ABSTRACTStaphylococcus aureusutilizes the two-component regulatory system VraSR to receive and relay environmental stress signals, and it is implicated in the development of bacterial resistance to several antibiotics through the upregulation of cell wall synthesis. VraS inhibition was shown to extend or restore the efficacy of several clinically used antibiotics. In this work, we study the enzymatic activity of the VraS intracellular domain (GST-VraS) to determine the kinetic parameters of the ATPase reaction and characterize the inhibition of NH125 underin vitroand microbiological settings. The rate of the autophosphorylation reaction was determined at different GST-VraS concentrations (0.95 to 9.49 μM) and temperatures (22 to 40°C) as well as in the presence of different divalent cations. The activity and inhibition by NH125, which is a known kinase inhibitor, were assessed in the presence and absence of the binding partner, VraR. The effects of inhibition on the bacterial growth kinetics and gene expression levels were determined. The GST-VraS rate of autophosphorylation increases with temperature and with the addition of VraR, with magnesium being the preferred divalent cation for the metal-ATP substrate complex. The mechanism of inhibition of NH125 was noncompetitive in nature and was attenuated in the presence of VraR. The addition of NH125 in the presence of sublethal doses of the cell wall-targeting antibiotics carbenicillin and vancomycin led to the complete abrogation ofStaphylococcus aureusNewman strain growth and significantly decreased the gene expression levels ofpbpB,blaZ, andvraSRin the presence of the antibiotics.IMPORTANCEThis work characterizes the activity and inhibition of VraS, which is a key histidine kinase in a bacterial two-component system that is involved inStaphylococcus aureusantibiotic resistance. The results show the effect of temperature, divalent ions, and VraR on the activity and the kinetic parameters of ATP binding. The value of theKMof ATP is vital in designing screening assays to discover potent and effective VraS inhibitors with high translational potential. We report the ability of NH125 to inhibit VraSin vitroin a noncompetitive manner and investigate its effect on gene expression and bacterial growth kinetics in the presence and absence of cell wall-targeting antibiotics. NH125 effectively potentiated the effects of the antibiotics on bacterial growth and altered the expression of the genes that are regulated by VraS and are involved in mounting a resistance to antibiotics.