A FRET study of PhoR-PhoB two component system in E.coli
The PhoR-PhoB (PhoR/B) two-component regulatory system in E.coli responds to environmental inorganic phosphate (Pi). A signal for environmental Pi insufficiency is propagated across the membrane to the cytosolic domain of the signaling histidine kinase PhoR via the ABC (ATP binding cassette) transporter and the Pst (P i-specific transport) system, in the absence of transport by protein-protein interactions, in both the membrane and the cytoplasm of the cell. Cross regulation also exists between PhoR/B two-component system and key regulatory systems in E.coli, including the ArcB/A two-component system for redox signaling and the sugar transporting system. Such cross regulation among key signaling system is thought to be important for integration of diverse cellular pathways in the control of cell growth and for survival under conditions of stress. Knowledge of these interactions is necessary to understand the dynamics of the response of the Pi signaling network to environmental signals and to understand interactions of the PhoR/B and other signaling systems. Here we have employed Förster Energy Transfer (FRET) techniques coupled with specific knock-out mutants to investigate in vivo interactions between ECFP-labelled members of the PhoR/B system and protein targets with which they are thought to interact, such as the osuccinylbenzoyl-CoA synthase, MenC (Venus-tagged) and a permease in sugar transporting phosphotransferase system, crr (Venus-labelled). We will also show a strong enhancement of the FRET signal through the use of knock-out mutants. We will discuss results of this study in terms of their implication on the dynamics of the PhoR/B two-component signal transporting system.^
Ken P. Ritchie, Purdue University.