Recent advances in the Phos-tag technique focused on the analysis of phosphoproteins in a bacterial two-component...

In a bacterial two-component system (TCS), signals are generally conveyed by means of a His-Asp phosphorelay. Each system consists of a histidine kinase (HK) and its cognate response regulator (RR). The His- and Asp-bound phosphate groups are extremely unstable under acidic conditions easily to be hydrolyzed within a few hours. Because of the labile nature of phosphorylated His and Asp residues, few approaches are available that permit a quantitative analysis of their phosphorylation states in the TCS. Here, we describe that Phos-tag technique is suitable for the quantitative analysis of His- and Asp-phosphorylated proteins. The dynamics of the His-Asp phosphorelay of recombinant TCS derived from Escherichia coli, was examined by Phos-tag SDS-PAGE or Phos-tag fluorescent dye gel staining. The technique permitted not only the quantitative monitoring of the autophosphorylation reactions of HK and RR in the presence of ATP or acetyl phosphate, respectively, but also that of the phosphotransfer reaction from HK to RR in the presence of ATP. Furthermore, we demonstrate profiling of waldiomycin, an HK inhibitor, by using the Phos-tag fluorescent dye gel staining. Consequently, Phos-tag technique provides a simple and convenient approach for screening of HK inhibitors that have potential as new antimicrobial agents. SIGNIFICANCE: Bacterial cells have unique phosphotransfer signaling mechanisms known as two-component systems (TCSs) that permit the organism to sense and respond to various environmental conditions. Each system consists of a histidine kinase (HK) and a response regulator (RR). A typical HK contains an invariant His residue that is autophosphorylated in an ATP-dependent manner. A typical RR has a conserved Asp residue that can acquire a phosphoryl group from its cognate HK. In general, TCS has this type of a His-Asp phosphorelay scheme. Because TCS is also involved in the virulence of pathogens, it is potential targets for novel antibiotics and antivirulence agents. It is, thus, very important to determine HK activity in the bacterial TCS. We believe that our Phos-tag technique provides a simple and convenient approach for drug discovery targeting the bacterial TCS.