Purification and characterization of VanR and the cytosolic domain of VanS: a two-component regulatory system required for vancomycin resistance in Enterococcus faecium BM4147.
Resistance to the glycopeptide antibiotic vancomycin requires five genes. Two of these, vanR and vanS, have sequence homology to cytoplasmic response regulatory (VanR) and transmembrane sensory (VanS) proteins of two-component regulatory systems used to sense and transduce environmental signals. We report the overproduction and purification to homogeneity of VanR (27 kDa) and of a fusion protein of VanS (residues 95-374, the cytosolic domain) to the maltose binding protein (MBP), yielding a MBP-VanS protein of 76 kDa. The MBP-VanS fusion protein displayed an ATP-dependent autophosphorylation on a histidine residue with a rate of 0.17 min-1 and a phosphorylation stoichiometry of 10-15%. 32P-PhosphoMBP-VanS transferred the phosphoryl group to VanR. 32P-Phospho VanR showed chemical stability anticipated for an aspartyl phosphate and was relatively stable to hydrolysis (t1/2 = 10-12 h). Thus, the vancomycin resistance operon appears to have collected and specifically tailored the His kinase and Asp phosphoryl receptor of two-component signal transduction logic for sensing extracellular vancomycin and turning on structural genes, vanA and vanH, to make altered peptidoglycan structures such that vancomycin does not bind.
Wright
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Holman
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Walsh
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D018528:ATP-Binding Cassette Transporters; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004352:Drug Resistance, Microbial / Q000235:genetics*; D016984:Enterococcus faecium / Q000187:drug effects / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D006639:Histidine / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014169:Transformation, Bacterial; D014640:Vancomycin / Q000494:pharmacology*
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C461599:maltose transport system, E coli; C073902:VanR protein, bacteria; C073901:VanS protein, Enterococcus; D006639:Histidine; D000244:Adenosine Diphosphate; D014640:Vancomycin; D000255:Adenosine Triphosphate; D011494:Protein Kinases