Biomedical subjects

Matthew I Hutchings

Publications and source records attributed to Matthew I Hutchings.

10 recordsLinked to original sources

PubMed2006

MtrAB-LpqB: a conserved three-component system in actinobacteria?

Streptomyces coelicolor is the model organism for the actinobacteria, a group of high-GC Gram-positive bacteria with members that are notable both for their industrial importance as antibiotic producers and for their pathogenicity. The S. coelicolor genome encodes a subgroup of sensor kinases that is genetically linked to lipoprotein genes, at least one of which functions as an accessory protein to its co-translated kinase. Another member of this subgroup, MtrB, is widely conserved in the actinobacteria, along with its presumed cognate response regulator MtrA and the lipoprotein LpqB. Here, we postulate a possible role for LpqB in the MtrAB signal transduction pathway. We discuss what is known about this pathway in the actinobacteria and offer insights into why an essential response regulator does not necessarily need its cognate sensor kinase for activation.

ATP-Binding Cassette Transporters↗

PubMed2006

The vancomycin resistance VanRS two-component signal transduction system of Streptomyces coelicolor.

We took advantage of the vancomycin-dependent phenotype of Streptomyces coelicolor femX null mutants to isolate a collection of spontaneous, drug-independent femX suppressor mutants that expressed the vancomycin-resistance (van) genes constitutively. All of the suppressor mutations were in vanS but, unexpectedly, many were predicted to be loss-of-function mutations. Confirming this interpretation, a constructed vanS deletion mutation also resulted in constitutive expression of the van genes, suggesting that VanS negatively regulated VanR function in the absence of drug. In contrast, a vanS pta ackA triple mutant, which should not be able synthesize acetyl phosphate, failed to express the van genes, whereas a pta ackA double mutant showed wild-type, regulated induction of the van genes. These results suggest that in the absence of vancomycin, acetyl phosphate phosphorylates VanR, and VanS acts as a phosphatase to suppress the levels of VanR approximately P. On exposure to vancomycin, VanS activity switches from a phosphatase to a kinase and vancomycin resistance is induced. In S. coelicolor, the van genes are induced by both vancomycin and the glycopeptide A47934, whereas in Streptomyces toyocaensis (the A47934 producer) resistance is induced by A47934 but not by vancomycin. We exploited this distinction to replace the S. coelicolor vanRS genes with the vanRS genes from S. toyocaensis. The resulting strain acquired the inducer profile of S. toyocaensis, providing circumstantial evidence that the VanS effector ligand is the drug itself, and not an intermediate in cell wall biosynthesis that accumulates as result of drug action. Consistent with this suggestion, we found that non-glycopeptide inhibitors of the late steps in cell wall biosynthesis such as moenomycin A, bacitracin and ramoplanin were not inducers of the S. coelicolor VanRS system, in contrast to results obtained in enterococcal VanRS systems.

Alleles↗

PubMed2006

The sigma(E) cell envelope stress response of Streptomyces coelicolor is influenced by a novel lipoprotein, CseA.

We have investigated the role of CseA in the sigma(E) cell envelope stress response of the gram-positive bacterium Streptomyces coelicolor. sigma(E) is an extracytoplasmic function RNA polymerase sigma factor required for normal cell envelope integrity in S. coelicolor. sigma(E) is encoded within a four-gene operon that also encodes CseA, a protein of unknown function, CseB, a response regulator and CseC, a transmembrane sensor histidine kinase (Cse represents control of sigma E). Previous work has shown that transcription of the sigE gene is completely dependent on the CseBC two-component system and that the CseBC-sigma(E) signal transduction system is induced by a wide variety of cell-wall-damaging agents. Here we address the role of CseA, a protein with no homologues outside the streptomycetes. We show that CseA is a novel lipoprotein localized to the extracytoplasmic face of the cell membrane and that loss of CseA results in upregulation of the sigE promoter.

Adaptation, Physiological↗

PubMed2005

The role of the novel Fem protein VanK in vancomycin resistance in Streptomyces coelicolor.

