. Furthermore, the work shows that subtilin biosynthesis in B. subtilis W168 is regulated by the two-component regulatory system SpaRK and strictly relies on subtilin itself as inducer to fulfill the autoregulatory circuit. In addition, by incorporating the subtilin-producing system (spa-locus) and subtilin-reporting system (PpsdA-lux) together, we developed “online” reporter strains to efficiently monitor the dynamics of subtilin biosynthesis.ConclusionsWithin this study, the model organism B. subtilis W168 was successfully established as a novel platform for subtilin biosynthesis and the underlying regulatory mechanism was comprehensively characterized. This work will not only facilitate genetic (engineering) studies on subtilin, but also pave the way for its industrial production. More broadly, this work will shed new light on the heterologous production of other lantibiotics.

The Epipeptide Biosynthesis Locus epeXEPAB Is Widely Distributed in Firmicutes and Triggers Intrinsic Cell Envelope Stress

The epeXEPAB (formerly yydFGHIJ) locus of Bacillus subtilis encodes a minimalistic biosynthetic pathway for a linear antimicrobial epipeptide, EpeX, which is ribosomally produced and post-translationally processed by the action of the radical-SAM epimerase, EpeE, and a membrane-anchored signal 2 peptide peptidase, EpeP. The ABC transporter EpeAB provides intrinsic immunity against self-produced EpeX, without conferring resistance against extrinsically added EpeX. EpeX specifically targets, and severely perturbs the integrity of the cytoplasmic membrane, which leads to the induction of the Lia-dependent envelope stress response. Here, we provide new insights into the distribution, expression, and regulation of the minimalistic epeXEPAB locus of B. subtilis, as well as the biosynthesis and biological efficiency of the produced epipeptide EpeX*. A comprehensive comparative genomics study demonstrates that the epe-locus is restricted to but widely distributed within the phylum Firmicutes. The gene products of epeXEP are necessary and sufficient for the production of the mature antimicrobial peptide EpeX*. In B. subtilis, the epeXEPAB locus is transcribed from three different promoters, one upstream of epeX (P_epeX) and two within epeP (P_epeA1 and P_epeA2). While the latter two are mostly constitutive, P_epeX shows a growth phase-dependent induction at the onset of stationary phase. We demonstrate that this regulation is the result of the antagonistic action of two global regulators: The transition state regulator AbrB keeps the epe locus shut off during exponential growth by direct binding. This tight repression is relieved by the master regulator of sporulation, Spo0A, which counteracts the AbrB-dependent repression of epeXEPAB expression during the transition to stationary phase. The net result of these three ­promoters is an expression pattern that ensures EpeAB-dependent autoimmunity prior to EpeX* production. In the absence of EpeAB, the general envelope stress response proteins LiaIH can compensate for the loss of specific autoimmunity by providing sufficient protection against the membrane-perturbating action of EpeX*. Hence, the transcriptional regulation of epe expression and the resulting intrinsic induction of the two corresponding resistance functions, encoded by epeAB and liaIH, are well balanced to provide a need-based immunity against mature EpeX*.

Synthesis and succinylation of subtilin-like lantibiotics are strongly influenced by glucose and transition state regulator AbrB.

Subtilin and the closely related entianin are class I lantibiotics produced by different subspecies of Bacillus subtilis. Both molecules are ribosomally synthesized peptide antibiotics with unusual ring structures. Subtilin-like lantibiotics develop strong antibiotic activities against various Gram-positive organisms with an efficiency similar to that of nisin from Lactococcus lactis. In contrast to nisin, subtilin-like lantibiotics partially undergo an additional posttranslational modification, where the N-terminal tryptophan residue becomes succinylated, resulting in drastically reduced antibiotic activities. A highly sensitive high-performance liquid chromatography (HPLC)-based quantification method enabled us to determine entianin and succinylated entianin (S-entianin) concentrations in the supernatant during growth. We show that entianin synthesis and the degree of succinylation drastically change with culture conditions. In particular, increasing glucose concentrations resulted in higher entianin amounts and lower proportions of S-entianin in Landy-based media. In contrast, no succinylation was observed in medium A with 10% glucose. Interestingly, glucose retarded the expression of entianin biosynthesis genes. Furthermore, deletion of the transition state regulator AbrB resulted in a 6-fold increased entianin production in medium A with 10% glucose. This shows that entianin biosynthesis in B. subtilis is strongly influenced by glucose, in addition to its regulation by the transition state regulator AbrB. Our results suggest that the mechanism underlying the succinylation of subtilin-like lantibiotics is enzymatically catalyzed and occurs in the extracellular space or at the cellular membrane.

