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Gerard D. Wright - One of the best experts on this subject based on the ideXlab platform.

  • phylogenetic reconciliation reveals the natural history of Glycopeptide Antibiotic biosynthesis and resistance
    Nature microbiology, 2019
    Co-Authors: Nicholas Waglechner, Andrew G Mcarthur, Gerard D. Wright
    Abstract:

    Glycopeptide Antibiotics are produced by Actinobacteria through biosynthetic gene clusters that include genes supporting their regulation, synthesis, export and resistance. The chemical and biosynthetic diversities of Glycopeptides are the product of an intricate evolutionary history. Extracting this history from genome sequences is difficult as conservation of the individual components of these gene clusters is variable and each component can have a different trajectory. We show that Glycopeptide biosynthesis and resistance in Actinobacteria maps to approximately 150–400 million years ago. Phylogenetic reconciliation reveals that the precursors of Glycopeptide biosynthesis are far older than other components, implying that these clusters arose from a pre-existing pool of genes. We find that resistance appeared contemporaneously with biosynthetic genes, raising the possibility that the mechanism of action of Glycopeptides was a driver of diversification in these gene clusters. Our results put Antibiotic biosynthesis and resistance into an evolutionary context and can guide the future discovery of compounds possessing new mechanisms of action, which are especially needed as the usefulness of the Antibiotics available at present is imperilled by human activity. Phylogenetic reconciliation traces the evolution of Glycopeptide Antibiotic biosynthesis gene clusters from pre-existing gene pools to 150–400 million years ago; resistance to these Antibiotics also arose contemporaneously.

  • trichlorination of a teicoplanin type Glycopeptide Antibiotic by the halogenase stai evades resistance
    Antimicrobial Agents and Chemotherapy, 2018
    Co-Authors: Wenliang Wang, Andrew C Pawlowski, Gerard D. Wright
    Abstract:

    Glycopeptide Antibiotics (GPAs) include clinically important drugs used for the treatment of infections caused by Gram positive pathogens. These Antibiotics are specialized metabolites produced by several genera of actinomycetes bacteria. While many GPAs are highly chemically modified, A47934 is a relatively unadorned GPA lacking sugar or acyl modifications, common to other members of the class, but which is chlorinated at three distinct sites. The biosynthesis of A47934 is encoded by a 68 kb gene cluster in Streptomyces toyocaensis NRRL 15009. The cluster includes all the necessary genes for the synthesis of A47934 including two predicted halogenase genes, staI and staK . In this study, we report that only one of the halogenase genes, staI , is necessary and essential for A47934 biosynthesis. Chlorination of the A47934 scaffold is important for Antibiotic activity as assessed by binding affinity for the target N-acyl-D-Ala-D-Ala. Surprisingly, chlorination is also vital to avoid activation of enterococcal and Streptomyces VanB-type GPA resistance through induction of resistance genes. Phenotypic assays showed stronger induction of GPA resistance by the dechlorinated compared to the chlorinated GPA. Correspondingly, the relative expression of enterococcal vanA resistance gene was shown to be increased by the dechlorinated compared to the chlorinated compound. These results provide insight into the biosynthesis of GPAs and the biological function of GPA chlorination for this medically important class of Antibiotic.

  • Glycopeptide Antibiotic biosynthesis
    The Journal of Antibiotics, 2013
    Co-Authors: Maulik N. Thaker, Kalinka Koteva, Gerard D. Wright
    Abstract:

    Glycopeptides such as vancomycin, teicoplanin and telavancin are essential for treating infections caused by Gram-positive bacteria. Unfortunately, the dwindled pipeline of new Antibiotics into the market and the emergence of Glycopeptide-resistant enterococci and other resistant bacteria are increasingly making effective Antibiotic treatment difficult. We have now learned a great deal about how bacteria produce Antibiotics. This information can be exploited to develop the next generation of antimicrobials. The biosynthesis of Glycopeptides via nonribosomal peptide assembly and unusual amino acid synthesis, crosslinking and tailoring enzymes gives rise to intricate chemical structures that target the bacterial cell wall. This review seeks to describe recent advances in our understanding of both biosynthesis and resistance of these important Antibiotics.

