Expression, purification and activities of the entire family of intact membrane sensor kinases fromEnterococcus faecalis

Abstract

Two-component signal transduction systems are the main mechanism by which bacteria sense and respond to their environment, and their membrane-located histidine protein kinases generally constitute the sensory components of these systems. Relatively little is known about their fundamental mechanisms and precise nature of the molecular signals sensed, because of the technical challenges of producing sufficient quantities of these hydrophobic membrane proteins. This study evaluated the heterologous production, purification and activities of the 16 intact membrane sensor kinases of Enterococcus faecalis. Following the cloning of the genes into expression plasmid pTTQ18His, all but one kinase was expressed successfully in Escherichia coli inner membranes. Purification of the hexa-histidine 'tagged' recombinant proteins was achieved for 13, and all but one were verified as intact. Thirteen intact kinases possessed autophosphorylation activity with no added signal when assayed in membrane vesicles or as purified proteins. Signal testing of two functionally-characterized kinases, FsrC and VicK, was successful examplifying the potential use of in vitro activity assays of intact proteins for systematic signal identification. Intact FsrC exhibited an approximately 10-fold increase in activity in response to a two-fold molar excess of synthetic GBAP pheromone, whilst glutathione, and possibly redox potential, were identified for the first time as direct modulators of VicK activity in vitro. The impact of DTT on VicK phosphorylation resulted in increased levels of phosphorylated VicR, the downstream response regulator, thereby confirming the potential of this in vitro approach for investigations of modulator effects on the entire signal transduction process of two-component systems.

Kinetic Characterization of the WalRKSpn(VicRK) Two-Component System ofStreptococcus pneumoniae: Dependence of WalKSpn(VicK) Phosphatase Activity on Its PAS Domain

“…This result contrasts with a recent report that addition of reducing reagent increased the autokinase activity of full-length WalK Efa from E. faecalis (52). Different numbers and locations of cysteine residues in WalK Spn and WalK Efa may underlie this difference.…”

“…Autophosphorylation of WalK and phosphoryl group transfer between WalKϳP and WalR was demonstrated for homologues from B. subtilis (34,35,91,96) and Enterococcus faecalis (52). In addition, phosphoryl transfer that reflects physiologically relevant cross talk was detected between PhoR Bsu ϳP and WalR Bsu (34,35).…”

Hydrodynamics of the VanA-type VanS histidine kinase: an extended solution conformation and first evidence for interactions with vancomycin

“…Here we provide direct in vitro evidence that vancomycin binds to the purified A-type VanS and causes changes to the state of the sensor. The study employs an in vitro approach developed previously in our laboratory for the routine production of purified intact and active membrane sensor kinases of bacteria including the enterococci2930. Heterologously-expressed, His 6 -tagged VanS was purified as an intact membrane protein and verified as intact by mass spectrometry, N-terminal sequencing and Western blotting.…”

“…Heterologously-expressed, His 6 -tagged VanS was purified as an intact membrane protein and verified as intact by mass spectrometry, N-terminal sequencing and Western blotting. In common with most purified intact sensor kinases, it was active in the absence of ligand as revealed using in vitro autophosphorylation activity assays30. We consider the oligomeric state in aqueous solution (supplemented with 20% glycerol to ensure the protein remains stable and active) using sedimentation velocity (SEDFIT analysis, ref.…”

Enterococcus faecalis Virulence Regulator FsrA Binding to Target Promoters

“…Accumulation of this peptide in the extracellular space is sensed by the FsrC membrane histidine kinase, leading to the activation of the response regulator and transcription factor FsrA. Stimulation of FsrC activity in vivo and in vitro by a chemically synthesized GBAP peptide has been demonstrated (16,20). The FsrABDC proteins are necessary for autoregulation at a promoter located upstream of fsrB and for the expression of two E. faecalis virulence-related proteases, gelatinase (GelE) and serine protease (SprE), from a promoter located upstream of the gelE gene (26).…”