scholarly journals Self-Resistance and Cell Wall Composition in the Glycopeptide Producer Amycolatopsis balhimycina

2011 ◽  
Vol 55 (9) ◽  
pp. 4283-4289 ◽  
Author(s):  
Till F. Schäberle ◽  
Waldemar Vollmer ◽  
Hans-Jörg Frasch ◽  
Stephan Hüttel ◽  
Andreas Kulik ◽  
...  
Keyword(s):  
Cell Wall ◽  
Resistance Genes ◽  
Streptomyces Coelicolor ◽  
Resistance Mechanism ◽  
Biosynthetic Gene Cluster ◽  
Resistant Cell ◽  
Glycopeptide Resistance ◽  
Content Type ◽  
Constitutive Synthesis ◽  
Gene Orthologs

ABSTRACTThe prevailing resistance mechanism against glycopeptides in Gram-positive pathogens involves reprogramming the biosynthesis of peptidoglycan precursors, resulting ind-alanyl-d-lactate depsipeptide termini.Amycolatopsis balhimycinaproduces the vancomycin-like glycopeptide balhimycin and therefore has to protect itself from the action of the glycopeptide. We studied the roles of the accessory resistance gene orthologsvanYb,vnlRb, andvnlSb, which are part of the balhimycin biosynthetic gene cluster (represented by the subscript “b”). The VanYbcarboxypeptidase cleaved the terminald-Ala from peptidoglycan precursors, and its heterologous expression enhanced glycopeptide resistance inStreptomyces coelicolor. The VanRS-like two component system VnlRSbwas not involved in glycopeptide resistance or in the expression of thevanHAXglycopeptide resistance genes. MatureA. balhimycinapeptidoglycan contained mainly tri- and tetrapeptides, with only traces of thed-Ala-d-Ala-ending pentapeptides that are binding sites for the antibiotic produced. The structure of the peptidoglycan precursor is consistent with the presence ofvanHAXgenes, which were identified outside the balhimycin synthesis cluster. Both wild-type and non-antibiotic-producing mutant strains synthesized peptidoglycan precursors ending mainly withd-Lac, indicating constitutive synthesis of a resistant cell wall.A. balhimycinacould provide a model for an ancestral glycopeptide producer with constitutively expressed resistance genes.

scholarly journals Relationship between Glycopeptide Production and Resistance in the Actinomycete Nonomuraea sp. ATCC 39727

2014 ◽  
Vol 58 (9) ◽  
pp. 5191-5201 ◽  
Author(s):  
Giorgia Letizia Marcone ◽  
Elisa Binda ◽  
Lucia Carrano ◽  
Mervyn Bibb ◽  
Flavia Marinelli
Keyword(s):  
Antibiotic Resistance ◽  
Gene Dosage ◽  
Antibiotic Production ◽  
Resistance Mechanism ◽  
Biosynthetic Gene Cluster ◽  
Penicillin G ◽  
Glycopeptide Resistance ◽  
Content Type ◽  
High Level

ABSTRACTGlycopeptides and β-lactams inhibit bacterial peptidoglycan synthesis in Gram-positive bacteria; resistance to these antibiotics is studied intensively in enterococci and staphylococci because of their relevance to infectious disease. Much less is known about antibiotic resistance in glycopeptide-producing actinomycetes that are likely to represent the evolutionary source of resistance determinants found in bacterial pathogens.Nonomuraeasp. ATCC 39727, the producer of A40926 (the precursor for the semisynthetic dalbavancin), does not harbor the canonicalvanHAXgenes. Consequently, we investigated the role of the β-lactam-sensitived,d-peptidase/d,d-carboxypeptidase encoded byvanYn, the onlyvan-like gene found in the A40926 biosynthetic gene cluster, in conferring immunity to the antibiotic inNonomuraeasp. ATCC 39727. Taking advantage of the tools developed recently to genetically manipulate this uncommon actinomycete, we variedvanYngene dosage and expressedvanHatAatXatfrom the teicoplanin producerActinoplanes teichomyceticusinNonomuraeasp. ATCC 39727. Knocking outvanYn, complementing avanYnmutant, or duplicatingvanYnhad no effect on growth but influenced antibiotic resistance and, in the cases of complementation and duplication, antibiotic production.Nonomuraeasp. ATCC 39727 was found to be resistant to penicillins, but its glycopeptide resistance was diminished in the presence of penicillin G, which inhibits VanYnactivity. The heterologous expression ofvanHatAatXatincreased A40926 resistance inNonomuraeasp. ATCC 39727 but did not increase antibiotic production, indicating that the level of antibiotic production is not directly determined by the level of resistance. ThevanYn-based self-resistance inNonomuraeasp. ATCC 39727 resembles the glycopeptide resistance mechanism described recently in mutants ofEnterococcus faeciumselectedin vitrofor high-level resistance to glycopeptides and penicillins.

