Biomedical subjects

Mark S B Paget

Publications and source records attributed to Mark S B Paget.

10 recordsLinked to original sources

PubMed2006

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.

Amino Acid Motifs↗

PubMed2006

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.

Antibiotics, Antitubercular↗

PubMed2005

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.

Amino Acid Motifs↗

PubMed2004

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.

Amino Acid Sequence↗

PubMed2003

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.

Alkylation↗

PubMed2003

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.

Amino Acid Sequence↗

PubMed2003

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.

Bacterial Proteins↗

PubMed2003

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.

Animals↗

PubMed2002

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.

Bacterial Proteins↗

PubMed2002

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.

Anti-Bacterial Agents↗