Reversal by a specific peptide (diacetyl-alpha gamma-L-diaminobutyryl-D-alanyl-D-alanine) of vancomycin inhibition in intact bacteria and cell-free preparations.
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ABSTRACT: Vancomycin inhibited the growth of Bacillus megaterium, Staphylococcus aureus and Micrococcus lysodeikticus, and in cell-free preparations from B. megaterium it inhibited the formation of mucopeptide and enhanced the accumulation of the lipid intermediate in the biosynthetic pathway. All these inhibitory processes were reversed by the presence of a synthetic peptide analogous to un-cross-linked mucopeptide side chains, namely diacetyl-l-diaminobutyryl-d-alanyl-d-alanine. A considerable amount of vancomycin was found in recovering cells, whether recovery was caused by peptide or took place naturally because a low initial concentration of antibiotic was used. In cell-free preparations pretreated with vancomycin, continued inhibition of mucopeptide synthesis depended on the presence of cell-wall material. This inhibition was also reversible by added peptide.
Project description:Electrometric and spectrophotometric titrations showed vancomycin to contain groups having pK values of about 2.9, 7.2, 8.6, 9.6, 10.5 and 11.7. Of these the four last-named were phenolic. Titration above pH11 and below pH1 was irreversible and antibiotic potency was destroyed. Combination with the specific peptide diacetyl-l-lysyl-d-alanyl-d-alanine hindered the titration of the first three phenolic groups. Spectrophotometric titration of iodovancomycin showed that the phenolic group with pK 9.6 was the one iodinated. The stability of the vancomycin-peptide complex in the range pH1-13 showed that complex-formation occurred only when carboxyl groups were ionized and the phenolic groups were non-ionized. The complex was formed in concentrations of urea up to 8m, of potassium chloride up to 4m, of sodium dodecyl sulphate up to 1%, and at temperatures up to 60 degrees C. From titration curves, organic chlorine and iodine analysis, and combination with peptide, a minimum molecular weight for vancomycin of 1700-1800 was estimated. Optical-rotatory-dispersion and circular-dichroism experiments suggested that vancomycin has only limited conformational flexibility. Both vancomycin and its complexes with peptide exhibited properties suggesting aggregation. Vancomycin and iodovancomycin can be fractionated into a main fraction and at least three minor components. The isolation of these fractions salt-free is described and their antibiotic properties are shown to correlate with their ability to form complexes with peptide.
Project description:The peptidoglycan composition in lactic acid bacteria dictates vancomycin resistance. Vancomycin binds relatively poorly to peptidoglycan ending in d-alanyl-d-lactate and binds with high affinity to peptidoglycan ending in d-alanyl-d-alanine (d-Ala-d-Ala), which results in vancomycin resistance and sensitivity, respectively. The enzyme responsible for generating these peptidoglycan precursors is dipeptide ligase (Ddl). A single amino acid in the Ddl active site, phenylalanine or tyrosine, determines depsipeptide or dipeptide activity, respectively. Here, we established that heterologous expression of dipeptide ligase in vancomycin-resistant lactobacilli increases their sensitivity to vancomycin in a dose-dependent manner and overcomes the effects of the presence of a native d-Ala-d-Ala dipeptidase. We incorporated the dipeptide ligase gene on a suicide vector and demonstrated that it functions as a counterselection marker (CSM) in lactobacilli; vancomycin selection allows only those cells to grow in which the suicide vector has been lost. Subsequently, we developed a liquid-based approach to identify recombinants in only 5 days, which is approximately half the time required by conventional approaches. Phylogenetic analysis revealed that Ddl serves as a marker to predict vancomycin resistance and consequently indicated the broad applicability of the use of Ddl as a counterselection marker in the genus Lactobacillus Finally, our system represents the first "plug and play" counterselection system in lactic acid bacteria that does not require prior genome editing and/or synthetic medium.IMPORTANCE The genus Lactobacillus contains more than 200 species, many of which are exploited in the food and biotechnology industries and in medicine. Prediction of intrinsic vancomycin resistance has thus far been limited to selected Lactobacillus species. Here, we show that heterologous expression of the enzyme Ddl (dipeptide ligase)-an essential enzyme involved in peptidoglycan synthesis-increases sensitivity to vancomycin in a dose-dependent manner. We exploited this to develop a counterselection marker for use in vancomycin-resistant lactobacilli, thereby expanding the poorly developed genome editing toolbox that is currently available for most strains. Also, we showed that Ddl is a phylogenetic marker that can be used to predict vancomycin resistance in Lactobacillus; 81% of Lactobacillus species are intrinsically resistant to vancomycin, which makes our tool broadly applicable.
