Competition between VanUG Repressor and VanRG Activator Leads to Rheostatic Control of vanG Vancomycin Resistance Operon Expression

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 P-YG resistance promoter is inducible and, upstream from these genes, there is an unusual three-component regulatory system encoded by the vanURS(G) operon from the P-UG regulatory promoter. In contrast to the other van operons in enterococci, the vanG operon possesses the additional vanU(G) gene which encodes a transcriptional regulator whose role remains unknown. We show by DNase I footprinting, RT-qPCR, and reporter proteins activities that VanU(G), but not VanR(G), binds to P-UG and negatively autoregulates the vanURSG operon and that it also represses PYG where it overlaps with VanR(G) for binding. In clinical isolate BM4518, the transcription level of the resistance genes was dependent on vancomycin concentration whereas, in a Delta vanUG mutant, resistance was expressed at a maximum level even at low concentrations of the inducer. The binding competition between VanU(G) and VanR(G) on the P-YG resistance promoter allowed rheostatic activation of the resistance operon depending likely on the level of VanR(G) phosphorylation by the VanS(G) sensor. In addition, there was cross-talk between VanS(G) and VanR'(G), a VanR(G) homolog, encoded elsewhere in the chromosome indicating a sophisticated and subtle regulation of vancomycin resistance expression by a complex two-component system.