Translational Control of the SigR-Directed Oxidative Stress Response in Streptomyces via IF3-Mediated Repression of a Noncanonical GTC Start Codon.

The major oxidative stress response in Streptomyces is controlled by the sigma factor SigR and its cognate antisigma factor RsrA, and SigR activity is tightly controlled through multiple mechanisms at both the transcriptional and posttranslational levels. Here we show that sigR has a highly unusual GTC start codon and that this leads to another level of SigR regulation, in which SigR translation is repressed by translation initiation factor 3 (IF3). Changing the GTC to a canonical start codon causes SigR to be overproduced relative to RsrA, resulting in unregulated and constitutive expression of the SigR regulon. Similarly, introducing IF3* mutations that impair its ability to repress SigR translation has the same effect. Thus, the noncanonical GTC sigR start codon and its repression by IF3 are critical for the correct and proper functioning of the oxidative stress regulatory system. sigR and rsrA are cotranscribed and translationally coupled, and it had therefore been assumed that SigR and RsrA are produced in stoichiometric amounts. Here we show that RsrA can be transcribed and translated independently of SigR, present evidence that RsrA is normally produced in excess of SigR, and describe the factors that determine SigR-RsrA stoichiometry.IMPORTANCE In all sigma factor-antisigma factor regulatory switches, the relative abundance of the two proteins is critical to the proper functioning of the system. Many sigma-antisigma operons are cotranscribed and translationally coupled, leading to a generic assumption that the sigma and antisigma factors are produced in a fixed 1:1 ratio. In the case of sigR-rsrA, we show instead that the antisigma factor is produced in excess over the sigma factor, providing a buffer to prevent spurious release of sigma activity. This excess arises in part because sigR has an extremely rare noncanonical GTC start codon, and as a result, SigR translation initiation is repressed by IF3. This finding highlights the potential significance of noncanonical start codons, very few of which have been characterized experimentally. It also emphasizes the limitations of predicting start codons using bioinformatic approaches, which rely heavily on the assumption that ATG, GTG, and TTG are the only permissible start codons.

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.

The sigmaR regulon of Streptomyces coelicolor A32 reveals a key role in protein quality control during disulphide stress.

Diamide is an artificial disulphide-generating electrophile that mimics an oxidative shift in the cellular thiol-disulphide redox state (disulphide stress). The Gram-positive bacterium Streptomyces coelicolor senses and responds to disulphide stress through the sigma(R)-RsrA system, which comprises an extracytoplasmic function (ECF) sigma factor and a redox-active anti-sigma factor. Known targets that aid in the protection and recovery from disulphide stress include the thioredoxin system and genes involved in producing the major thiol buffer mycothiol. Here we determine the global response to diamide in wild-type and sigR mutant backgrounds to understand the role of sigma(R) in this response and to reveal additional regulatory pathways that allow cells to cope with disulphide stress. In addition to thiol oxidation, diamide was found to cause protein misfolding and aggregation, which elicited the induction of the HspR heat-shock regulon. Although this response is sigma(R)-independent, sigma(R) does directly control Clp and Lon ATP-dependent AAA(+) proteases, which may partly explain the reduced ability of a sigR mutant to resolubilize protein aggregates. sigma(R) also controls msrA and msrB methionine sulphoxide reductase genes, implying that sigma(R)-RsrA is responsible for the maintenance of both cysteine and methionine residues during oxidative stress. This work shows that the sigma(R)-RsrA system plays a more significant role in protein quality control than previously realized, and emphasizes the importance of controlling the cellular thiol-disulphide redox balance.

The zinc-responsive regulator Zur controls expression of the coelibactin gene cluster in Streptomyces coelicolor.

Streptomyces coelicolor mutants lacking the zinc-responsive Zur repressor are conditionally defective in sporulation, presumably due to the overexpression of one or more Zur target genes. Gene disruption analyses revealed that deregulation of previously known Zur targets was not responsible for the sporulation phenotype. We used microarrays to identify further Zur targets and discovered that Zur controls a cluster of genes predicted to direct synthesis of an uncharacterized siderophore-related non-ribosomally encoded peptide designated coelibactin. Disruption of a key coelibactin biosynthetic gene suppressed the Zur sporulation phenotype, suggesting that deregulation of coelibactin synthesis inhibits sporulation.

Zinc-responsive regulation of alternative ribosomal protein genes in Streptomyces coelicolor involves zur and sigmaR.

Streptomyces coelicolor contains paralogous versions of seven ribosomal proteins (S14, S18, L28, L31, L32, L33, and L36), which differ in their potential to bind structural zinc. The paralogues are termed C(+) or C(-) on the basis of the presence or absence of putative cysteine ligands. Here, mutational studies suggest that the C(-) version of L31 can functionally replace its C(+) paralogue only when expressed at an artificially elevated level. We show that the level of expression of four transcriptional units encoding C(-) proteins is elevated under conditions of zinc deprivation. Zur controls the expression of three transcriptional units (including rpmG2, rpmE2, rpmB2, rpsN2, rpmF2, and possibly rpsR2). Zur also controls the expression of the znuACB operon, which is predicted to encode a high-affinity zinc transport system. Surprisingly, the zinc-responsive control of the rpmG3-rpmJ2 operon is dictated by sigma(R), a sigma factor that was previously shown to control the response to disulfide stress in S. coelicolor. The induction of sigma(R) activity during zinc limitation establishes an important link between thiol-disulfide metabolism and zinc homeostasis.