Structure and DNA-Binding Traits of the Transition State Regulator AbrB

The AbrB protein from Bacillus subtilis is a DNA-binding global regulator controlling the onset of a vast array of protective functions under stressful conditions. Such functions include biofilm formation, antibiotic production, competence development, extracellular enzyme production, motility, and sporulation. AbrB orthologs are known in a variety of prokaryotic organisms, most notably in all infectious strains of Clostridia, Listeria, and Bacilli. Despite its central role in bacterial response and defense, its structure has been elusive because of its highly dynamic character. Orienting its N- and C-terminal domains with respect to one another has been especially problematic. Here, we have generated astructure of full-length, tetrameric AbrB using nuclear magnetic resonance, chemical crosslinking, and mass spectrometry. We note that AbrB possesses a strip of positive electrostatic potential encompassing its DNA-binding region and that its C-terminal domain aids in DNA binding.

The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism.

Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter.

Behavior of transition state regulator AbrB in batch cultures of Bacillus thuringiensis.

The transition state regulator AbrB is involved in the regulation of various cellular functions such as exponential growth, transition state and sporulation onset, due to its ability to activate, suppress or prevent the inappropriate expression of various genes in Bacillus subtilis. In order to understand combined behavior in batch cultures of AbrB in Bacillus thuringiensis, we cloned and expressed the abrB gene of B. thuringiensis in Escherichia coli. The deduced sequence of abrB gene coded for a protein consisting of 94 amino acids with ~10.5kDa protein that shares 100 and 85% identity with those from Bacillus cereus and Bacillus subtilis. The recombinant AbrB protein was used as antigen for the production of rabbit polyclonal antibodies anti-AbrB. Two media cultures with carbon: nitrogen ratios of 7.0, but varying access to nutrients were tested in batch cultures. In the case of both media, AbrB accumulation occurred from the beginning of the process and was maximal during early exponential growth. Thereafter, the level of AbrB decreased when there were no nutrient limitations and coincided with a decreased value in specific growth rate, although growth continued exponentially. Nonetheless, sporulation onset was determined 3 h and 4 h later, in media with highly metabolizable nutrients clean medium and Farrera medium, respectively. Hence, the maximal level of AbrB accumulation in batch cultures of B. thuringiensis is not influenced by limiting nutrients; however, nutrient availability affects the required time lapse for transition state regulator accumulation.

Substitutional analysis of the C-terminal domain of AbrB revealed its essential role in DNA-binding activity.

The global transition state regulator AbrB controls more than 100 genes of the Bacillus relatives and is known to interact with varying DNA-sequences. The DNA-binding domain of the AbrB-like proteins was proposed to be located exclusively within the amino-terminal ends. However, the recognition of DNA, and specificity of the binding mechanism, remains elusive still in view of highly differing recognition sites. Here we present a substitutional analysis to examine the role of the carboxy-terminal domain of AbrB from Bacillus subtilis and Bacillus amyloliquefaciens. Our results demonstrate that the carboxy-terminal domains of AbrB affect the DNA-binding properties of the tetrameric AbrB. Most likely, the C-termini are responsible for the cooperative character observed for AbrB interaction with some DNA targets like tycA and phyC.

Cis -Acting Elements That Control Expression of the Master Virulence Regulatory Gene atxA in Bacillus anthracis