  • A vancomycin photoprobe identifies the histidine kinase VanSsc as a vancomycin receptor
    Nature Chemical Biology, 2010
    Co-Authors: Kalinka Koteva, Heejeon Hong, Xiao Dong Wang, Ishac Nazi, Donald Hughes, Mike J Naldrett, Mark J Buttner, Gerard D. Wright
    Abstract:

    Expression of vancomycin resistance genes is known to be controlled by the two-component regulatory system VanRS, but the identity of the VanS receptor ligand has been controversial. Synthesis of a vancomycin photoaffinity probe has now revealed that vancomycin directly binds VanS to induce the expression of resistance genes. Inducible resistance to the Glycopeptide Antibiotic vancomycin requires expression of vanH , vanA and vanX , controlled by a two-component regulatory system consisting of a receptor histidine kinase, VanS, and a response regulator, VanR. The identity of the VanS receptor ligand has been debated. Using a synthesized vancomycin photoaffinity probe, we show that vancomycin directly binds Streptomyces coelicolor VanS (VanSsc) and this binding is correlated with resistance and required for vanH , vanA and vanX gene expression.

  • crystal structure of stal a Glycopeptide Antibiotic sulfotransferase from streptomyces toyocaensis
    Journal of Biological Chemistry, 2007
    Co-Authors: Sherry S Lamb, Gerard D. Wright, Sathesh Bhat, Traian Sulea, Allan Matte, Miroslaw Cygler
    Abstract:

    Abstract Over the past decade, antimicrobial resistance has emerged as a major public health crisis. Glycopeptide Antibiotics such as vanco-mycin and teicoplanin are clinically important for the treatment of Gram-positive bacterial infections. StaL is a 3′-phosphoadenosine 5′-phosphosulfate-dependent sulfotransferase capable of sulfating the cross-linked heptapeptide substrate both in vivo and in vitro, yielding the product A47934, a unique teicoplanin-class Glycopeptide Antibiotic. The sulfonation reaction catalyzed by StaL constitutes the final step in A47934 biosynthesis. Here we report the crystal structure of StaL and its complex with the cofactor product 3′-phosphoadenosine 5′-phosphate. This is only the second prokaryotic sulfotransferase to be structurally characterized. StaL belongs to the large sulfotransferase family and shows higher similarity to cytosolic sulfotransferases (ST) than to the bacterial ST (Stf0). StaL has a novel dimerization motif, different from any other STs that have been structurally characterized. We have also applied molecular modeling to investigate the binding mode of the unique substrate, desulfo-A47934. Based on the structural analysis and modeling results, a series of residues was mutated and kinetically characterized. In addition to the conserved residues (Lys12, His67, and Ser98), molecular modeling, fluorescence quenching experiments, and mutagenesis studies identified several other residues essential for substrate binding and/or activity, including Trp34, His43, Phe77, Trp132, and Glu205.

Max J Cryle - One of the best experts on this subject based on the ideXlab platform.

  • the biosynthetic implications of late stage condensation domain selectivity during Glycopeptide Antibiotic biosynthesis
    Chemical Science, 2019
    Co-Authors: Max J Cryle, Madeleine Peschke, Melanie Schoppet, Roderich D Sussmuth, Evi Stegmann, Anja Kirchberg, Vincent Wiebach
    Abstract:

