Mark S. B. Paget

Bacterial redox sensors

Redox reactions pervade living cells. They are central to both anabolic and catabolic metabolism. The ability to maintain redox balance is therefore vital to all organisms. Various regulatory sensors continually monitor the redox state of the internal and external environments and control the processes that work to maintain redox homeostasis. In response to redox imbalance, new metabolic pathways are initiated, the repair or bypassing of damaged cellular components is coordinated and systems that protect the cell from further damage are induced. Advances in biochemical analyses are revealing a range of elegant solutions that have evolved to allow bacteria to sense different redox signals.

RsrA, an anti‐sigma factor regulated by redox change

SigR (σR) is a sigma factor responsible for inducing the thioredoxin system in response to oxidative stress in the antibiotic‐producing, Gram‐positive bacterium Streptomyces coelicolor A3(2). Here we identify a redox‐sensitive, σR‐specific anti‐sigma factor, RsrA, which binds σR and inhibits σR‐directed transcription in vitro only under reducing conditions. Exposure to H2O2 or to the thiol‐specific oxidant diamide caused the dissociation of the σR–RsrA complex, thereby allowing σR‐dependent transcription. This correlated with intramolecular disulfide bond formation in RsrA. Thioredoxin was able to reduce oxidized RsrA, suggesting that σR, RsrA and the thioredoxin system comprise a novel feedback homeostasis loop that senses and responds to changes in the intracellular thiol–disulfide redox balance.

σR, an RNA polymerase sigma factor that modulates expression of the thioredoxin system in response to oxidative stress in Streptomyces coelicolor A3(2)

We have identified an RNA polymerase sigma factor, σR, that is part of a system that senses and responds to thiol oxidation in the Gram‐positive, antibiotic‐producing bacterium Streptomyces coelicolor A3(2). Deletion of the gene (sigR) encoding σR caused sensitivity to the thiol‐specific oxidant diamide and to the redox cycling compounds menadione and plumbagin. This correlated with reduced levels of disulfide reductase activity and an inability to induce this activity on exposure to diamide. The trxBA operon, encoding thioredoxin reductase and thioredoxin, was found to be under the direct control of σR. trxBA is transcribed from two promoters, trxBp1 and trxBp2, separated by 5–6 bp. trxBp1 is transiently induced at least 50‐fold in response to diamide treatment in a sigR‐dependent manner. Purified σR directed transcription from trxBp1 in vitro, indicating that trxBp1 is a target for σR. Transcription of sigR itself initiates at two promoters, sigRp1 and sigRp2, which are separated by 173 bp. The sigRp2 transcript was undetectable in a sigR‐null mutant, and purified σR could direct transcription from sigRp2 in vitro, indicating that sigR is positively autoregulated. Transcription from sigRp2 was also transiently induced (70‐fold) following treatment with diamide. We propose a model in which σR induces expression of the thioredoxin system in response to cytoplasmic disulfide bond formation. Upon reestablishment of normal thiol levels, σR activity is switched off, resulting in down‐regulation of trxBA and sigR. We present evidence that the σR system also functions in the actinomycete pathogen Mycobacterium tuberculosis.

A novel sensor of NADH/NAD+ redox poise in Streptomyces coelicolor A3(2)

We describe the identification of Rex, a novel redox‐sensing repressor that appears to be widespread among Gram‐positive bacteria. In Streptomyces coelicolor Rex binds to operator (ROP) sites located upstream of several respiratory genes, including the cydABCD and rex‐hemACD operons. The DNA‐binding activity of Rex appears to be controlled by the redox poise of the NADH/NAD+ pool. Using electromobility shift and surface plasmon resonance assays we show that NADH, but not NAD+, inhibits the DNA‐binding activity of Rex. However, NAD+ competes with NADH for Rex binding, allowing Rex to sense redox poise over a range of NAD(H) concentrations. Rex is predicted to include a pyridine nucleotide‐binding domain (Rossmann fold), and residues that might play key structural and nucleotide binding roles are highly conserved. In support of this, the central glycine in the signature motif (GlyXGlyXXGly) is shown to be essential for redox sensing. Rex homologues exist in most Gram‐positive bacteria, including human pathogens such as Staphylococcus aureus, Listeria monocytogenes and Streptococcus pneumoniae.

Defining the disulphide stress response in Streptomyces coelicolor A3(2): identification of the sigmaR regulon.

