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.

SummaryWe 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 ∼ ∼ ∼ ∼ 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.

The spread of vancomycin resistance among pathogenic bacteria is an important public health concern. Ever since vancomycin-resistant strains of pathogenic Enterococcus faecalis and Enterococcus faecium (VRE) first emerged in the late 1980s, the intergeneric transfer of vancomycin resistance from these strains to methicillin-resistant Staphylococcus aureus, a major killer in hospital-acquired infections, has been widely anticipated. This recently became a reality with the first reports of clinical isolates of vancomycin-resistant Staphylococcus aureus (VRSA) 1 from hospitals in the United States (1-4). Vancomycin and other glycopeptide antibiotics inhibit cell wall biosynthesis in Gram-positive bacteria but not in Gramnegative bacteria because they cannot penetrate the outer membrane permeability barrier. They bind the D-alanyl-D-alanine (D-Ala-D-Ala) terminus of lipid-attached peptidoglycan precursors on the outside of the cytoplasmic membrane (5, 6), and this interaction blocks the formation of mature peptidoglycan, principally by denying transpeptidase access to its substrate, thus preventing formation of the peptide cross-links between polysaccharide strands that give the cell wall its structural rigidity. Because of the distinctive mode of action of vancomycin, mutations in transpeptidase cannot give rise to drug resistance. For this reason, it was originally suggested that pathogens might never acquire resistance to vancomycin because it would require them to remodel the peptidoglycan biosynthetic pathway itself. In the late 1980s, however, the first clinical isolates of VRE appeared and were found to repro- We have shown previously that the non-pathogen Streptomyces coelicolor carries a gene cluster conferring inducible, high-level resistance to vancomycin (13). S. coelicolor is the model species of a genus of Gram-positive, mycelial soil bacteria responsible for the production of two-thirds of the commercially important antibiotics. S. coelicolor itself does not make a glycopeptide, but all of the known glycopeptide antibiotics are produced by actinomycetes, the family to which the streptomycetes belong. Because most non-pathogenic actinomycetes live in the soil, it seems likely that S. coelicolor encounters glycopeptide producers and that the van gene cluster therefore confers a selective advantage. Further, it is widely believed that all glycopeptide resistance genes are ultimately derived from actinomycete glycopeptide producers (14), which must carry these genes to avoid autotoxicity. Consistent with this idea, the S. coelicolor resistance genes are clearly associated with a laterally acquired DNA element. 2The S. coelicolor cluster consists of seven genes, vanSR-JKHAX ( Fig. 1) (13). vanHAX are orthologous to the genes found in VRE strains. vanR and vanS encode a two-component signal transduction system that mediates transcriptional in-* This work was funded by Biotechnology and Biological Sciences Research Council Grant 208/P20040 (to H-J. H. and M. J. B.) and by a grant-in-aid to the John Innes C...

SummaryWe have investigated a signal transduction system proposed to allow Streptomyces coelicolor to sense and respond to changes in the integrity of its cell envelope. The system consists of four proteins, encoded in an operon: s E , an RNA polymerase s factor; CseA (formerly ORF202), a protein of unknown function; CseB, a response regulator; and CseC, a sensor histidine protein kinase with two predicted transmembrane helices (Cse stands for control of sigma E). To develop a sensitive bioassay for inducers of the sigE system, the promoter of the sigE operon (sigEp) was fused to a reporter gene conferring resistance to kanamycin. Antibiotics that acted as inducers of the sigE signal transduction system were all inhibitors of intermediate and late steps in peptidoglycan biosynthesis, including ramoplanin, moenomycin A, bacitracin, several glycopeptides and some b-lactams. The cell wall hydrolytic enzyme lysozyme also acted as an inducer. These data suggest that the CseB-CseC signal transduction system may be activated by the accumulation of an intermediate in peptidoglycan biosynthesis or degradationa. A computer-based searching method was used to identify a s E target operon of 12 genes (the cwg operon), predicted to specify the biosynthesis of a cell wall glycan. In low-Mg 2+ medium, transcription of the cwg operon was induced by vancomycin in a sigE-dependent manner but, in high-Mg 2+ medium, there was substantial cwg transcription in a sigE null mutant, and this sigE-independent activity was also induced by vancomycin. Based on these data, we propose a model for the regulation and function of the s E signal transduction system.

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.