title: Silencing of glycopeptide resistance in Enterococcus faecalis BM4405 by novobiocin.
abstract: Enterococcus faecalis BM4405-1, a susceptible derivative of the VanE-type vancomycin-resistant E. faecalis strain BM4405, was obtained after growth in the presence of novobiocin, an inhibitor of the GyrB subunit of DNA gyrase. In contrast to findings for BM4405, UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala (pentapeptide[D-Ala]) was the only peptidoglycan precursor found in BM4405-1, and no VanXY(E) D,D-peptidase or VanT serine racemase activities were detected in that strain, even after induction by subinhibitory concentrations of vancomycin. Sequencing of the vanE operon of BM4405-1 revealed two mutations leading to substitutions in VanE (D200N) and in the C-terminal amino acid of VanR(E) (Y225F). Cloning of the vanE, vanXY(E), and vanT(E) genes of BM4405-1 into the susceptible E. faecalis strain JH2-2 conferred resistance to vancomycin, indicating that the mutation in vanE was not responsible for susceptibility. Transcriptional analysis of the vanE operon in BM4405 by quantitative reverse transcription-PCR indicated that novobiocin did not affect the expression level of the vanE operon. Sequencing of the gyrB gene of BM4405-1 revealed a mutation responsible for substitution of a residue (K337Y) required for ATPase activity and thus implicated in DNA supercoiling. Cloning of the gyrB gene of BM4405 restored vancomycin resistance to BM4405-1. Taken together, these data suggest that alteration of DNA supercoiling following a mutation in GyrB was responsible for lack of expression of the vanE operon and thus for vancomycin susceptibility in BM4405-1.
authors.affiliation: Unité des Agents Antibactériens, Institut Pasteur, 25, Rue du Dr. Roux, 75724 Paris cedex 15, France.
authors.forename: Lorena Abadía
authors.initials: LA
authors.lastname: Patiño
authors.forename: Marc
authors.initials: M
authors.lastname: Chippaux
authors.forename: Patrice
authors.initials: P
authors.lastname: Courvalin
authors.forename: Bruno
authors.initials: B
authors.lastname: Périchon
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D027081:DNA Gyrase / Q000235:genetics; D004278:DNA, Superhelical / Q000378:metabolism; D013293:Enterococcus faecalis / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009675:Novobiocin / Q000494:pharmacology*; D009876:Operon; D014640:Vancomycin / Q000494:pharmacology; D020713:Vancomycin Resistance / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004278:DNA, Superhelical; D009675:Novobiocin; D014640:Vancomycin; D027081:DNA Gyrase; D008025:Ligases; C469907:VanE protein, Enterococcus faecalis