Assignment of the zinc ligands in RsrA, a redox-sensing ZAS protein from Streptomyces coelicolor.

ZAS proteins are widespread bacterial zinc-containing anti-sigma factors that regulate the activity of sigma factors in response to diverse cues. One of the best characterized ZAS proteins is RsrA from Streptomyces coelicolor, which responds to disulfide stress. Zn-RsrA binds and represses the transcriptional activity of sigmaR in the reducing environment of the cytoplasm but undergoes reversible, intramolecular disulfide bond formation during oxidative stress. This expels the single metal ion and causes dramatic structural changes in RsrA that result in its dissociation from sigmaR, leaving the sigma factor free to activate the transcription of antioxidant genes. We showed recently that Zn2+ serves a critical role in modulating the redox activity of RsrA thiols but uncertainty remains as to how the metal ion is coordinated in RsrA and related ZAS proteins. Using a combination of random and site-specific mutagenesis with zinc K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy, we have assigned unambiguously the metal ligands in RsrA, thereby distinguishing between the different ligation models that have been proposed. The data show that the zinc site in RsrA is comprised of Cys11, His37, Cys41, and Cys44. Three of these residues are part of a conserved ZAS-specific sequence motif (H37xxxC41xxC44), with the fourth ligand, Cys11, found in a subset of ZAS proteins. Cys11 and Cys44 form the trigger disulfide in RsrA, explaining why the metal ion is expelled during oxidation. We discuss these data in the context of redox sensing by RsrA and the sensory mechanisms of other ZAS proteins.

The RNA polymerase-binding protein RbpA confers basal levels of rifampicin resistance on Streptomyces coelicolor.

RbpA is an RNA polymerase-binding protein that occurs in the actinomycete family of bacteria and is regulated by the disulphide stress-response sigma factor, sigma(R), in Streptomyces coelicolor. Here we demonstrate that rbpA null mutants exhibit a slow-growth phenotype and are particularly sensitive to the transcription inhibitor rifampicin. Strikingly, transcription mapping experiments revealed that rbpA expression is induced upon exposure of S. coelicolor to rifampicin and that this, in part, involves an increase in the activity of sigma(R). In contrast, the ribosomal RNA operon promoter rrnDp3, which is recognized by the vegetative sigma factor sigma(HrdB), was strongly inhibited by rifampicin. Reconstitution of RNAP from an rbpA null mutant with purified RbpA revealed that RbpA stimulates transcription from rrnDp3, even in the presence of rifampicin. The data presented suggest that RbpA confers basal levels of rifampicin resistance and is a novel regulator of rRNA synthesis in S. coelicolor.

X-ray structure of a Rex-family repressor/NADH complex insights into the mechanism of redox sensing.

The redox-sensing repressor Rex regulates transcription of respiratory genes in response to the intra cellular NADH/NAD(+) redox poise. As a step toward elucidating the molecular mechanism of NADH/NAD(+) sensing, the X-ray structure of Thermus aquaticus Rex (T-Rex) bound to effector NADH has been determined at 2.9 A resolution. The fold of the C-terminal domain of T-Rex is characteristic of NAD(H)-dependent enzymes, whereas the N-terminal domain is similar to a winged helix DNA binding motif. T-Rex dimerization is primarily mediated by "domain-swapped" alpha helices. Each NADH molecule binds to the C-terminal domain near the dimer interface. In contrast to NAD(H)-dependent enzymes, the nicotinamide is deeply buried within a hydrophobic pocket that appears to preclude substrate entry. We show that T-Rex binds to the Rex operator, and NADH but not NAD(+) inhibits T-Rex/DNA binding activity. A mechanism for redox sensing by Rex family members is proposed by analogy with domain closure of NAD(H)-dependent enzymes.

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.

Thiol-based regulatory switches.

Thiol-based regulatory switches play central roles in cellular responses to oxidative stress, nitrosative stress, and changes in the overall thiol-disulfide redox balance. Protein sulfhydryls offer a great deal of flexibility in the different types of modification they can undergo and the range of chemical signals they can perceive. For example, recent work on OhrR and OxyR has clearly established that disulfide bonds are not the only cysteine oxidation products that are likely to be relevant to redox sensing in vivo. Furthermore, different stresses can result in distinct modifications to the same protein; in OxyR it seems that distinct modifications can occur at the same cysteine, and in Yap1 a partner protein ensures that the disulfide bond induced by peroxide stress is different from the disulfide bond induced by other stresses. These kinds of discoveries have also led to the intriguing suggestion that different modifications to the same protein can create multiple activation states and thus deliver discrete regulatory outcomes. In this review, we highlight these issues, focusing on seven well-characterized microbial proteins controlled by thiol-based switches, each of which exhibits unique regulatory features.