Transcription of the Bacillus anthracis structural genes for the anthrax toxin proteins and biosynthetic operon for capsule is positively regulated by AtxA, a transcription regulator with unique properties. Consistent with the role of atxA in virulence factor expression, a B. anthracis atxA-null mutant is avirulent in a murine model for anthrax. In culture, multiple signals impact atxA transcript levels, and the timing and steady-state level of atxA expression are critical for optimal toxin and capsule synthesis. Despite the apparent complex control of atxA transcription, only one trans-acting protein, the transition state regulator AbrB, has been demonstrated to interact directly with the atxA promoter. Here we employ 5' and 3' deletion analysis and site-directed mutagenesis of the atxA control region to demonstrate that atxA transcription from the major start site P1 is dependent upon a consensus sequence for the housekeeping sigma factor SigA and an A+T-rich upstream element for RNA polymerase. We also show that an additional trans-acting protein(s) binds specifically to atxA promoter sequences located between -13 and +36 relative to P1 and negatively impacts transcription. Deletion of this region increases promoter activity up to 15-fold. Site-directed mutagenesis of a 9-bp palindromic sequence within the region prevents binding of the trans-acting protein(s), increasing promoter activity 7-fold and resulting in a corresponding increase in AtxA and anthrax toxin production. Notably, an atxA promoter mutant that produced elevated levels of AtxA and toxin proteins during culture was unaffected for virulence in a murine model for anthrax.

Transcriptome profiling of degU expression reveals unexpected regulatory patterns in Bacillus megaterium and discloses new targets for optimizing expression

The first whole transcriptome assessment of a Bacillus megaterium strain provides unanticipated insights into the degSU regulon considered to be of central importance for exo-enzyme production. Regulatory patterns as well as the transcription of degSU itself deviate from the model organism Bacillus subtilis; the number of DegU-regulated secretory enzymes is rather small. Targets for productivity optimization, besides degSU itself, arise from the unexpected DegU-dependent induction of the transition-state regulator AbrB during exponential growth. Induction of secretion-assisting factors, such as the translocase subunit SecY or the signal peptidase SipM, promote hypersecretion. B. megaterium DegSU transcriptional control is advantageous for production purposes, since the degU32 constitutively active mutant conferred hypersecretion of a heterologous Bacillus amyloliquefaciens amylase without the detrimental rise, as for B. subtilis and Bacillus licheniformis, in extracellular proteolytic activities.

1H, 13C, and 15N resonance assignments and secondary structure prediction of the full-length transition state regulator AbrB from Bacillus anthracis

The AbrB protein is a transcription factor that regulates the expression of numerous essential genes during the cells transition phase state. AbrB from Bacillus anthracis is, nototriously, the principal protein responsible for anthrax toxin gene expression and is highly homologous to the much-studied AbrB protein from Bacillus subtilis having 85% sequence identity and the ability to regulate the same target promoters. Here we report backbone and sidechain resonance assignments and secondary structure prediction for the full-length AbrB protein from B. anthracis.

Genome-wide binding profiles of the Bacillus subtilis transition state regulator AbrB and its homolog Abh reveals their interactive role in transcriptional regulation

AbrB is a global transcriptional regulator of Bacillus subtilis that represses the expression of many genes during exponential growth. Here, we demonstrate that AbrB and its homolog Abh bind to hundreds of sites throughout the entire B. subtilis genome during exponential growth. Comparison of regional binding of AbrB and Abh in wild-type, ΔabrB and Δabh backgrounds revealed that they bind as homomer and/or heteromer forms with different specificities and affinities. We found four AbrB and Abh binding patterns were major. Three of these contain pairs of TGGNA motifs connected by A/T-rich sequences, differing in arrangement and spacing. We also assessed the direct involvement of these complexes in the control of gene expression. Our data indicate that AbrB usually acts as a repressor, and that the ability of Abh to act as a transcriptional regulator was limited. We found that changes to AbrB/Abh levels affect their binding at several promoters and consequently transcriptional regulation. Surprisingly, most AbrB/Abh binding events had no impact on transcription, suggesting an interesting possibility that AbrB/Abh binding is analogous to nucleoid-associated protein binding in Escherichia coli.

The master transcription factor Spo0A is required for poly(3-hydroxybutyrate) (PHB) accumulation and expression of genes involved in PHB biosynthesis in Bacillus thuringiensis

Bacillus thuringiensis is a gram-positive spore-forming bacterium that can accumulate poly(3-hydroxybutyrate) (PHB) as a carbon and energy storage substance in response to nutritional stress. The regulatory mechanism for PHB biosynthesis in B. thuringiensis and diverse Bacillus species is still poorly understood. We now report that disruption of the sigH gene or the gene encoding the master sporulation transcription factor Spo0A severely impaired PHB accumulation in B. thuringiensis. Complementation of the spo0A mutation with the spo0A gene restored PHB accumulation. We have found that the requirement of Spo0A for PHB accumulation is independent of the transition state regulator AbrB and of loss of sporulation ability. We also show that Spo0A is required for the expression of three genes involved in PHB biosynthesis. These findings have uncovered a new role of Spo0A in the regulation of stationary-phase-associated cellular events.

