Single-cell analysis of glycopeptide resistance gene expression in teicoplanin-resistant mutants of a VanB-type Enterococcus faecalis

The vane gene cluster confers resistance to vancomycin but not to the relat ed antibiotic teicoplanin, as the VanR(B)S(B) two-component regulatory syst em triggers expression of the glycopeptide resistance genes only in respons e to vancomycin. The VanR(B) regulator activates promoters P-RB and P-YB fo r transcription of the regulatory (vanR(B)S(B)) and resistance (vanY(B) WHB BXB) genes respectively. The gfpmut1 gene encoding a green fluorescent pro tein was fused to P-YB to analyse promoter activation in single cells by fl uorescence microscopy and flow cytometry. Characterization of 17 teicoplani n-resistant mutants indicated that amino acid substitutions on either side of the VanS(B) autophosphorylation site led to a constitutive phenotype. Su bstitutions in the membrane-associated domain resulted in a gain of functio n, as they allowed induction by teicoplanin. A vanS(B) null mutant expresse d gfpmut1 at various levels under non-inducing conditions, and the majority of the bacteria were not fluorescent. Bacteria grown in the presence of va ncomycin or teicoplanin were homogeneously fluorescent. The increase in the number of fluorescent bacteria resulted from induction in negative cells r ather than from selection of a resistant subpopulation, indicating that Van R(B) was activated by cross-talk. Transglycosylase inhibition was probably the stimulus for the heterologous kinase, as moenomycin was also an inducer .