Protein exchange dynamics at chemoreceptor clusters in Escherichia coli

Abstract

Signal processing in bacterial chemotaxis relies on large sensory complexes consisting of thousands of protein molecules. These clusters create a scaffold that increases the efficiency of pathway reactions and amplifies and integrates chemotactic signals. The cluster core in Escherichia coli comprises a ternary complex composed of receptors, kinase CheA, and adaptor protein CheW. All other chemotaxis proteins localize to clusters by binding either directly to receptors or to CheA. Here, we used fluorescence recovery after photobleaching (FRAP) to investigate the turnover of chemotaxis proteins at the cluster and their mobility in the cytoplasm. We found that cluster exchange kinetics were proteinspecific and took place on several characteristic time scales that correspond to excitation, adaptation, and cell division, respectively. We further applied analytical and numerical data fitting to analyze intracellular protein diffusion and to estimate the rate constants of cluster equilibration in vivo. Our results indicate that the rates of protein turnover at the cluster have evolved to ensure optimal performance of the chemotaxis pathway.T he relatively simple chemotaxis signaling pathway in Escherichia coli, with analogues of its components-receptors, kinase, phosphatase, and adaptation system-common to many other networks, is an ideal model system for studying general principles of signal transduction (1-3). In E. coli, allosteric interactions among receptors in chemosensory arrays or clusters (Fig. 1), where receptors of different ligand specificities are intermixed (4, 5), integrate and amplify chemotactic stimuli. The networked receptors regulate the autophosphorylation activity of an associated kinase, CheA, which in turn controls the phosphorylation state of a small response regulator protein, CheY, to modulate the cell's flagellar motors. The signaling pathway also includes CheZ, a phosphatase of CheY-P. Excitatory signaling is rapid: changes in CheY phosphorylation level upon repellent or attractant stimulation take place in several hundreds of milliseconds (6-9).In addition, the pathway includes an adaptation system, comprising methyltransferase CheR and methylesterase CheB, that adjusts the activity and sensitivity of the sensory complex by methylating and demethylating receptors. The adaptation system uses feedback from receptor and kinase activity to return CheY phosphorylation to a preset level even in the presence of high levels of chemoeffectors. The time course of the adaptation process depends on stimulus strength (10, 11), varying from several seconds for weak stimuli to several minutes for strong stimuli.Most of the reaction rates and binding constants for chemotaxis proteins have been measured in vitro, and the average intracellular protein concentrations under standard growth conditions were determined (12,13). This abundance of biochemical data has inspired multiple attempts at detailed kinetic analysis of the chemotaxis pathway (9, 13-17), making it the most thoroughly modeled signaling pathw...

Molecular architecture of chemoreceptor arrays revealed by cryoelectron tomography of Escherichia coli minicells

“…The simplest explanation is that virtually all MCPs are captured in arrays, whereas a substantial portion of CheW and CheA is free in solution and able to exchange with their counterparts within the arrays. In fact, fluorescence recovery after photobleaching (FRAP) experiments have indicated an average lifetime at 20°C of only about 12 min for both CheA and CheW within an E. coli receptor patch in vivo (39). In contrast, the receptors themselves persist within a patch for more than a cell generation (39).…”

“…In fact, fluorescence recovery after photobleaching (FRAP) experiments have indicated an average lifetime at 20°C of only about 12 min for both CheA and CheW within an E. coli receptor patch in vivo (39). In contrast, the receptors themselves persist within a patch for more than a cell generation (39).…”

Caulobacter PopZ forms an intrinsically disordered hub in organizing bacterial cell poles

“…The dynamic behavior of CckA-GFP was increased in Caulobacter relative to E. coli cells, possibly because of competition with other proteins for binding to PopZ. Both of these proteins were more dynamic than McpA-GFP, a transmembrane chemoreceptor that forms stable clusters (27).…”

A dynamic-signaling-team model for chemotaxis receptors in Escherichia coli

“…Fluorescent imaging (16)(17)(18) and cryoEM tomography (19)(20)(21) reveal large polar or lateral clusters of receptors, with cryoEM revealing a slightly disordered honeycomb lattice of trimers. Receptors within clusters are relatively stable, as demonstrated by fluorescence recovery after photobleaching (22) and by receptor cross-linking (9).…”