scholarly journals In VivoStudies Suggest that Induction of VanS-Dependent Vancomycin Resistance Requires Binding of the Drug to d-Ala-d-Ala Termini in the Peptidoglycan Cell Wall

2013 ◽  
Vol 57 (9) ◽  
pp. 4470-4480 ◽  
Author(s):  
Min Jung Kwun ◽  
Gabriela Novotna ◽  
Andrew R. Hesketh ◽  
Lionel Hill ◽  
Hee-Jeon Hong
Keyword(s):  
Cell Wall ◽  
Transcriptional Activation ◽  
Streptomyces Coelicolor ◽  
Constitutive Expression ◽  
Transcriptional Response ◽  
Vancomycin Resistance ◽  
Content Type ◽  
Expression Of Genes ◽  
Peptidoglycan Cell Wall

ABSTRACTVanRS two-component regulatory systems are key elements required for the transcriptional activation of inducible vancomycin resistance genes in bacteria, but the precise nature of the ligand signal that activates these systems has remained undefined. Using the resistance system inStreptomyces coelicoloras a model, we have undertaken a series ofin vivostudies which indicate that the VanS sensor kinase in VanB-type resistance systems is activated by vancomycin in complex with thed-alanyl-d-alanine (d-Ala-d-Ala) termini of cell wall peptidoglycan (PG) precursors. Complementation of an essentiald-Ala-d-Ala ligase activity by constitutive expression ofvanAencoding a bifunctionald-Ala-d-Ala andd-alanyl-d-lactate (d-Ala-d-Lac) ligase activity allowed construction of strains that synthesized variable amounts of PG precursors containingd-Ala-d-Ala. Assays quantifying the expression of genes under VanRS control showed that the response to vancomycin in these strains correlated with the abundance ofd-Ala-d-Ala-containing PG precursors; strains producing a lower proportion of PG precursors terminating ind-Ala-d-Ala consistently exhibited a lower response to vancomycin. Pretreatment of wild-type cells with vancomycin or teicoplanin to saturate and mask thed-Ala-d-Ala binding sites in nascent PG also blocked the transcriptional response to subsequent vancomycin exposure, and desleucyl vancomycin, a vancomycin analogue incapable of interacting withd-Ala-d-Ala residues, failed to inducevangene expression. Activation of resistance by a vancomycin–d-Ala-d-Ala PG complex predicts a limit to the proportion of PG that can be derived from precursors terminating ind-Ala-d-Lac, a restriction also enforced by the bifunctional activity of the VanA ligase.

scholarly journals Peptidoglycan Cross-Linking in Glycopeptide-Resistant Actinomycetales

2014 ◽  
Vol 58 (3) ◽  
pp. 1749-1756 ◽  
Author(s):  
Jean-Emmanuel Hugonnet ◽  
Nabila Haddache ◽  
Carole Veckerlé ◽  
Lionel Dubost ◽  
Arul Marie ◽  
...  
Keyword(s):  
Streptomyces Coelicolor ◽  
Residual Activity ◽  
Resistance Mechanisms ◽  
Strain Atcc ◽  
Glycopeptide Resistance ◽  
Content Type ◽  
Peptidoglycan Structure ◽  
Cross Links ◽  
Acyl Donors ◽  
Hydrolysis Of