    Non-ribosomal peptide synthesis is a highly important biosynthetic pathway for the formation of many secondary metabolites of medical relevance. Due to the challenges associated with the chemical synthesis of many of the products of these assembly lines, understanding the activity and selectivity of non-ribosomal peptide synthetase (NRPS) machineries is an essential step towards the redesign of such machineries to produce new bioactive peptides. Whilst the selectivity of the adenylation domains responsible for amino acid activation during NRPS synthesis has been widely studied, the selectivity of the essential peptide bond forming domains – known as condensation domains – is not well understood. Here, we present the results of a combination of in vitro and in vivo investigations into the final condensation domain from the NRPS machinery that produces the Glycopeptide Antibiotics (GPAs). Our results show that this condensation domain is tolerant for a range of peptide substrates and even those with unnatural stereochemistry of the peptide C-terminus, which is in contrast to the widely ascribed role of these domains as a stereochemical gatekeeper during NRPS synthesis. Furthermore, we show that this condensation domain has a significant preference for linear peptide substrates over crosslinked peptides, which indicates that the GPA crosslinking cascade targets the heptapeptide bound to the final module of the NRPS machinery and reinforces the role of the unique GPA X-domain in this process. Finally, we demonstrate that the peptide bond forming activity of this condensation domain is coupled to the rate of amino acid activation performed by the subsequent adenylation domain. This is a significant result with implications for NRPS redesign, as it indicates that the rate of amino acid activation of modified adenylation domains must be maintained to prevent unwanted peptide hydrolysis from the NRPS due to a loss of the productive coupling of amino acid selection and peptide bond formation. Taken together, our results indicate that assessing condensation domain activity is a vital step in not only understanding the biosynthetic logic and timing of NRPS-mediated peptide assembly, but also the rules which redesign efforts must obey in order to successfully produce functional, modified NRPS assembly lines.

  • investigating cytochrome p450 specificity during Glycopeptide Antibiotic biosynthesis through a homologue hybridization approach
    Journal of Inorganic Biochemistry, 2018
    Co-Authors: Clara Brieke, Anja Greule, M Tarnawski, Max J Cryle
    Abstract:

    Accepted manuscript: Investigating Cytochrome P450 specificity during Glycopeptide Antibiotic biosynthesis through a homologue hybridization approach Clara Brieke, Miroslaw Tarnawski, Anja Greule and Max J. CryleJournal of Inorganic BiochemistryCytochrome P450 enzymes perform an impressive range of oxidation reactions against diverse substrate scaffolds whilst generally maintaining a conserved tertiary structure and active site chemistry. Within secondary metabolism, P450 enzymes play widespread and important roles in performing crucial modifications of precursor molecules, with one example of the importance of such reactions being found in the biosynthesis of the Glycopeptide Antibiotics (GPAs). In GPA biosynthesis P450s, known as Oxy enzymes, are key players in the cyclization of the linear GPA peptide precursor, which is a process that is both essential for their Antibiotic activity and is the source of the synthetic challenge of these important Antibiotics. In this work, we developed chimeric P450 enzymes from GPA biosynthesis based on two homologues from different GPA biosynthesis pathways – vancomycin and teicoplanin – as an approach to explore the divergent catalytic behavior of the two parental homologues. We could generate, crystalize and explore the activity of new hybrid P450 enzymes from GPA biosynthesis and show that the unusual in vitro behavior of the vancomycin OxyB homologue does not stem from the major regions of the P450 active site, and that additional regions in and around the P450 active site must contribute to the unusual properties of this P450 enzyme. Our results further show that it is possible to successfully transplant entire regions of secondary structure between such P450s and retain P450 expression and activity, which opens the door to use such targeted approaches to generate and explore novel biosynthetic P450 enzymes.

  • a route to diastereomerically pure phenylglycine thioester peptides crucial intermediates for investigating Glycopeptide Antibiotic biosynthesis
    Chemical Communications, 2018
    Co-Authors: Julien Tailhades, Max J Cryle, Clara Brieke, Madeleine Peschke, Melanie Schoppet, Anja Greule
    Abstract:

    Non-ribosomal peptides contain an array of amino acid building blocks that can present challenges for the synthesis of important intermediates. Here, we report the synthesis of Glycopeptide Antibiotic (GPA) thioester peptides that retains the crucial stereochemical purity of the terminal phenylglycine residue, which we show is essential for the enzymatic GPA cyclisation cascade.

