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

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.

Functional Dissection of the CroRS Two-Component System Required for Resistance to Cell Wall Stressors in Enterococcus faecalis

“…CisR was recently implicated in cross talk in another E. faecalis lineage which carries a Van G gene cluster controlled by the VanRS G TCS for vancomycin resistance. In this E. faecalis isolate, CisR was found to promote the production of the VanT G racemase in the absence of VanR G , a process which requires the HK VanS G and vancomycin (52). Thus, it remains unclear if CisRS cross talk is relevant to the biology of wild-type E. faecalis or if this phenomenon only manifests when cognate HK-RR pairs are disrupted.…”

The Enterococcus: a Model of Adaptability to Its Environment

“…They encode a (serine) racemase (VanT), a D-Ala-D-Ser ligase (VanC), a combined dipeptidase-carboxypeptidase(VanXY), and the products of the regulatory genes vanR and vanS. The vanG operon has an additional carboxypeptidase, an analogue of VanW from the vanB operon, and an additional regulatory gene (vanU) (174).…”

Molecular mechanisms of vancomycin resistance

“…The structure of VanU G determined in two crystal forms (PDB codes 3TYR and 3TYS) reveals a tight dimer with each chain adopting a canonical helix‐turn‐helix motif featuring a recognition helix rich in basic residues (Figure ). The role of this protein in regulation of the vancomycin resistance was not understood until a recent study showing that VanU G negatively regulates the expression from both P UG and P YG (the resistance) promoters thereby repressing transcription of all genes in this Van cassette . Notably, phospho‐VanR G was shown to not bind to P UG region but instead to compete with VanU G for binding to P YG .…”

“…The role of this protein in regulation of the vancomycin resistance was not understood until a recent study showing that VanU G negatively regulates the expression from both P UG and P YG (the resistance) promoters thereby repressing transcription of all genes in this Van cassette . Notably, phospho‐VanR G was shown to not bind to P UG region but instead to compete with VanU G for binding to P YG . Thus, it was suggested that VanU G fine‐tunes the expression levels of the vancomycin resistance genes by dampening the activation by phospho‐VanR G .…”

“…Notably, phospho‐VanR G was shown to not bind to P UG region but instead to compete with VanU G for binding to P YG . Thus, it was suggested that VanU G fine‐tunes the expression levels of the vancomycin resistance genes by dampening the activation by phospho‐VanR G . This model requires further investigation, such as a comparison of the in vitro affinity of VanU G vs. VanR G for P YG , studies on the oligomeric assembly of VanU G with DNA, or crystal structure determination of VanU G in complex with DNA.…”