Bacterial Histidine Kinases: Overexpression, Purification, and Inhibitor Screen

Abstract

Bacterial histidine kinases are promising targets for new antimicrobial agents. In antibacterial therapy such agents could inhibit bacterial growth by targeting essential two-component regulatory systems or resensitize bacteria to known antibiotics by blocking stress responses like the cell wall stress response. However, (1) activity assays using the truncated phosphorylation domains have been shown to produce artifacts and (2) the purification of the full-length histidine kinases is complicated. Here, we describe a standard protocol for the recombinant expression and purification of functional full-length histidine kinases and other membrane proteins from gram-positive bacteria that do not harbor more than two trans-membrane domains using an Escherichia coli host. This guide also presents in vitro phosphorylation assays to screen for new antimicrobial compounds that target bacterial histidine kinases using radioactively labeled ATP and, as a novel approach, Phos-tag acrylamide gel electrophoresis to detect phosphorylated proteins by mobility shift in the polyacrylamide gel.

The Role of β-Glycosylated Wall Teichoic Acids in the Reduction of Vancomycin Susceptibility in Vancomycin-Intermediate Staphylococcus aureus

“…The phosphorylation of VraR and the VraR(M54T) variant with the cognate kinase VraS was performed essentially as described in Gajdiss et al ( 51 ). Afterward, the phosphorylation was evaluated by 12.5% Phos-Tag (Wako Chemicals, Neuss, Germany) SDS-PAGE ( 52 ).…”

YycH and YycI Regulate Expression of Staphylococcus aureus Autolysins by Activation of WalRK Phosphorylation

“…A detailed protocol used for overexpression and purification has been described previously [33]. Briefly, E. coli strains containing the expression plasmids were incubated in LB broth supplemented with 100 mg/L ampicillin and 50 mg/L kanamycin at 37 • C in a shaking water bath until exponential growth phase.…”

“…Phosphorylation assays of the liposomes containing the reconstituted proteins were performed as previously described [33]. Proteoliposomes were mixed with 1 µCi/µL [γ 32 P]-ATP in phosphorylation buffer (50 mM HEPES, 500 mM KCl, 25 mM MgCl 2 , 0.5 mM DTT, 3.5% (v/v) glycerol, pH 8) and incubated at 15 • C for 30 min.…”

“…After 120 min of lysis, the suspension was spun down and the supernatant was used for the phosphorylation assays with WalK and WalR. Phosphorylation assays with detergent-micelles were performed using the Phos-tag SDS-PAGE method for histidine kinases as previously described [33]. Prior to addition of ATP, the kinase was incubated for 10 min with 500 µM N-acetylglucosamine, 500 µM N-acetylmuramic acid, 500 µM of the peptidoglycan pentapeptide (Ala-d-γ-Glu-Lys-d-Ala-D-Ala), 1 mM d-alanine (all from Sigma), 1.5 to 6 µL of the lysed peptidoglycan, or 2 µL of purified WTA as described in the WTA extraction and quantification section.…”

Quantitative monitoring of His and Asp phosphorylation in a bacterial signaling system by using Phos‐tag Magenta/Cyan fluorescent dyes

“…Consequently, Phos‐tag SDS‐PAGE permits the absolute quantification of phosphorylated and nonphosphorylated forms in purified single protein molecules. This electrophoretic approach has been used by many researchers in the field of bacteriology . In the fluorometric approach, a tetramethylrhodamine‐labeled Phos‐tag derivative has recently been used as a stain for SDS‐PAGE gels to permit the selective detection of certain phosphoproteins .…”