Project description:Rationale: Rapid and facile detection of pathogenic bacteria is challenging due to the requirement of large-scale instruments and equipment in conventional methods. We utilize D-amino acid as molecules to selectively target bacteria because bacteria can incorporate DADA in its cell wall while mammalian cells or fungi cannot. Methods: We show a broad-spectrum bacterial detection system based on D-amino acid-capped gold nanoparticles (AuNPs). AuNPs serve as the signal output that we can monitor without relying on any complex instruments. Results: In the presence of bacteria, the AuNPs aggregate and the color of AuNPs changes from red to blue. This convenient color change can distinguish between Staphylococcus aureus (S. aureus) and methicillin-resistant Staphylococcus aureus (MRSA). This system can be applied for detection of ascites samples from patients. Conclusion: These D-amino acid-modified AuNPs serve as a promising platform for rapid visual identification of pathogens in the clinic.
Project description:Vancomycin forms complexes with peptides terminating in d-alanyl-d-alanine that are analogous to the biosynthetic precursors of bacterial mucopeptides. The specificity of complex-formation has been studied by means of many synthetic peptides, prepared by both solid-phase and conventional methods. The following conclusions can be drawn: (a) three amide linkages are required to form a stable complex; (b) the terminal carboxyl group must be free; (c) the carboxyl terminal and subterminal residues must be either glycine or of the d-configuration; (d) the size of the side chain in these residues greatly influences the affinity for vancomycin, a methyl group being the optimum in each case; (e) the nature of the side chain in the third and fourth residues has a smaller effect on complex-formation, but an l-configuration was somewhat better than a d-configuration in the third position. In addition to acyl-d-alanyl-d-alanine, other peptides that occur in bacterial cell walls will combine with vancomycin, although less strongly, e.g. acyl-d-alanyl-d-alpha-amino acid (where the terminal d-residue may form the cross-link in mucopeptide structure) and acyl-l-alanyl-d-glutamylglycine (a sequence found in the mucopeptide of Micrococcus lysodeikticus and related organisms). These results throw some light on the specificity of the uptake of vancomycin by living bacteria.
Project description:Fragments of erm(E2), otrA, and aph(6) shorter than 400 bp and producer strain-specific rRNA genes were amplified from various antibiotics. The amount of genetic material and the sizes of amplicons recovered from murine feces after oral administration of a beta-lactamase-encoding plasmid indicated substantial DNA degradation in the mammalian gastrointestinal tract. These observations imply that antibiotics are no major source for horizontal resistance gene transfer in clinical settings.
Project description:Determining transmural mechanical properties in the heart provides a foundation to understand physiological and pathophysiological cardiac mechanics. Although work on mechanical characterisation has begun in isolated cells and permeabilised samples, the mechanical profile of living individual cardiac layers has not been examined. Myocardial slices are 300 ?m-thin sections of heart tissue with preserved cellular stoichiometry, extracellular matrix, and structural architecture. This allows for cardiac mechanics assays in the context of an intact in vitro organotypic preparation. In slices obtained from the subendocardium, midmyocardium and subepicardium of rats, a distinct pattern in transmural contractility is found that is different from that observed in other models. Slices from the epicardium and midmyocardium had a higher active tension and passive tension than the endocardium upon stretch. Differences in total myocyte area coverage, and aspect ratio between layers underlined the functional readouts, while no differences were found in total sarcomeric protein and phosphoprotein between layers. Such intrinsic heterogeneity may orchestrate the normal pumping of the heart in the presence of transmural strain and sarcomere length gradients in the in vivo heart.