In the Gram‐positive, antibiotic‐producing bacterium Streptomyces coelicolor A3(2), the thiol‐disulphide status of the hyphae is controlled by a novel regulatory system consisting of a sigma factor, σR, and its cognate anti‐sigma factor, RsrA. Oxidative stress induces intramolecular disulphide bond formation in RsrA, which causes it to lose affinity for σR, thereby releasing σR to activate transcription of the thioredoxin operon, trxBA. Here, we exploit a preliminary consensus sequence for σR target promoters to identify 27 new σR target genes and operons, thereby defining the global response to disulphide stress in this organism. Target genes related to thiol metabolism encode a second thioredoxin (TrxC), a glutaredoxin‐like protein and enzymes involved in the biosynthesis of the low‐molecular‐weight thiol‐containing compounds cysteine and molybdopterin. In addition, the level of the major actinomycete thiol buffer, mycothiol, was fourfold lower in a sigR null mutant, although no candidate mycothiol biosynthetic genes were identified among the σR targets. Three σR target genes encode ribosome‐associated products (ribosomal subunit L31, ppGpp synthetase and tmRNA), suggesting that the translational machinery is modified by disulphide stress. The product of another σR target gene was found to be a novel RNA polymerase‐associated protein, RbpA, suggesting that the transcriptional machinery may also be modified in response to disulphide stress. We present DNA sequence evidence that many of the targets identified in S. coelicolor are also under the control of the σR homologue in the actinomycete pathogen Mycobacterium tuberculosis.

Mutational analysis of RsrA, a zinc‐binding anti‐sigma factor with a thiol–disulphide redox switch

In the Gram‐positive bacterium, Streptomyces coelicolor A3(2), expression of the thioredoxin system is modulated by a sigma factor called σR in response to changes in the cytoplasmic thiol–disulphide status, and the activity of σR is controlled post‐translationally by an anti‐sigma factor, RsrA. In vitro, the anti‐sigma factor activity of RsrA, which contains seven cysteines, correlates with its thiol–disulphide redox status. Here, we investigate the function of RsrA in vivo. A constructed rsrA null mutant had very high constitutive levels of disulphide reductase activity and σR‐dependent transcription, confirming that RsrA is a negative regulator of σR and a key sensor of thiol–disulphide status. Targeted mutagenesis revealed that three of the seven cysteines in RsrA (C11, C41 and C44) were essential for anti‐sigma factor activity and that a mutant RsrA protein containing only these three cysteines was active and still redox sensitive in vivo. We also show that RsrA is a metalloprotein, containing near‐stoichiometric amounts of zinc. On the basis of these data, we propose that a thiol–disulphide redox switch is formed between two of C11, C41 and C44, and that all three residues play an essential role in anti‐sigma factor activity in their reduced state, perhaps by acting as ligands for zinc. Unexpectedly, rsrA null mutants were blocked in sporulation, probably as a consequence of an increase in the level of free σR.

Characterization of an inducible vancomycin resistance system in Streptomyces coelicolor reveals a novel gene (vanK) required for drug resistance

Vancomycin is the front‐line therapy for treating problematic infections caused by methicillin‐resistant Staphylococcus aureus (MRSA), and the spread of vancomycin resistance is an acute problem. Vancomycin blocks cross‐linking between peptidoglycan intermediates by binding to the d‐Ala‐d‐Ala termini of bacterial cell wall precursors, which are the substrate of transglycosylase/transpeptidase. We have characterized a cluster of seven genes (vanSRJKHAX) in Streptomyces coelicolor that confers inducible, high‐level vancomycin resistance. vanHAX are orthologous to genes found in vancomycin‐resistant enterococci that encode enzymes predicted to reprogramme peptidoglycan biosynthesis such that cell wall precursors terminate in d‐Ala‐d‐Lac rather than d‐Ala‐d‐Ala. vanR and vanS encode a two‐component signal transduction system that mediates transcriptional induction of the seven van genes. vanJ and vanK are novel genes that have no counterpart in previously characterized vancomycin resistance clusters from pathogens. VanK is a member of the Fem family of enzymes that add the cross‐bridge amino acids to the stem pentapeptide of cell wall precursors, and vanK is essential for vancomycin resistance. The van genes are organized into four transcription units, vanRS, vanJ, vanK and vanHAX, and these transcripts are induced by vancomycin in a vanR‐dependent manner. To develop a sensitive bioassay for inducers of the vancomycin resistance system, the promoter of vanJ was fused to a reporter gene conferring resistance to kanamycin. All the inducers identified were glycopeptide antibiotics, but teicoplanin, a membrane‐anchored glycopeptide, failed to act as an inducer. Analysis of mutants defective in the vanRS and cseBC cell envelope signal transduction systems revealed significant cross‐talk between the two pathways.