  • The Thioesterase Domain in Glycopeptide Antibiotic Biosynthesis Is Selective for Cross-Linked Aglycones
    ACS Chemical Biology, 2017
    Co-Authors: Madeleine Peschke, Michael Heimes, Clara Brieke, Max J Cryle
    Abstract:

    The biosynthesis of the Glycopeptide Antibiotics (GPAs)—which include teicoplanin and vancomycin—is a complex enzymatic process relying on the interplay of nonribosomal peptide synthesis and a cytochrome P450-mediated cyclization cascade. This unique cyclization cascade generates the highly cross-linked state of these nonribosomal peptides, which is crucial for their antimicrobial activity. Given that these essential oxidative transformations occur while the peptide remains bound to the terminal module of the nonribosomal peptide synthetase (NRPS) machinery, it is important to assess the selectivity of the terminal thioesterase (TE) domain and how this domain contributes to the maintenance of an efficient biosynthetic pathway while at the same time ensuring GPA maturation is completed. In this study, we report the in vitro characterization of the thioesterase domain from teicoplanin biosynthesis, the first GPA thioesterase to be characterized. Our results show that the activity of this TE domain relies on...

  • Chlorinated Glycopeptide Antibiotic Peptide Precursors Improve Cytochrome P450-Catalyzed Cyclization Cascade Efficiency
    Biochemistry, 2017
    Co-Authors: Madeleine Peschke, Robert J. A. Goode, Clara Brieke, Ralf B. Schittenhelm, Max J Cryle
    Abstract:

    The activity of Glycopeptide Antibiotics (GPAs) depends upon important structural modifications to their precursor heptapeptide backbone: specifically, the cytochrome P450-catalyzed oxidative cross-linking of aromatic side chains as well as the halogenation of specific residues within the peptide. The timing of halogenation and its effect on the cyclization of the peptide are currently unclear. Our results show that chlorination of peptide precursors improves their processing by P450 enzymes in vitro, which provides support for GPA halogenation occurring prior to peptide cyclization during nonribosomal peptide synthesis. We could also determine that the activity of the second enzyme in the oxidative cyclization cascade, OxyA, remains higher for chlorinated peptide substrates even when the biosynthetic GPA product possesses an altered chlorination pattern, which supports the role of the chlorine atoms in orienting the peptide substrate in the active site of these enzymes.

Flavia Marinelli - One of the best experts on this subject based on the ideXlab platform.

  • new molecular tools for regulation and improvement of a40926 Glycopeptide Antibiotic production in nonomuraea gerenzanensis atcc 39727
    Frontiers in Microbiology, 2020
    Co-Authors: Oleksandr Yushchuk, Flavia Marinelli, Andres Andreovidal, Giorgia Letizia Marcone, Mervyn J Bibb, Elisa Binda
    Abstract:

    : Genome sequencing has revealed that Nonomuraea spp. represent a still largely unexplored source of specialized metabolites. Nonomuraea gerenzanensis ATCC 39727 is the most studied representative species since it produces the Glycopeptide Antibiotic (GPA) A40926 - the precursor of the clinically relevant Antibiotic dalbavancin, approved by the FDA in 2014 for the treatment of acute skin infections caused by multi-drug resistant Gram-positive pathogens. The clinical relevance of dalbavancin has prompted increased attention on A40926 biosynthesis and its regulation. In this paper, we investigated how to enhance the genetic toolkit for members of the Nonomuraea genus, which have proved quite recalcitrant to genetic manipulation. By constructing promoter-probe vectors, we tested the activity of 11 promoters (heterologous and native) using the GusA reporter system in N. gerenzanensis and in Nonomuraea coxensis; this latter species is phylogenetically distant from N. gerenzanesis and also possesses the genetic potential to produce A40926 or a very similar GPA. Finally, the strongest constitutive promoter analyzed in this study, aac(3)IVp, was used to overexpress the cluster-situated regulatory genes controlling A40926 biosynthesis (dbv3 and dbv4 from N. gerenzanensis and nocRI from N. coxensis) in N. gerenzanensis, and the growth and productivity of the best performing strains were assessed at bioreactor scale using an industrial production medium. Overexpression of positive pathway-specific regulatory genes resulted in a significant increase in the level of A40926 production in N. gerenzanensis, providing a new knowledge-based approach to strain improvement for this valuable Glycopeptide Antibiotic.