Project description:The VanX protein is a D-alanyl-D-alanine (D-Ala-D-Ala) dipeptidase essential for resistance to the glycopeptide antibiotic vancomycin. While this enzymatic activity has been typically associated with vancomycin- and teicoplainin-resistant enterococci, we now report the identification of a D-Ala-D-Ala dipeptidase in the gram-negative species Salmonella enterica. The Salmonella enzyme is only 36% identical to VanX but exhibits a similar substrate specificity: it hydrolyzes D-Ala-D-Ala, DL-Ala-DL-Phe, and D-Ala-Gly but not the tripeptides D-Ala-D-Ala-D-Ala and DL-Ala-DL-Lys-Gly or the dipeptides L-Ala-L-Ala, N-acetyl-D-Ala-D-Ala, and L-Leu-Pro. The Salmonella dipeptidase gene, designated pcgL, appears to have been acquired by horizontal gene transfer because pcgL-hybridizing sequences were not detected in related bacterial species and the G+C content of the pcgL-containing region (41%) is much lower than the overall G+C content of the Salmonella chromosome (52%). In contrast to wild-type Salmonella, a pcgL mutant was unable to use D-Ala-D-Ala as a sole carbon source. The pcgL gene conferred D-Ala-D-Ala dipeptidase activity upon Escherichia coli K-12 but did not allow growth on D-Ala-D-Ala. The PcgL protein localizes to the periplasmic space of Salmonella, suggesting that this dipeptidase participates in peptidoglycan metabolism.
Project description:Mutants of a diaminopimelic acid (Dap)-requiring strain of Escherichia coli were isolated which failed to grow on media in which Dap was replaced by the cell wall murein tripeptide, L-alanyl-gamma-D-glutamyl-mesodiaminopimelate. In one such mutant, which is oligopeptide permease (Opp) positive, we have identified a new gene product, designated MppA (murein peptide permease A), that is about 46% identical to OppA, the periplasmic binding protein for Opp. A plasmid carrying the wild-type mppA gene allows the mutant to grow on tripeptide. Two other mutants that failed to grow on tripeptide were resistant to triornithine toxicity, indicating a defect in the opp operon. An E. coli strain whose entire opp operon was deleted but which carried the mppA locus was unable to grow on murein tripeptide unless it was provided with oppBCDF genes in trans. Our data suggest a model whereby the periplasmic MppA binds the murein tripeptide, which is then transported into the cytoplasm via membrane-bound and cytoplasmic OppBCDF. In assessing the affinity of MppA for non-cell wall peptides, we have found that proline auxotrophy can be satisfied with the peptide Pro-Phe-Lys, which utilizes either MppA or OppA in conjunction with OppBCDF for its uptake. Thus, MppA, OppA, and perhaps the third OppA paralog revealed by the E. coli genome sequence may each bind a particular family of peptides but interact with common membrane-associated components for transport of their bound ligands into the cell. As to the physiological function of MppA, the possibility that it may be involved in signal transduction pathway(s) is discussed.
Project description:Plants establish mutualistic association with beneficial microbes while deploy the immune system to defend against pathogens. Little is known about the interplay between mutualism and immunity and about the mediator molecules. Here we show that plants respond differently to a bacterial volatile compound through integral modulation of the immune system and the phosphate starvation response (PSR) system, resulting in either mutualism or immunity. We found that the same exposure of a recognized plant growth-promoting rhizobacterium unexpectedly causes either beneficial or deleterious effects to plants. The beneficial-to-deleterious transition is dependent on plant nutrition of phosphorus (P) and is mediated by diacetyl (DA), a bacterial volatile compound. In P-sufficient plants, DA partially suppresses plant production of reactive oxygen species (ROS) and enhances symbiont colonization without compromising disease resistance. In P-deficient plants, DA elevates phytohormone-mediated immunity and consequently causes plant hypersensitivity to P deficiency. Therefore, DA affects the types of relation between plants and certain rhizobacteria in a way that depends on plant PSR system and phytohormone-mediated immunity.
Project description:d-Alanyl-d-alanine carboxypeptidase DacC is important for synthesis and stabilization of the peptidoglycan layer of Escherichia coli. In this work, dacC of E. coli BL21 (DE3) was successfully deleted, and the effects of this deletion on extracellular protein production in E. coli were investigated. The extracellular activities and fluorescence value of recombinant amylase, green fluorescent protein, and α-galactosidase of the deletion mutants were increased by 82.3, 29.1, and 37.7%, respectively, compared with that of control cells. The outer membrane permeability and intracellular soluble peptidoglycan accumulation of deletion mutant were also enhanced compared with those of control cells, respectively. Based on fluorescence-assisted cell sorting analyses, we found that the morphology of the E. coli deletion mutant cells was altered compared with that of control cells. Local transparent bulges in the poles of the E. coli mutant with deletion of the dacC gene were found by transmission electron microscopy analysis. These bulges in the poles could explain the improvement in the production of extracellular protein by the E. coli mutant with deletion of the dacC gene. These findings provide important insights into the extracellular production of proteins using E. coli as microbial cell factories.