A signal transduction system in Streptomyces coelicolor that activates the expression of a putative cell wall glycan operon in response to vancomycin and other cell wall-specific antibiotics

We have investigated a signal transduction system proposed to allow Streptomyces coelicolor to sense and respond to changes in the integrity of its cell envelope. The system consists of four proteins, encoded in an operon: σE, an RNA polymerase σ factor; CseA (formerly ORF202), a protein of unknown function; CseB, a response regulator; and CseC, a sensor histidine protein kinase with two predicted transmembrane helices (Cse stands for control of sigma E). To develop a sensitive bioassay for in‐ducers of the sigE system, the promoter of the sigE operon (sigEp) was fused to a reporter gene conferring resistance to kanamycin. Antibiotics that acted as inducers of the sigE signal transduction system were all inhibitors of intermediate and late steps in peptidoglycan biosynthesis, including ramoplanin, moenomycin A, bacitracin, several glycopeptides and some β‐lactams. The cell wall hydrolytic enzyme lysozyme also acted as an inducer. These data suggest that the CseB–CseC signal transduction system may be activated by the accumulation of an intermediate in peptidoglycan biosynthesis or degradationa. A computer‐based searching method was used to identify a σE target operon of 12 genes (the cwg operon), predicted to specify the biosynthesis of a cell wall glycan. In low‐Mg2+ medium, transcription of the cwg operon was induced by vancomycin in a sigE‐dependent manner but, in high‐Mg2+ medium, there was substantial cwg transcription in a sigE null mutant, and this sigE‐independent activity was also induced by vancomycin. Based on these data, we propose a model for the regulation and function of the σE signal transduction system.

A putative two-component signal transduction system regulates sigmaE, a sigma factor required for normal cell wall integrity in Streptomyces coelicolor A3(2).

The extracytoplasmic function (ECF) sigma factor, σE, is required for normal cell wall integrity in Streptomyces coelicolor. We have investigated the regulation of σE through a transcriptional and mutational analysis of sigE and the surrounding genes. Nucleotide sequencing identified three genes located downstream of sigE ; orf202, cseB and cseC (cse, control of sigE ). cseB and cseC encode a putative response regulator and a putative transmembrane sensor histidine protein kinase respectively. Although most sigE transcription appeared to be monocistronic, sigE was also transcribed as part of a larger operon, including at least orf202. sigE null mutants are sensitive to cell wall lytic enzymes, have an altered peptidoglycan muropeptide profile, and on medium deficient in Mg2+ they overproduce actinorhodin, sporulate poorly and form crenellated colonies. A constructed cseB null mutant appeared to have the same phenotype as a sigE null mutant, which was accounted for by the observed absolute dependence of the sigE promoter on cseB. It is likely that the major role of cseB is to regulate sigE transcription because expression of sigE alone from a heterologous promoter suppressed the cseB mutation. Mg2+ suppresses the CseB/SigE phenotype, probably by stabilizing the cell envelope, and sigE transcript levels were consistently higher in Mg2+‐deficient cultures than in high Mg2+‐grown cultures. We propose a model in which the CseB/CseC two‐component system modulates activity of the sigE promoter in response to signals from the cell envelope.

Structural Basis for NADH/NAD+ Redox Sensing by a Rex Family Repressor

Nicotinamide adenine dinucleotides have emerged as key signals of the cellular redox state. Yet the structural basis for allosteric gene regulation by the ratio of reduced NADH to oxidized NAD(+) is poorly understood. A key sensor among Gram-positive bacteria, Rex represses alternative respiratory gene expression until a limited oxygen supply elevates the intracellular NADH:NAD(+) ratio. Here we investigate the molecular mechanism for NADH/NAD(+) sensing among Rex family members by determining structures of Thermus aquaticus Rex bound to (1) NAD(+), (2) DNA operator, and (3) without ligand. Comparison with the Rex/NADH complex reveals that NADH releases Rex from the DNA site following a 40 degrees closure between the dimeric subunits. Complementary site-directed mutagenesis experiments implicate highly conserved residues in NAD-responsive DNA-binding activity. These rare views of a redox sensor in action establish a means for slight differences in the nicotinamide charge, pucker, and orientation to signal the redox state of the cell.