The Role of zinc in the disulphide stress-regulated anti-sigma factor RsrA from Streptomyces coelicolor.

The regulation of disulphide stress in actinomycetes such as Streptomyces coelicolor is known to involve the zinc-containing anti-sigma factor RsrA that binds and inactivates the redox-regulated sigma factor sigmaR. However, it is not known how RsrA senses disulphide stress nor what role the metal ion plays. Using in vitro assays, we show that while zinc is not required for sigmaR binding it is required for functional anti-sigma factor activity, and that it plays a critical role in modulating the reactivity of RsrA cysteine thiol groups towards oxidation. Apo-RsrA is easily oxidised and, while the Zn-bound form is relatively resistant, the metal ion is readily expelled when the protein is treated with strong oxidants such as diamide. We also show, using a combination of proteolysis and mass spectrometry, that the first critical disulphide to form in RsrA involves Cys11 and one of either Cys41 or Cys44, all previously implicated in metal binding. Circular dichroism spectroscopy was used to follow structural changes during oxidation of RsrA, which indicated that concomitant with formation of this critical disulphide bond is a major restructuring of the protein where its alpha-helical content increases. Our data demonstrate that RsrA can only bind sigmaR in the reduced state and that this state is stabilised by zinc. Redox stress induces disulphide bond formation amongst zinc-ligating residues, expelling the metal ion and stabilising a structure incapable of binding the sigma factor.

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.

The sigma70 family of sigma factors.

Members of the sigma70 family of sigma factors are components of the RNA polymerase holoenzyme that direct bacterial or plastid core RNA polymerase to specific promoter elements that are situated 10 and 35 base-pairs upstream of transcription-initiation points. Members of the sigma70 family also function as contact points for some activator proteins, such as PhoB and lambda(cl), and play a role in the initiation process itself. The primary sigma factor, which is essential for general transcription in exponentially growing cells, is reversibly associated with RNA polymerase and can be replaced by alternative sigma factors that co-ordinately express genes involved in diverse functions, such as stress responses, morphological development and iron uptake. On the basis of gene structure and function, members of the sigma70 family can broadly be divided into four main groups. Sequence alignments of the sigma70 family members reveal that they have four conserved regions, although the highest conservation is found in regions 2 and 4, which are involved in binding to RNA polymerase, recognizing promoters and separating DNA strands (so-called 'DNA melting'). The division of the linear sequence of sigma70 factors into four regions is largely supported by recent structural data indicating that primary sigma factors have three stable domains that incorporate regions 2, 3 and 4. Furthermore, structures of the RNA polymerase holoenzyme have revealed that these domains of sigma70 are spread out across one face of RNA polymerase. These structural data are starting to illuminate the mechanistic role of sigma factors in transcription initiation.

Identification and structure of the anti-sigma factor-binding domain of the disulphide-stress regulated sigma factor sigma(R) from Streptomyces coelicolor.

The extracytoplasmic function (ECF) sigma factor sigma(R) is a global regulator of redox homeostasis in the antibiotic-producing bacterium Streptomyces coelicolor, with a similar role in other actinomycetes such as Mycobacterium tuberculosis. Normally maintained in an inactive state by its bound anti-sigma factor RsrA, sigma(R) dissociates in response to intracellular disulphide-stress to direct core RNA polymerase to transcribe genes, such as trxBA and trxC that encode the enzymes of the thioredoxin disulphide reductase pathway, that re-establish redox homeostasis. Little is known about where RsrA binds on sigma(R) or how it suppresses sigma(R)-dependent transcriptional activity. Using a combination of proteolysis, surface-enhanced laser desorption ionisation mass spectrometry and pull-down assays we identify an N-terminal, approximately 10kDa domain (sigma(RN)) that encompasses region 2 of sigma(R) that represents the major RsrA binding site. We show that sigma(RN) inhibits transcription by an unrelated sigma factor and that this inhibition is relieved by RsrA binding, reaffirming that region 2 is involved in binding to core RNA polymerase but also demonstrating that the likely mechanism by which RsrA inhibits sigma(R) activity is by blocking this association. We also report the 2.4A resolution crystal structure of sigma(RN) that reveals extensive structural conservation with the equivalent region of sigma(70) from Escherichia coli as well as with the cyclin-box, a domain-fold found in the eukaryotic proteins TFIIB and cyclin A. sigma(RN) has a propensity to aggregate, due to steric complementarity of oppositely charged surfaces on the domain, but this is inhibited by RsrA, an observation that suggests a possible mode of action for RsrA which we compare to other well-studied sigma factor-anti-sigma factor systems.

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: sigmaE, an RNA polymerase 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 inducers 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 beta-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 sigmaE target operon of 12 genes (the cwg operon), predicted to specify the biosynthesis of a cell wall glycan. In low-Mg(2+) medium, transcription of the cwg operon was induced by vancomycin in a sigE-dependent manner but, in high-Mg(2+) 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 sigmaE signal transduction system.