ABSTRACTSynthesis of peptidoglycan precursors ending ind-lactate (d-Lac) is thought to be responsible for glycopeptide resistance in members of the orderActinomycetalesthat produce these drugs and in related soil bacteria. More recently, the peptidoglycan of several members of the orderActinomycetaleswas shown to be cross-linked byl,d-transpeptidases that use tetrapeptide acyl donors devoid of the target of glycopeptides. To evaluate the contribution of these resistance mechanisms, we have determined the peptidoglycan structure ofStreptomyces coelicolorA(3)2, which harbors avanHAXgene cluster for the production of precursors ending ind-Lac, andNonomuraeasp. strain ATCC 39727, which is devoid ofvanHAXand produces the glycopeptide A40296. Vancomycin retained residual activity againstS. coelicolorA(3)2 despite efficient incorporation ofd-Lac into cytoplasmic precursors. This was due to ad,d-transpeptidase-catalyzed reaction that generated a stem pentapeptide recognized by glycopeptides by the exchange ofd-Lac ford-Ala and Gly. The contribution ofl,d-transpeptidases to resistance was limited by the supply of tetrapeptide acyl donors, which are essential for the formation of peptidoglycan cross-links by these enzymes. In the absence of a cytoplasmic metallo-d,d-carboxypeptidase, the tetrapeptide substrate was generated by hydrolysis of the C-terminald-Lac residue of the stem pentadepsipeptide in the periplasm in competition with the exchange reaction catalyzed byd,d-transpeptidases. InNonomuraeasp. strain ATCC 39727, the contribution ofl,d-transpeptidases to glycopeptide resistance was limited by the incomplete conversion of pentapeptides into tetrapeptides despite the production of a cytoplasmic metallo-d,d-carboxypeptidase. Since the level of drug production exceeds the level of resistance, we propose thatl,d-transpeptidases merely act as a tolerance mechanism in this bacterium.

scholarly journals Oritavancin Activity against Vancomycin-Susceptible and Vancomycin-Resistant Enterococci with Molecularly Characterized Glycopeptide Resistance Genes Recovered from Bacteremic Patients, 2009-2010

2011 ◽  
Vol 56 (3) ◽  
pp. 1639-1642 ◽  
Author(s):  
Rodrigo E. Mendes ◽  
Leah N. Woosley ◽  
David J. Farrell ◽  
Helio S. Sader ◽  
Ronald N. Jones
Keyword(s):  
Resistance Genes ◽  
Glycopeptide Resistance ◽  
Content Type ◽  
Vancomycin Resistant Enterococci ◽  
Vancomycin Resistant

ABSTRACTOritavancin exhibited potent activity against vancomycin-susceptible (MIC50and MIC90, 0.015/0.03 μg/ml) andvanB-carryingE. faecalisisolates (MIC50and MIC90, 0.015 and 0.015 μg/ml). Higher (16- to 32-fold) MIC50s and MIC90s forvanA-harboringE. faecaliswere noted (MIC50and MIC90, 0.25 and 0.5 μg/ml), although oritavancin inhibited all strains at ≤0.5 μg/ml. Vancomycin-susceptible andvanB-carryingE. faeciumstrains (MIC50and MIC90, ≤0.008 and ≤0.008 μg/ml for both) were very susceptible to oritavancin, as were VanA-producing isolates (MIC50and MIC90, 0.03 and 0.06 μg/ml). Oritavancin exhibited goodin vitropotency against this collection of organisms, including vancomycin-resistant enterococci.

scholarly journals Substrate Inhibition of VanA by d-Alanine Reduces Vancomycin Resistance in a VanX-Dependent Manner

2016 ◽  
Vol 60 (8) ◽  
pp. 4930-4939 ◽  
Author(s):  
Lizah T. van der Aart ◽  
Nicole Lemmens ◽  
Willem J. van Wamel ◽  
Gilles P. van Wezel
Keyword(s):  
Cell Wall ◽  
Streptomyces Coelicolor ◽  
Substrate Inhibition ◽  
Model Organism ◽  
Vancomycin Resistance ◽  
Dependent Manner ◽  
Wild Type ◽  
Content Type ◽  
Lipid Ii ◽  
Vancomycin Resistant

