Dynamic map of protein interactions in the Escherichia coli chemotaxis pathway

Abstract

Protein-protein interactions play key roles in virtually all cellular processes, often forming complex regulatory networks. A powerful tool to study interactions in vivo is fluorescence resonance energy transfer (FRET), which is based on the distance-dependent energy transfer from an excited donor to an acceptor fluorophore. Here, we used FRET to systematically map all protein interactions in the chemotaxis signaling pathway in Escherichia coli, one of the most studied models of signal transduction, and to determine stimulation-induced changes in the pathway. Our FRET analysis identified 19 positive FRET pairs out of the 28 possible protein combinations, with 9 pairs being responsive to chemotactic stimulation. Six stimulation-dependent and five stimulation-independent interactions were direct, whereas other interactions were apparently mediated by scaffolding proteins. Characterization of stimulation-induced responses revealed an additional regulation through activity dependence of interactions involving the adaptation enzyme CheB, and showed complex rearrangement of chemosensory receptors. Our study illustrates how FRET can be efficiently employed to study dynamic protein networks in vivo.

Chemotactic signaling via carbohydrate phosphotransferase systems in Escherichia coli

“…Glucose signals to the chemotaxis system both through the periplasmic glucose/galactose binding protein (GBP, gene mglB), which binds to the receptor Trg (4,14), and via the PTS (6). To address the mechanism of signal transduction from the glucose-specific PTS to the chemotaxis pathway, we first tested in vivo all possible binary interactions or immediate proximities between components of the two systems using acceptor photobleaching FRET (15). In this screen, fusions of chemotaxis and PTS proteins to cyan and yellow fluorescent proteins (CFP and YFP, respectively) were coexpressed pairwise in wild-type E. coli cells, and FRET signals were detected by selectively photobleaching YFP (FRET acceptor) and following ensuing changes in the CFP (FRET donor) emission (Fig.…”

Spatiotemporal modelling of CheY complexes in Escherichia coli chemotaxis

“…In their experiments, high CheZ expression was induced with 0.01% arabinose from plasmid pVS54; in Kentner and Sourjik this level of induction was reported to give a total CheZ concentration of 11.7 mM (Kentner and Sourjik, 2009), which is w3 fold higher than the wild-type level of 3.8 mM. Low levels of expression refer to arabinose concentrations of less than 0.0003%.…”

Oligomeric Sensor Kinase DcuS in the Membrane of Escherichia coli and in Proteoliposomes: Chemical Cross-linking and FRET Spectroscopy

“…In this study, the oligomerization of full-length DcuS was examined in vivo in growing bacteria and in bacterial membranes and in vitro after isolation and reconstitution in liposomes by chemical crosslinking and fluorescence resonance energy transfer (FRET) spectroscopy. FRET techniques have been used widely to study intermolecular interactions of biological molecules (1,4,18,21,23,34). The sensitivity of fluorescence allows experiments at low concentrations of native proteins, and genetically generated fusions of DcuS with fluorescent proteins ensure site-specific labeling of DcuS for noninvasive and nondestructive measurements in living cells.…”