The master transcription factor Spo0A is required for poly(3-hydroxybutyrate) (PHB) accumulation and expression of genes involved in PHB biosynthesis inBacillus thuringiensis

Bacillus thuringiensis is a gram-positive spore-forming bacterium that can accumulate poly(3-hydroxybutyrate) (PHB) as a carbon and energy storage substance in response to nutritional stress. The regulatory mechanism for PHB biosynthesis in B. thuringiensis and diverse Bacillus species is still poorly understood. We now report that disruption of the sigH gene or the gene encoding the master sporulation transcription factor Spo0A severely impaired PHB accumulation in B. thuringiensis. Complementation of the spo0A mutation with the spo0A gene restored PHB accumulation. We have found that the requirement of Spo0A for PHB accumulation is independent of the transition state regulator AbrB and of loss of sporulation ability. We also show that Spo0A is required for the expression of three genes involved in PHB biosynthesis. These findings have uncovered a new role of Spo0A in the regulation of stationary-phase-associated cellular events.

Cereulide synthesis in emetic Bacillus cereus is controlled by the transition state regulator AbrB, but not by the virulence regulator PlcR

Cereulide, a depsipeptide structurally related to the antibiotic valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that cereulide is produced non-ribosomally by the plasmid-encoded peptide synthetase Ces. Using deletion mutants of the emetic reference strain B. cereus F4810/72, the influence of the well-known transcription factors PlcR, Spo0A and AbrB on cereulide production and on the transcription of the cereulide synthetase gene cluster was investigated. Our data demonstrate that cereulide synthesis is independent of the B. cereus specific virulence regulator PlcR but belongs to the Spo0A-AbrB regulon. Although cereulide production turned out to be independent of sporulation, it required the activity of the sporulation factor Spo0A. The sigma(A)-promoted transcription of spo0A was found to be crucial for cereulide production, while the sigma(H)-driven transcription of spo0A did not affect cereulide synthesis. Overexpression of the transition state factor AbrB in B. cereus F4810/72 resulted in a non-toxic phenotype. Moreover, AbrB was shown to bind efficiently to the main promoter region of the ces operon, indicating that AbrB acts as a repressor of cereulide production by negatively affecting ces transcription.

Transition State Regulator AbrB Inhibits Transcription of Bacillus amyloliquefaciens FZB45 Phytase through Binding at Two Distinct Sites Located within the Extended phyC Promoter Region

We have previously identified the phyC gene of Bacillus amyloliquefaciens FZB45, encoding extracellular phytase, as a member of the PhoP regulon, which is expressed only during phosphate starvation. Its sigma(A)-dependent promoter is positively and negatively regulated by the phosphorylated PhoP response regulator in a phosphate-dependent manner (O. Makarewicz, S. Dubrac, T. Msadek, and R. Borriss, J. Bacteriol. 188:6953-6965, 2006). Here, we provide experimental evidence that the transcription of phyC underlies a second control mechanism exerted by the global transient-phase regulator protein, AbrB, which hinders its expression during exponential growth. Gel mobility shift and DNase I footprinting experiments demonstrated that AbrB binds to two different regions in the phyC promoter region that are separated by about 200 bp. One binding site is near the divergently orientated yodU gene, and the second site is located downstream of the phyC promoter and extends into the coding region of the phyC gene. Cooperative binding to the two distant binding regions is necessary for the AbrB-directed repression of phyC transcription. AbrB does not affect the transcription of the neighboring yodU gene.

An AbrB-Like protein regulates the expression of the bidirectional hydrogenase in Synechocystis sp. strain PCC 6803.