ABSTRACTThe increasing resistance of clinical pathogens against the glycopeptide antibiotic vancomycin, a last-resort drug against infections with Gram-positive pathogens, is a major problem in the nosocomial environment. Vancomycin inhibits peptidoglycan synthesis by binding to thed-Ala–d-Ala terminal dipeptide moiety of the cell wall precursor lipid II. Plasmid-transferable resistance is conferred by modification of the terminal dipeptide into the vancomycin-insensitive variantd-Ala–d-Lac, which is produced by VanA. Here we show that exogenousd-Ala competes withd-Lac as a substrate for VanA, increasing the ratio of wild-type to mutant dipeptide, an effect that was augmented by several orders of magnitude in the absence of thed-Ala–d-Ala peptidase VanX. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that high concentrations ofd-Ala led to the production of a significant amount of wild-type cell wall precursors, whilevanX-null mutants produced primarily wild-type precursors. This enhanced the efficacy of vancomycin in the vancomycin-resistant model organismStreptomyces coelicolor, and the susceptibility of vancomycin-resistant clinical isolates ofEnterococcus faecium(VRE) increased by up to 100-fold. The enhanced vancomycin sensitivity ofS. coelicolorcells correlated directly to increased binding of the antibiotic to the cell wall. Our work offers new perspectives for the treatment of diseases associated with vancomycin-resistant pathogens and for the development of drugs that target vancomycin resistance.

scholarly journals An Alternative and Conserved Cell Wall Enzyme That Can Substitute for the Lipid II Synthase MurG

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
L. Zhang ◽  
K. Ramijan ◽  
V. J. Carrión ◽  
L. T. van der Aart ◽  
J. Willemse ◽  
...  
Keyword(s):  
Cell Wall ◽  
Sequence Similarity ◽  
Streptomyces Coelicolor ◽  
Genomic Analysis ◽  
Environmental Harm ◽  
Cell Wall Synthesis ◽  
Precursor Molecule ◽  
Content Type ◽  
Lipid Ii ◽  
A Cell

ABSTRACT The cell wall is a stress-bearing structure and a unifying trait in bacteria. Without exception, synthesis of the cell wall involves formation of the precursor molecule lipid II by the activity of the essential biosynthetic enzyme MurG, which is encoded in the division and cell wall synthesis (dcw) gene cluster. Here, we present the discovery of a cell wall enzyme that can substitute for MurG. A mutant of Kitasatospora viridifaciens lacking a significant part of the dcw cluster, including murG, surprisingly produced lipid II and wild-type peptidoglycan. Genomic analysis identified a distant murG homologue, which encodes a putative enzyme that shares only around 31% amino acid sequence identity with MurG. We show that this enzyme can replace the canonical MurG, and we therefore designated it MglA. Orthologues of mglA are present in 38% of all genomes of Kitasatospora and members of the sister genus Streptomyces. CRISPR interference experiments showed that K. viridifaciens mglA can also functionally replace murG in Streptomyces coelicolor, thus validating its bioactivity and demonstrating that it is active in multiple genera. All together, these results identify MglA as a bona fide lipid II synthase, thus demonstrating plasticity in cell wall synthesis. IMPORTANCE Almost all bacteria are surrounded by a cell wall, which protects cells from environmental harm. Formation of the cell wall requires the precursor molecule lipid II, which in bacteria is universally synthesized by the conserved and essential lipid II synthase MurG. We here exploit the unique ability of an actinobacterial strain capable of growing with or without its cell wall to discover an alternative lipid II synthase, MglA. Although this enzyme bears only weak sequence similarity to MurG, it can functionally replace MurG and can even do so in organisms that naturally have only a canonical MurG. The observation that MglA proteins are found in many actinobacteria highlights the plasticity in cell wall synthesis in these bacteria and demonstrates that important new cell wall biosynthetic enzymes remain to be discovered.

scholarly journals Cell Wall Recycling-Linked Coregulation of AmpC and PenB β-Lactamases throughampDMutations in Burkholderia cenocepacia