  • specificity of induction of Glycopeptide Antibiotic resistance in the producing actinomycetes
    The Journal of Antibiotics, 2018
    Co-Authors: Elisa Binda, Flavia Marinelli, Pamela Cappelletti, Giorgia Letizia Marcone
    Abstract:

    Glycopeptide Antibiotics are drugs of last resort for treating severe infections caused by Gram-positive pathogens. It is widely believed that Glycopeptide-resistance determinants (van genes) are ultimately derived from the producing actinomycetes. We hereby investigated the relationship between the antimicrobial activity of vancomycin and teicoplanins and their differential ability to induce van gene expression in Actinoplanes teichomyceticus—the producer of teicoplanin—and Nonomuraea gerenzanensis—the producer of the teicoplanin-like A40926. As a control, we used the well-characterized resistance model Streptomyces coelicolor. The enzyme activities of a cytoplasmic-soluble d,d-dipeptidase and of a membrane-associated d,d-carboxypeptidase (corresponding to VanX and VanY respectively) involved in resistant cell wall remodeling were measured in the actinomycetes grown in the presence or absence of subinhibitory concentrations of vancomycin, teicoplanin, and A40926. Results indicated that actinomycetes possess diverse self-resistance mechanisms, and that each of them responds differently to Glycopeptide induction. Gene swapping among teicoplanins-producing actinomycetes indicated that cross-talking is possible and provides useful information for predicting the evolution of future resistance gene combinations emerging in pathogens.

  • streptomyces spp as efficient expression system for a d d peptidase d d carboxypeptidase involved in Glycopeptide Antibiotic resistance
    BMC Biotechnology, 2013
    Co-Authors: Elisa Binda, Giorgia Letizia Marcone, Loredano Pollegioni, Francesca Berini, Flavia Marinelli
    Abstract:

    Background: VanYn, encoded by the dbv7 gene (also known as vanYn) of the biosynthetic cluster devoted to A40926 production, is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp. ATCC 39727. This filamentous actinomycete is an uncommon microorganism, difficult-to-handle but biotechnologically valuable since it produces the Glycopeptide Antibiotic A40926, which is the precursor of the second-generation dalbavancin in phase III of clinical development. In order to investigate VanYn role in Glycopeptide resistance in the producer actinomycete an appropriate host-vector expression system is required. Results: The cloning strategy of vanYn gene (G-C ratio 73.3%) in the expression vector pIJ86 yielded a recombinant protein with a tag encoding for a histidine hexamer added at the C-terminus (C-His6-vanYn) or at the N-terminus (N-His6-vanYn). These plasmids were used to transform three Streptomyces spp., which are genetically-treatable high G-C content Gram-positive bacteria taxonomically related to the homologous producer Nonomuraea sp.. Highest yield of protein expression and purification (12 mg of protein per liter of culture at 3 L bioreactor-scale) was achieved in Streptomyces venezuelae ATCC 10595, that is a fast growing streptomyces susceptible to Glycopeptides. VanYn is a transmembrane protein which was easily detached and recovered from the cell wall fraction. Purified C-His6-VanYn showed D,D-carboxypeptidase and D,D-dipeptidase activities on synthetic analogs of bacterial peptidoglycan (PG) precursors. C-His6-VanYn over-expression conferred Glycopeptide resistance to S. venezuelae. On the contrary, the addition of His6-tag at the N-terminus of the protein abolished its biological activity either in vitro or in vivo assays. Conclusions: Heterologous expression of vanYn from Nonomuraea sp. ATCC 39727 in S. venezuelae was successfully achieved and conferred the host an increased level of Glycopeptide resistance. Cellular localization of recombinant VanYn together with its enzymatic activity as a D,D-peptidase/D,D-carboxypeptidase agree with its role in removing the last D-Ala from the pentapeptide PG precursors and reprogramming cell wall biosynthesis, as previously reported in Glycopeptide resistant pathogens.

