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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.
| Abbreviations | ||
| DDM | = | dodecyl-β-D-maltoside |
| DTT | = | dithiothreitol |
| EDTA | = | ethylenediamine tetraacetic acid |
| ESI-MS | = | electrospray ionization mass spectrometry |
| GBAP | = | gelatinase biosynthesis-activating pheromone |
| GSH | = | reduced glutathione |
| GSSG | = | oxidized glutathione |
| HK | = | histidine kinase |
| IPTG | = | isopropyl -D-1-thiogalactopyranoside |
| LB | = | Luria-Bertani |
| LDAO | = | lauryldimethylamine-oxide |
| Ni2+-NTA | = | nickel-nitrilotriacetic acid |
| PAS | = | Per-Arnt-Sim |
| RR | = | response regulator |
| SDS-PAGE | = | sodium dodecyl sulphate polyacrylamide gel electrophoresis |
| TCS | = | two-component systems |
| TM | = | transmembrane |
| Abbreviations | ||
| DDM | = | dodecyl-β-D-maltoside |
| DTT | = | dithiothreitol |
| EDTA | = | ethylenediamine tetraacetic acid |
| ESI-MS | = | electrospray ionization mass spectrometry |
| GBAP | = | gelatinase biosynthesis-activating pheromone |
| GSH | = | reduced glutathione |
| GSSG | = | oxidized glutathione |
| HK | = | histidine kinase |
| IPTG | = | isopropyl -D-1-thiogalactopyranoside |
| LB | = | Luria-Bertani |
| LDAO | = | lauryldimethylamine-oxide |
| Ni2+-NTA | = | nickel-nitrilotriacetic acid |
| PAS | = | Per-Arnt-Sim |
| RR | = | response regulator |
| SDS-PAGE | = | sodium dodecyl sulphate polyacrylamide gel electrophoresis |
| TCS | = | two-component systems |
| TM | = | transmembrane |