In the unicellular cyanobacterium Synechocystis sp. strain PCC 6803, the pentameric bidirectional Ni-Fe hydrogenase (HoxEFUYH) is the sole enzyme involved in hydrogen metabolism. Recent investigations implicated the transcription factor LexA in the regulation of the hox genes in this cyanobacterium, suggesting the factor to work as an activator. In this work, we show evidence that LexA cannot account exclusively for the regulation of the hox genes in this cyanobacterium. Therefore, we investigated which additional transcription factors interact in and may regulate the expression of the hox genes in Synechocystis sp. strain PCC 6803. By using DNA affinity assays, a transcription factor with similarity to the transition state regulator AbrB from Bacillus subtilis was isolated. Electrophoretic mobility shift assays showed that the AbrB-like protein specifically interacts with the promoter region of the hox genes as well as with its own promoter region. In addition, results obtained with two genetically modified strains of Synechocystis sp. strain PCC 6803, one with a not fully segregated inactivation mutation of the abrB-like gene and the other overexpressing the same abrB-like gene, suggest that this transcription factor functions as a regulator of hox gene expression.

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.

Novel Methods for Genetic Transformation of Natural Bacillus subtilis Isolates Used To Study the Regulation of the Mycosubtilin and Surfactin Synthetases

Natural isolates of Bacillus subtilis are often difficult to transform due to their low genetic competence levels. Here we describe two methods that stimulate natural transformation. The first method uses plasmid pGSP12, which expresses the competence transcription factor ComK and stimulates competence development about 100-fold. The second method stimulates Campbell-type recombination of DNA ligation mixtures in B. subtilis by the addition of polyethylene glycol. We employed these novel methods to study the regulation of the synthetases for the lipopeptide antibiotics mycosubtilin (myc) and surfactin (srfA) in B. subtilis strain ATCC 6633. By means of lacZ reporter fusions, it was shown that the expression of srfA is >100 times lower in strain ATCC 6633 than in the laboratory strain B. subtilis 168. Expression of the myc operon was highest in rich medium, whereas srfA expression reached maximal levels in minimal medium. Further genetic analyses showed that the srfA operon is mainly regulated by the response regulator ComA, while the myc operon is primarily regulated by the transition-state regulator AbrB. Although there is in vitro evidence for a synergistic activity of mycosubtilin and surfactin, the expression of both lipopeptide antibiotics is clearly not coordinated.

The Alternative Sigma Factor σHIs Required for Toxin Gene Expression byBacillus anthracis

Expression of the structural genes for the anthrax toxin proteins is coordinately controlled by host-related signals, such as elevated CO(2), and the trans-acting positive regulator AtxA. In addition to these requirements, toxin gene expression is under growth phase regulation. The transition state regulator AbrB represses atxA expression to influence toxin synthesis. During the late exponential phase of growth, when AbrB levels begin to decrease, toxin synthesis increases. Here we report that toxin gene expression also requires the presence of sigH, a gene encoding the RNA polymerase sigma factor associated with development in Bacillus subtilis. In the well-studied B. subtilis system, sigma(H) is required for sporulation and other post-exponential-phase processes and is part of a feedback control pathway for abrB expression. Our data indicate that a Bacillus anthracis sigH-null mutant is asporogenous and toxin deficient. Yet the sigma factor is required for toxin gene expression in a manner that is independent of the pathway leading to post-exponential-phase gene expression. Sigma(H) positively controls atxA in an AbrB-independent manner. These findings, combined with previous observations, suggest that the steady-state level of atxA expression is critical for optimal toxin gene transcription. We propose a model whereby, under toxin-inducing growth conditions, control of toxin gene expression is fine-tuned by the independent effects of sigma(H) and AbrB on the expression of atxA.

Revised structure of the AbrB N-terminal domain unifies a diverse superfamily of putative DNA-binding proteins

New relationships found in the process of updating the structural classification of proteins (SCOP) database resulted in the revision of the structure of the N-terminal, DNA-binding domain of the transition state regulator AbrB. The dimeric AbrB domain shares a common fold with the addiction antidote MazE and the subunit of uncharacterized protein MraZ implicated in cell division and cell envelope formation. It has a detectable sequence similarity to both MazE and MraZ thus providing an evolutionary link between the two proteins. The putative DNA-binding site of AbrB is found on the same face as the DNA-binding site of MazE and appears similar, both in structure and sequence, to the exposed conserved region of MraZ. This strongly suggests that MraZ also binds DNA and allows for a consensus model of DNA recognition by the members of this novel protein superfamily.