The non-pathogenic, non-glycopeptide-producing actinomycete Streptomyces coelicolor carries a cluster of seven genes (vanSRJKHAX) that confers inducible, high level resistance to vancomycin. The vanK gene has no counterpart in previously characterized vancomycin resistance clusters, yet vanK is required for vancomycin resistance in S. coelicolor. VanK belongs to the Fem family of enzymes, which add the branch amino acid(s) to the stem pentapeptide of peptidoglycan precursors. Upon exposure to vancomycin, the VanRS two-component system switches on expression of all seven van genes, and the VanHAX enzymes reprogram the cell wall such that precursors terminate D-Ala-D-lactate (Lac) rather than D-Ala-D-Ala, thus conferring resistance to vancomycin, which only binds D-Ala-D-Ala-containing precursors. Here we provide biochemical and genetic evidence that VanK is required for vancomycin resistance because the constitutively expressed FemX enzyme, encoded elsewhere on the chromosome, cannot recognize D-Lac-containing precursors as a substrate, whereas VanK can. Consistent with this view, D-Lac-containing precursors carrying the Gly branch are present in the wild type transiently exposed to vancomycin but are undetectable in a vanK mutant treated in the same way. Further, femX null mutants are viable in the presence of vancomycin but die in its absence. Because only VanK can recognize D-Lac-containing precursors, vancomycin-induced expression of VanHAX in a vanK mutant is lethal, and so vanK is required for vancomycin resistance.

Amino Acids↗

PubMed2004

Sensing and responding to diverse extracellular signals? Analysis of the sensor kinases and response regulators of Streptomyces coelicolor A3(2).

Streptomyces coelicolor is a Gram-positive soil bacterium that undergoes a complex developmental life cycle. The genome sequence of this organism was recently completed and has revealed the presence of over 60 sigma factors and a multitude of other transcriptional regulators, with a significant number of these being putative two-component signal transduction proteins. The authors have used the criteria established by Hoch and co-workers (Fabret et al., 1999, J Bacteriol 181, 1975-1983) to identify sensor kinase and response regulator genes encoded within the S. coelicolor genome. This analysis has revealed the presence of 84 sensor kinase genes, 67 of which lie adjacent to genes encoding response regulators. This strongly suggests that these paired genes encode two-component systems. In addition there are 13 orphan response regulators encoded in the genome, several of which have already been characterized and are implicated in development and antibiotic production, and 17 unpaired and as yet uncharacterized sensor kinases. This article attempts to infer useful information from sequence analysis and reviews what is currently known about the two-component systems, unpaired sensor kinases and orphan response regulators of S. coelicolor from both published reports and the authors' own unpublished data.

Adaptation, Physiological↗

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↗

PubMed2004

Expression of matrix metalloproteinase-2 and -14 persists during early resolution of experimental liver fibrosis and might contribute to fibrolysis.

BACKGROUND/AIMS: Resolution of liver fibrosis is possible but the identity of the matrix metalloproteinases (MMPs) which degrade the accumulated collagens is uncertain. We examined MMP-2 and MMP-14 expression in established and resolving fibrosis to assess their role in resolution of liver fibrosis. METHODS: MMP and tissue inhibitor of metalloproteinase (TIMP)-2 expression in liver extracts was examined by ribonuclease protection assay, Western blotting and gelatin zymography. MMP activity was examined by (14)C gelatin degradation. RESULTS: In human cirrhotic liver, MMP-14 mRNA was increased to 230-330% of normal liver expression. Both 63 kDa proenzyme and 60 kDa activated form were present. Cirrhotic livers had 270-320% of normal liver expression of MMP-2 protein with 20-25% being the 62 Da activated form. Protein and mRNA for MMP-2 and MMP-14 progressively increased during 8 weeks of CCl(4) treatment in rats. Between 3 and 7 days of resolution from CCl(4) liver fibrosis, MMP-2 and MMP-14 persisted at elevated levels. Gelatinolytic activity in liver homogenates peaked at 7 days of recovery, being 140% above that in livers at peak fibrosis. CONCLUSIONS: Increased expression and activation of MMP-2 and -14 occurs even under conditions of elevated TIMPs during liver fibrogenesis. During liver fibrosis resolution, as TIMP expression decays, the persistence of MMP-2 and MMP-14 may permit collagen degradation.

