Cross Regulation in Signaling Networks: A FRET Study of the PhoB-PhoR Two-Component System in E-Coli

Abstract

The signal for the PhoR-PhoB two-component regulatory system of E. coli is environmental inorganic phosphate (Pi). A signal for environmental Pi insufficiency is propagated across the membrane to the cytosolic domain of the signalling histidine kinase PhoR via the ABC (ATP-binding cassette) transporter, the Pst (Pi-specific transport) system, in the absence of transport by protein-protein interactions, both in the membrane and the cytoplasm of the cell. Cross regulation also exists between PhoR-PhoB two-component system and key regulatory systems in E. coli, including the ArcB-ArcA two-component system for redox signalling. Such cross regulation among key signalling systems 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 how these interactions is brought about necessary to understand fully the dynamics of the response of the Pi signalling network to environmental Pi insufficiency and to understand interactions of the PhoR-PhoB and other signalling systems. Here we have employed Forster resonance energy transfer (FRET) techniques (acceptor photobleaching and sensitized emission) coupled with specific knock-out mutants to investigate in live E. coli cells interactions between ECFP-labelled members of the PhoR-PhoB system and protein targets with which they are thought to interact such as the o-succinylbenzoyl-CoA synthase, MenC. We will show a strong enhancement of the FRET signal through the use of protein fusions and knock-out mutants. We will discuss results of this study in terms of their implication on the dynamics of the PhoR-PhoB two-component signal transduction system.