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

被引:34
作者
Baptista, M
Rodrigues, P
Depardieu, F
Courvalin, P
Arthur, M
机构
[1] Inst Pasteur, Unite Agents Antibacteriens, F-75724 Paris 15, France
[2] Inst Pasteur, Lab Retrovirus & Transfert Genet, F-75724 Paris, France
关键词
D O I
10.1046/j.1365-2958.1999.01308.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The vane gene cluster confers resistance to vancomycin but not to the related antibiotic teicoplanin, as the VanR(B)S(B) two-component regulatory system triggers expression of the glycopeptide resistance genes only in response to vancomycin. The VanR(B) regulator activates promoters P-RB and P-YB for transcription of the regulatory (vanR(B)S(B)) and resistance (vanY(B) WHB BXB) genes respectively. The gfpmut1 gene encoding a green fluorescent protein was fused to P-YB to analyse promoter activation in single cells by fluorescence microscopy and flow cytometry. Characterization of 17 teicoplanin-resistant mutants indicated that amino acid substitutions on either side of the VanS(B) autophosphorylation site led to a constitutive phenotype. Substitutions in the membrane-associated domain resulted in a gain of function, as they allowed induction by teicoplanin. A vanS(B) null mutant expressed gfpmut1 at various levels under non-inducing conditions, and the majority of the bacteria were not fluorescent. Bacteria grown in the presence of vancomycin or teicoplanin were homogeneously fluorescent. The increase in the number of fluorescent bacteria resulted from induction in negative cells rather than from selection of a resistant subpopulation, indicating that VanR(B) was activated by cross-talk. Transglycosylase inhibition was probably the stimulus for the heterologous kinase, as moenomycin was also an inducer.
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页码:17 / 28
页数:12
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