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Regulation of Glycopeptide Resistance Genes of Enterococcal Transposon Tn1546 by the VanR-VanS Two-Component Regulatory System, Page 1 of 2
< Previous page Next page > /docserver/preview/fulltext/10.1128/9781555818319/9781555810894_Chap24-1.gif /docserver/preview/fulltext/10.1128/9781555818319/9781555810894_Chap24-2.gifAbstract:
Glycopeptide antibiotics vancomycin and teicoplanin are used to treat severe infections caused by gram-positive cocci. Strains displaying the so-called VanA resistance phenotype are inducibly resistant to high levels of vancomycin and teicoplanin. Production of the depsipeptide D-Ala-D-Lac is controlled by the VanR-VanS two-component regulatory system that activates transcription of vancomycin resistance genes in response to the presence of glycopeptides in the culture medium. Response regulators (RRs) of this subclass regulate transcription at specific promoters thought to be recognized by the main form of RNA polymerase holoenzyme, corresponding to Eσ70 in Escherichia coli. The first 122 amino acids at the N terminus of VanS are not related in sequence to other HPKs. This region of VanS contains two clusters of hydrophobic amino acids that could correspond to membrane-spanning regions. Validation of the predicted roles of VanS and VanR in sequential phosphoryl group transfer was obtained by overproduction, purification, and assay of the two proteins. The vanR and vanS genes were introduced into the chromosome of a susceptible strain of Enterococcus faecalis using an integrative vector. trans-Activation of transcriptional fusions carried by plasmids were analyzed based on determination of chloramphenicol acetyltransferase (CAT) activity. Mapping of the 5’ end of mRNA by S1 nuclease protection and by primer extension assays identified one transcriptional start site in the vanS-vanH intergenic region. Cloning of vanR, vanS, vanH, vanA, and vanX upstream from the cat gene in a multicopy vector resulted in high-level transcription of the reporter gene.