  • characterization of vanyn a novel d d peptidase d d carboxypeptidase involved in Glycopeptide Antibiotic resistance in nonomuraea sp atcc 39727
    FEBS Journal, 2012
    Co-Authors: Elisa Binda, Giorgia Letizia Marcone, Loredano Pollegioni, Flavia Marinelli
    Abstract:

    VanYn is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp. ATCC 39727, which produces the Glycopeptide Antibiotic A40926, the precursor of the second-generation dalbavancin, which is in phase III of clinical development. VanYn (196 residues) is encoded by the dbv7 gene within the dbv biosynthetic cluster devoted to A40926 production. C-terminal His6-tagged VanYn was successfully expressed as a soluble and active protein in Escherichia coli. The analysis of the sequence suggests the presence of a hydrophobic transmembrane portion and two conserved sequences (SxHxxGxAxD and ExxH) in the extracytoplasmic domain that are potentially involved in coordination of Zn2+ and catalytic activity. The presence of these conserved sequences indicates a similar mechanism of action and substrate binding in VanYn as in VanY, VanX and VanXY Zn2+-dependent d,d-carboxypeptidases and d-Ala-d-Ala dipeptidases acting on peptidoglycan maturation and involved in Glycopeptide resistance in pathogens. On substrates mimicking peptidoglycan precursors, VanYn shows d,d-carboxypeptidase and d,d-dipeptidase activity, but lacks d,d-carboxyesterase ability on d-Ala-d-Lac-terminating peptides. VanYn belongs to the metallo-d,d-carboxypeptidase family, but it is inhibited by β-lactams. Its characterization provides new insights into the evolution and transfer of resistance determinants from environmental Glycopeptide-producing actinomycetes (such as Nonomuraea sp.) to Glycopeptide-resistant pathogens (enterococci and staphylococci). It may also contribute to an early warning system for emerging resistance mechanisms following the introduction into clinics of a second-generation Glycopeptide such as dalbavancin. Database The nucleotide sequence of vanYn is available in the GenBank data base under accession number CAD91202

  • valine influences production and complex composition of Glycopeptide Antibiotic a40926 in fermentations of nonomuraea sp atcc 39727
    The Journal of Antibiotics, 2004
    Co-Authors: Fabrizio Beltrametti, Enrico Selva, Luciano Gastaldo, S Jovetic, Marina Feroggio, Flavia Marinelli
    Abstract:

    In actinomycetes the catabolism products of branched chain amino acids provide biosynthetic precursors for the formation of several lipid-containing Antibiotics. We have determined in Nonomuraea sp. ATCC 39727 the effect of valine on production of Glycopeptide Antibiotic A40926, which is a complex of factors structurally differing in fatty acid moieties. Addition of valine to minimal medium increased A40926 production and modified complex composition towards a mono-component. Similar results were also obtained in a rich production medium.

C. G. Marshall - One of the best experts on this subject based on the ideXlab platform.

  • molecular mechanism of vanhst an alpha ketoacid dehydrogenase required for Glycopeptide Antibiotic resistance from a Glycopeptide producing organism
    Biochemistry, 1999
    Co-Authors: C. G. Marshall, M Zolli, Gerard D. Wright
    Abstract:

    The vancomycin resistance enzyme VanH is an α-ketoacid dehydrogenase that stereospecifically reduces pyruvate to d-lactate, which is required for the synthesis of the depsipeptide d-alanine-d-lactate. This compound then forms an integral part of the bacterial cell wall replacing the vancomycin target dipeptide d-alanine-d-alanine, thus the presence of VanH is essential for Glycopeptide resistance. In this work, the VanH homologue from the Glycopeptide Antibiotic producing organism Streptomyces toyocaensis NRRL 15009, VanHst, has been overexpressed in Escherichia coli and purified, and its substrate specificity and mechanism were probed by steady-state kinetic methods and site-directed mutagenesis. The enzyme is highly efficient at pyruvate reduction with kcat/Km = 1.3 × 105 M-1 s-1 and has a more restricted α-ketoacid substrate specificity than VanH from vancomycin resistant enterococci (VRE). Conversely, VanHst shows no preference between NADH and NADPH while VanH from VRE prefers NADPH. The kinetic mech...