2015 ◽  
Vol 59 (12) ◽  
pp. 7602-7610 ◽  
Author(s):  
Junghyun Hwang ◽  
Heenam Stanley Kim
Keyword(s):  
Cell Wall ◽  
Burkholderia Cepacia ◽  
Nucleotide Substitution ◽  
Resistance Mechanism ◽  
Gene Organization ◽  
Burkholderia Cenocepacia ◽  
Single Nucleotide ◽  
Content Type ◽  
Cephalosporin Antibiotics ◽  
Cell Wall Recycling

ABSTRACTIn many Gram-negative pathogens, mutations in the key cell wall-recycling enzyme AmpD (N-acetyl-anhydromuramyl-l-alanine amidase) affect the activity of the regulator AmpR, which leads to the expression of AmpC β-lactamase, conferring resistance to expanded-spectrum cephalosporin antibiotics.Burkholderia cepaciacomplex (Bcc) species also have these Amp homologs; however, the regulatory circuitry and the nature of causalampDmutations remain to be explored. A total of 92ampDmutants were obtained, representing four types of mutations: single nucleotide substitution (causing an amino acid substitution or antitermination of the enzyme), duplication, deletion, and IS element insertion. Duplication, which can go through reversion, was the most frequent type. Intriguingly, mutations inampDled to the induction of two β-lactamases, AmpC and PenB. Coregulation of AmpC and PenB inB. cenocepacia, and likely also in many Bcc species with the same gene organization, poses a serious threat to human health. This resistance mechanism is of evolutionary optimization in thatampDis highly prone to mutations allowing rapid response to antibiotic challenge, and many of the mutations are reversible in order to resume cell wall recycling when the antibiotic challenge is relieved.

scholarly journals Polydiglycosylphosphate Transferase PdtA (SCO2578) of Streptomyces coelicolor A3(2) Is Crucial for Proper Sporulation and Apical Tip Extension under Stress Conditions

2016 ◽  
Vol 82 (18) ◽  
pp. 5661-5672 ◽  
Author(s):  
Steffen Sigle ◽  
Nadja Steblau ◽  
Wolfgang Wohlleben ◽  
Günther Muth
Keyword(s):  
Cell Wall ◽  
Life Cycle ◽  
Vegetative Growth ◽  
Cell Envelope ◽  
Streptomyces Coelicolor ◽  
Morphological Differentiation ◽  
Spore Wall ◽  
Stress Conditions ◽  
Content Type ◽  
Tip Extension

ABSTRACTAlthough anionic glycopolymers are crucial components of the Gram-positive cell envelope, the relevance of anionic glycopolymers for vegetative growth and morphological differentiation ofStreptomyces coelicolorA3(2) is unknown. Here, we show that the LytR-CpsA-Psr (LCP) protein PdtA (SCO2578), a TagV-like glycopolymer transferase, has a dual function in theS. coelicolorA3(2) life cycle. Despite the presence of 10 additional LCP homologs, PdtA is crucial for proper sporulation. The integrity of the spore envelope was severely affected in apdtAdeletion mutant, resulting in 34% nonviable spores.pdtAdeletion caused a significant reduction in the polydiglycosylphosphate content of the spore envelope. Beyond that, apical tip extension and normal branching of vegetative mycelium were severely impaired on high-salt medium. This growth defect coincided with the mislocalization of peptidoglycan synthesis. Thus, PdtA itself or the polydiglycosylphosphate attached to the peptidoglycan by the glycopolymer transferase PdtA also has a crucial function in apical tip extension of vegetative hyphae under stress conditions.IMPORTANCEAnionic glycopolymers are underappreciated components of the Gram-positive cell envelope. They provide rigidity to the cell wall and position extracellular enzymes involved in peptidoglycan remodeling. AlthoughStreptomyces coelicolorA3(2), the model organism for bacterial antibiotic production, is known to produce two distinct cell wall-linked glycopolymers, teichulosonic acid and polydiglycosylphosphate, the role of these glycopolymers in theS. coelicolorA3(2) life cycle has not been addressed so far. This study reveals a crucial function of the anionic glycopolymer polydiglycosylphosphate for the growth and morphological differentiation ofS. coelicolorA3(2). Polydiglycosylphosphate is attached to the spore wall by the LytR-CpsA-Psr protein PdtA (SCO2578), a component of theStreptomycesspore wall-synthesizing complex (SSSC), to ensure the integrity of the spore envelope. Surprisingly, PdtA also has a crucial role in vegetative growth under stress conditions and is required for proper peptidoglycan incorporation during apical tip extension.

