Abstract
Enterococcus faecalis BM4518 is resistant to vancomycin by synthesis of peptidoglycan precursors ending in D-alanyl-D-serine. In the chromosomal vanG locus, transcription of the resistance genes from the PYG resistance promoter is inducible and, upstream from these genes, there is an unusual three-component regulatory system encoded by the vanURSG operon from the PUG regulatory promoter. In contrast to the other van operons in enterococci, the vanG operon possesses the additional vanUG gene which encodes a transcriptional regulator whose role remains unknown. We show by DNase I footprinting, RT-qPCR, and reporter proteins activities that VanUG, but not VanRG, binds to PUG and negatively autoregulates the vanURSG operon and that it also represses PYG where it overlaps with VanRG for binding. In clinical isolate BM4518, the transcription level of the resistance genes was dependent on vancomycin concentration whereas, in a ΔvanUG mutant, resistance was expressed at a maximum level even at low concentrations of the inducer. The binding competition between VanUG and VanRG on the PYG resistance promoter allowed rheostatic activation of the resistance operon depending likely on the level of VanRG phosphorylation by the VanSG sensor. In addition, there was cross-talk between VanSG and VanR'G, a VanRG homolog, encoded elsewhere in the chromosome indicating a sophisticated and subtle regulation of vancomycin resistance expression by a complex two-component system.
Highlights
Vancomycin-resistant enterococci are a major cause of nosocomial infections and an important public health problem because the treatment options for the infections they cause are very limited [1]
We show that in VanG-type vancomycin resistant Enterococcus faecalis a repressor (VanUG) allows rheostatic expression of a target
Vancomycin, which can be the only antibiotic effective against multiresistant clinical isolates, acts by binding to the C-terminal D-alanyl-D-alanine (D-Ala-D-Ala) residues of peptidoglycan precursors blocking the extracellular steps in peptidoglycan synthesis [2]
Summary
Vancomycin-resistant enterococci are a major cause of nosocomial infections and an important public health problem because the treatment options for the infections they cause are very limited [1]. Expression of the vancomycin resistance operons is regulated by VanS/VanR-type twocomponent signal transduction systems composed of a membrane-bound histidine kinase (VanS-type) and a cytoplasmic response regulator (VanR-type) that acts as a transcriptional activator [3]. In the presence of vancomycin, VanS acts primarily as a kinase that autophosphorylates and transfers its phosphate to VanR. Phosphorylated VanR binds to the promoters upstream from the vanRS regulatory and resistance operons leading to increased transcription of the regulatory and resistance genes [7,8,9]. The phosphatase activity of VanS-type sensors is required for negative regulation of the resistance genes in the absence of vancomycin preventing accumulation of VanRtype regulators phosphorylated by acetylphosphate or by kinases encoded by the host chromosome [7, 10]
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