  • Glycopeptide Antibiotic resistance genes in Glycopeptide-producing organisms
    Antimicrobial Agents and Chemotherapy, 1998
    Co-Authors: C. G. Marshall, I. A D Lessard, I. S. Park, G. D. Wright
    Abstract:

    The mechanism of high-level resistance to vancomycin in enterococci consists of the synthesis of peptidoglycan terminating in D-alanyl-D-lactate instead of the usual D-alanyl-D-alanine. This alternate cell wall biosynthesis pathway is ensured by the collective actions of three enzymes: VanH, VanA, and VanX. The origin of this resistance mechanism is unknown. We have cloned three genes encoding homologs of VanH, VanA, and VanX from two organisms which produce Glycopeptide Antibiotics: the A47934 producer Streptomyces toyocaensis NRRL 15009 and the vancomycin producer Amycolatopsis orientalis C329.2. The predicted amino acid sequences are highly similar to those found in VRE: 54 to 61% identity for VanH, 59 to 63% identity for VanA, and 61 to 64% identity for VanX. Furthermore, the orientations of the genes, vanH, vanA, and vanX, are identical to the orientations found in vancomycin-resistant enterococci. Southern analysis of total DNA from other Glycopeptide-producing organisms, A. orientalis 18098 (chloro-eremomycin producer), A. orientalis subsp. lurida (ristocetin producer), and Amycolatopsis coloradensis subsp. labeda (teicoplanin and avoparcin producer), with a probe derived from the vanH, vanA, and vanX cluster from A. orientalis C329.2 revealed cross-hybridizing DNA in all strains. In addition, the vanH, vanA, vanX cluster was amplified from all Glycopeptide-producing organisms by PCR with degenerate primers complementary to conserved regions in VanH and VanX. Thus, this gene sequence is common to all Glycopeptide producers tested. These results suggest that Glycopeptide-producing organisms may have been the source of resistance genes in vancomycin-resistant enterococci.

  • d ala d ala ligases from Glycopeptide Antibiotic producing organisms are highly homologous to the enterococcal vancomycin resistance ligases vana and vanb
    Proceedings of the National Academy of Sciences of the United States of America, 1997
    Co-Authors: C. G. Marshall, Greg Broadhead, B K Leskiw, Gerard D. Wright
    Abstract:

    The crisis in Antibiotic resistance has resulted in an increasing fear of the emergence of untreatable organisms. Resistance to the Glycopeptide Antibiotic vancomycin in the enterococci, and the spread of these pathogens throughout the environment, has shown that this scenario is a matter of fact rather than fiction. The basis for vancomycin resistance is the manufacture of the depsipeptide d-Ala-d-lactate, which is incorporated into the peptidoglycan cell wall in place of the vancomycin target d-Ala-d-Ala. Pivotal to the resistance mechanism is the production of a d-Ala-d-Ala ligase capable of ester formation. Two highly efficient depsipeptide ligases have been cloned from vancomycin-resistant enterococci: VanA and VanB. These ligases show high amino acid sequence similarity to each other (≈75%), but less so to other d-Ala-d-X ligases ( 60%) but not to other d-Ala-d-X ligases (<35%). The d-Ala-d-Ala ligase from S. toyocaensis shows d-Ala-d-lactate synthase activity in cell-free extracts of S. lividans transformed with the ddl gene and confirms the predicted enzymatic activity. These results imply a close evolutionary relationship between resistance mechanisms in the clinics and in drug-producing bacteria.