scholarly journals A Two-Step Mechanism for the Activation of Actinorhodin Export and Resistance in Streptomyces coelicolor

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Ye Xu ◽  
Andrew Willems ◽  
Catherine Au-yeung ◽  
Kapil Tahlan ◽  
Justin R. Nodwell
Keyword(s):  
Secondary Metabolites ◽  
Pathogenic Bacteria ◽  
Streptomyces Coelicolor ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Resistance Mechanisms ◽  
Biosynthetic Gene ◽  
Content Type

ABSTRACT Many microorganisms produce secondary metabolites that have antibiotic activity. To avoid self-inhibition, the producing cells often encode cognate export and/or resistance mechanisms in the biosynthetic gene clusters for these molecules. Actinorhodin is a blue-pigmented antibiotic produced by Streptomyces coelicolor. The actAB operon, carried in the actinorhodin biosynthetic gene cluster, encodes two putative export pumps and is regulated by the transcriptional repressor protein ActR. In this work, we show that normal actinorhodin yields require actAB expression. Consistent with previous in vitro work, we show that both actinorhodin and its 3-ring biosynthetic intermediates [e.g., (S)-DNPA] can relieve repression of actAB by ActR in vivo. Importantly, an ActR mutant that interacts productively with (S)-DNPA but not with actinorhodin responds to the actinorhodin biosynthetic pathway with the induction of actAB and normal yields of actinorhodin. This suggests that the intermediates are sufficient to trigger the export genes in actinorhodin-producing cells. We further show that actinorhodin-producing cells can induce actAB expression in nonproducing cells; however, in this case actinorhodin is the most important signal. Finally, while the “intermediate-only” ActR mutant permits sufficient actAB expression for normal actinorhodin yields, this expression is short-lived. Sustained culture-wide expression requires a subsequent actinorhodin-mediated signaling step, and the defect in this response causes widespread cell death. These results are consistent with a two-step model for actinorhodin export and resistance where intermediates trigger initial expression for export from producing cells and actinorhodin then triggers sustained export gene expression that confers culture-wide resistance. IMPORTANCE Understanding the links between antibiotic resistance and biosynthesis is important for our efforts to manipulate secondary metabolism. For example, many secondary metabolites are produced at low levels; our work suggests that manipulating export might be one way to enhance yields of these molecules. It also suggests that understanding resistance will be relevant to the generation of novel secondary metabolites through the creation of synthetic secondary metabolic gene clusters. Finally, these cognate resistance mechanisms are related to mechanisms that arise in pathogenic bacteria, and understanding them is relevant to our ability to control microbial infections clinically.

scholarly journals The Activity of Glycopeptide Antibiotics against Resistant Bacteria Correlates with Their Ability To Induce the Resistance System

2014 ◽  
Vol 58 (10) ◽  
pp. 6306-6310 ◽  
Author(s):  
Min Jung Kwun ◽  
Hee-Jeon Hong
Keyword(s):  
Resistance Genes ◽  
Streptomyces Coelicolor ◽  
Resistant Bacteria ◽  
Glycopeptide Antibiotics ◽  
Content Type ◽  
Vancomycin Resistant Enterococci ◽  
Resistance System ◽  
Vancomycin Resistant

ABSTRACTGlycopeptide antibiotics containing a hydrophobic substituent display the best activity against vancomycin-resistant enterococci, and they have been assumed to be poor inducers of the resistance system. Using a panel of 26 glycopeptide derivatives and the model resistance system inStreptomyces coelicolor, we confirmed this hypothesis at the level of transcription. Identification of the structural glycopeptide features associated with inducing the expression of resistance genes has important implications in the search for more effective antibiotic structures.

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