The glycopeptide antibiotics vancomycin and teicoplanin are vital components of modern anti-infective chemotherapy exhibiting outstanding activity against Gram-positive pathogens including members of the genera Streptococcus, Staphylococcus, and Enterococcus. These antibiotics also provide fascinating examples of the chemical and associated biosynthetic complexity exploitable in the synthesis of natural products by actinomycetes group of bacteria. We report the sequencing and annotation of the biosynthetic gene cluster for the glycopeptide antibiotic A47934 from Streptomyces toyocaensis NRRL15009, the first complete sequence for a teicoplanin class glycopeptide. The cluster includes 34 ORFs encompassing 68 kb and includes all of the genes predicted to be required to synthesize A47934 and regulate its biosynthesis. The gene cluster also contains ORFs encoding enzymes responsible for glycopeptide resistance. This role was confirmed by insertional inactivation of the D-Ala-D-lactate ligase, vanAst, which resulted in the predicted A47934-sensitive phenotype and impaired antibiotic biosynthesis. These results provide increased understanding of the biosynthesis of these complex natural products.G lycopeptide antibiotics (GPAs) have been mainstays of antimicrobial chemotherapy since their discovery in the mid 1950s. These antibiotics act exclusively on Gram-positive bacteria by forming a tight and specific noncovalent complex with the D-Ala-D-Ala terminus of the peptidoglycan, inhibiting cell wall growth and crosslinking (1, 2). Clinical resistance to GPAs was first described in the enterococci in 1988 (3), and resistance has now manifested itself in the more virulent streptococci and staphylococci (reviewed in ref. 4). GPA resistance is now a significant worldwide phenomenon that has severely impacted the health care sector both in increased mortality and morbidity, and economically (5). The predominant mechanism of resistance is the synthesis of cell wall peptidoglycan terminating in D-Ala-D-lactate, which dramatically decreases the affinity of these antibiotics for their target (6).GPAs are exclusively obtained through fermentation, yet despite their importance, their biosynthesis is not well understood. They are comprised of a heptapeptide core consisting of both common and unusual amino acids (4). Crosslinking of the amino acids through aryl ether and carbon-carbon bonds provides rigidity to the peptide. There are two major structural classes of GPAs based on the identity of the core peptide, and the two clinically used GPAs, vancomycin and teicoplanin, exemplify both classes (Fig. 1). An additional class of structurally homologous secondary metabolites with anticomplement activity is exemplified by complestatin (Fig. 1). Structural diversity in these natural products is achieved through changes in the peptide backbone, and selective amino acid halogenation, glycosylation, lipidation, methylation, and sulfonylation. In principle, understanding of the genetic and mechanistic basis of these modifications could lead ...

SummaryVancomycin 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, highlevel 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.

Glycopeptide antibiotics are integral components of the current antibiotic arsenal that is under strong pressures as a result of the emergence of a variety of resistance mechanisms over the past 15 years. Resistance has manifested itself largely through the expression of genes that encode proteins that reprogram cell wall biosynthesis and thus evade the action of the antibiotic in the enterococci, though recently new mechanisms have appeared that afford resistance and tolerance in the more virulent staphylococci and streptococci. Overcoming glycopeptide resistance will require innovative approaches to generate new antibiotics or otherwise to inhibit the action of resistance elements in various bacteria. The chemical complexity of the glycopeptides, the challenges of discovering and successfully exploiting new targets, and the growing number of distinct resistance types all increase the difficulty of the current problem we face as a result of the emergence of glycopeptide resistance.

StoPK-1, a serine/threonine protein kinase from the glycopeptide antibiotic producer Streptomyces toyocaensis NRRL 15009, affects oxidative stress response in signalling pathways sensitive to oxidative stress and/or glucose metabolism. These results broaden the roles of Ser/Thr protein kinases in bacteria and underscore the diversity of signal transduction mechanisms available to respond to various stimuli.