  • The Glycopeptide Antibiotic producer Streptomyces toyocaensis NRRL 15009 has both D-alanyl-D-alanine and D-alanyl-D-lactate ligases
    FEMS Microbiology Letters, 1997
    Co-Authors: C. G. Marshall, G. D. Wright
    Abstract:

    High level resistance to vancomycin and other Glycopeptide Antibiotics requires the synthesis of peptidoglycan terminating in the depsipeptide D- Ala-D-lactate, rather than the usual D-Ala-D-Ala. We report the purification and enzymatic characterization of two D-Ala ligases from Streptomyces toyocaensis NRRL 15009 which produces the Glycopeptide Antibiotic A47934. One of these enzymes catalyzes only D-Ala-D-Ala peptide formation and is recovered from mid-exponential phase cell cultures. The other enzyme is a D- Ala-D-lactate ligase which can be detected in actively Antibiotic producing stationary phase cultures or mid-exponential phase cultures grown in the presence of A47934. These results imply that peptidoglycan components of S. toyocaensis NRRL 15009 change upon induction of Antibiotic production and predict the existence of a VanX-like D-Ala-D-Ala DD-dipeptidase activity.

G. D. Wright - One of the best experts on this subject based on the ideXlab platform.

  • Glycopeptide Antibiotic resistance genes in Glycopeptide-producing organisms
    Antimicrobial Agents and Chemotherapy, 1998
    Co-Authors: C. G. Marshall, I. A D Lessard, I. S. Park, G. D. Wright
    Abstract:

    The mechanism of high-level resistance to vancomycin in enterococci consists of the synthesis of peptidoglycan terminating in D-alanyl-D-lactate instead of the usual D-alanyl-D-alanine. This alternate cell wall biosynthesis pathway is ensured by the collective actions of three enzymes: VanH, VanA, and VanX. The origin of this resistance mechanism is unknown. We have cloned three genes encoding homologs of VanH, VanA, and VanX from two organisms which produce Glycopeptide Antibiotics: the A47934 producer Streptomyces toyocaensis NRRL 15009 and the vancomycin producer Amycolatopsis orientalis C329.2. The predicted amino acid sequences are highly similar to those found in VRE: 54 to 61% identity for VanH, 59 to 63% identity for VanA, and 61 to 64% identity for VanX. Furthermore, the orientations of the genes, vanH, vanA, and vanX, are identical to the orientations found in vancomycin-resistant enterococci. Southern analysis of total DNA from other Glycopeptide-producing organisms, A. orientalis 18098 (chloro-eremomycin producer), A. orientalis subsp. lurida (ristocetin producer), and Amycolatopsis coloradensis subsp. labeda (teicoplanin and avoparcin producer), with a probe derived from the vanH, vanA, and vanX cluster from A. orientalis C329.2 revealed cross-hybridizing DNA in all strains. In addition, the vanH, vanA, vanX cluster was amplified from all Glycopeptide-producing organisms by PCR with degenerate primers complementary to conserved regions in VanH and VanX. Thus, this gene sequence is common to all Glycopeptide producers tested. These results suggest that Glycopeptide-producing organisms may have been the source of resistance genes in vancomycin-resistant enterococci.

  • The Glycopeptide Antibiotic producer Streptomyces toyocaensis NRRL 15009 has both D-alanyl-D-alanine and D-alanyl-D-lactate ligases
    FEMS Microbiology Letters, 1997
    Co-Authors: C. G. Marshall, G. D. Wright
    Abstract:

    High level resistance to vancomycin and other Glycopeptide Antibiotics requires the synthesis of peptidoglycan terminating in the depsipeptide D- Ala-D-lactate, rather than the usual D-Ala-D-Ala. We report the purification and enzymatic characterization of two D-Ala ligases from Streptomyces toyocaensis NRRL 15009 which produces the Glycopeptide Antibiotic A47934. One of these enzymes catalyzes only D-Ala-D-Ala peptide formation and is recovered from mid-exponential phase cell cultures. The other enzyme is a D- Ala-D-lactate ligase which can be detected in actively Antibiotic producing stationary phase cultures or mid-exponential phase cultures grown in the presence of A47934. These results imply that peptidoglycan components of S. toyocaensis NRRL 15009 change upon induction of Antibiotic production and predict the existence of a VanX-like D-Ala-D-Ala DD-dipeptidase activity.