Adult↗

PubMed2002

The NorR protein of Escherichia coli activates expression of the flavorubredoxin gene norV in response to reactive nitrogen species.

The Escherichia coli norVW genes encode a flavorubredoxin and NADH:(flavo)rubredoxin reductase, respectively, which are involved in nitric oxide detoxification under anaerobic growth conditions. Here it is shown that the norVW genes also have a role in protection against reactive nitrogen intermediates generated from nitroprusside. Transcription from the norV promoter is activated by the presence of nitroprusside in the growth medium; activation requires the product of a divergently transcribed regulatory gene, norR.

Base Sequence↗

PubMed2002

Transcription factor FnrP from Paracoccus denitrificans contains an iron-sulfur cluster and is activated by anoxia: identification of essential cysteine residues.

The Paracoccus denitrificans transcription factor FnrP has been characterized using artificial FNR-dependent promoter-lacZ fusion plasmids in Escherichia coli. FnrP can activate both class I and class II FNR-dependent promoters in response to anoxia but shows a marked preference for the class II promoter, where the FNR binding site is centered at -41.5 with respect to the transcription start site. FnrP was found to be inactive in an iscS mutant in vivo, demonstrating a requirement for cysteine desulfurase activity to assemble an iron-sulfur cluster in FnrP. Accordingly, an iron-sulfur cluster could be reconstituted into the purified protein in vitro using cysteine desulfurase, ferrous ions, and cysteine. Thus, FnrP is a true orthologue of FNR from E. coli and switches on target genes in response to anoxia. Inactivation of FnrP by oxygen very likely involves the oxidative disassembly of an iron-sulfur cluster. Possible ligands for the iron-sulfur cluster were identified by substituting each of the seven cysteine residues with serine and characterizing the altered proteins in vivo. Four substituted proteins showed activities less than 5% of the wild type, and so identify the four cysteines (Cys-14, Cys-17, Cys-25, and Cys-113) that are most likely to be involved in cluster ligation. The effects of N-oxides, NO-releasing compounds and a nitrosating agent on FNR and FnrP activity were investigated in vivo using the reporter system. Both proteins are very sensitive to the inclusion of sodium nitroprusside (a source of NO(+)) in defined growth media but are only moderately sensitive to those sources of NO that were tested.

Amino Acid Sequence↗

PubMed2000

The nitric oxide regulated nor promoter of Paracoccus denitrificans.

The promoter of the Paracoccus denitrificans nitric oxide reductase operon (norCBQDEF) has been characterized by primer extension and deletion analysis. A major transcript that is detectable only in anaerobically grown cells initiates 43.5 bp downstream of the centre of a putative binding site for the transcription factor NNR (nitrite and nitric oxide reductase regulator, which is known to regulate nor expression). A minor transcript initiates 121 bp upstream of the major transcript and is detectable in cells grown aerobically or anaerobically. Deletion derivatives of the nor promoter region were constructed and analysed in vivo using transcriptional fusions to the reporter gene lacZ. Expression patterns from promoter deletions in a wild-type strain and an nnr mutant confirmed that the minor transcript is NNR independent, and makes a small contribution to nor expression under both aerobic and anaerobic growth conditions. A deletion derivative truncated to within 7 bp of the putative NNR-binding site showed a near wild-type response to anaerobic growth, showing that no upstream DNA sequences are required for activation of the major promoter. Site-directed mutagenesis of the putative NNR-binding site confirmed that this is the major cis-acting sequence mediating the anaerobic inducibility of nor expression.

Base Sequence↗