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...

In bacterial chemotaxis, several types of receptors form mixed clusters. Receptor adaptation is shown to depend on the receptor's own conformational state rather than on the cluster's global activity, enabling cells to differentiate stimuli in complex environments.

Recent developments in fluorescence microscopy raise the demands for bright and photostable fluorescent tags for specific and background free labeling in living cells. Aside from fluorescent proteins and other tagging methods, labeling of SNAP-tagged proteins has become available thereby increasing the pool of potentially applicable fluorescent dyes for specific labeling of proteins. Here, we report on novel conjugates of benzylguanine (BG) which are quenched in their fluorescence and become highly fluorescent upon labeling of the SNAP-tag, the commercial variant of the human O(6)-alkylguanosyltransferase (hAGT). We identified four conjugates showing a strong increase, i.e., >10-fold, in fluorescence intensity upon labeling of SNAP-tag in vitro. Moreover, we screened a subset of nine BG-dye conjugates in living Escherichia coli and found them all suited for labeling of the SNAP-tag. Here, quenched BG-dye conjugates yield a higher specificity due to reduced contribution from excess conjugate to the fluorescence signal. We further extended the application of these conjugates by labeling a SNAP-tag fusion of the Tar chemoreceptor in live E. coli cells and the eukaryotic transcription factor STAT5b in NIH 3T3 mouse fibroblast cells. Aside from the labeling efficiency and specificity in living cells, we discuss possible mechanisms that might be responsible for the changes in fluorescence emission upon labeling of the SNAP-tag, as well as problems we encountered with nonspecific labeling with certain conjugates in eukaryotic cells.

Communication by means of diffusible signaling molecules facilitates higher-level organization of cellular populations. Gram-positive bacteria frequently use signaling peptides, which are either detected at the cell surface or 'probed' by intracellular receptors after being pumped into the cytoplasm. While the former type is used to monitor cell density, the functions of pump-probe networks are less clear. Here we show that pump-probe networks can, in principle, perform different tasks and mediate quorum-sensing, chronometric and ratiometric control. We characterize the properties of the prototypical PhrA-RapA system in Bacillus subtilis using FRET. We find that changes in extracellular PhrA concentrations are tracked rather poorly; instead, cells accumulate and strongly amplify the signal in a dosedependent manner. This suggests that the PhrA-RapA system, and others like it, have evolved to sense changes in the composition of heterogeneous populations and infer the fraction of signal-producing cells in a mixed population to coordinate cellular behaviors.

BackgroundExtracellular stimuli in chemotaxis of Escherichia coli and other bacteria are processed by large clusters of sensory complexes. The stable core of these clusters is formed by transmembrane receptors, a kinase CheA, and an adaptor CheW, whereas adaptation enzymes CheR and CheB dynamically associate with the clusters via interactions with receptors and/or CheA. Several biochemical studies have indicated the dependence of the sensory complex stability on the adaptive modification state of receptors and/or on temperature, which may potentially allow environment-dependent tuning of its signalling properties. However, the extent of such regulation in vivo and its significance for chemotaxis remained unclear.ResultsHere we used fluorescence recovery after photobleaching (FRAP) to confirm in vivo that the exchange of CheA and CheW shows a modest dependency on the level of receptor modification/activity. An even more dramatic effect was observed for the exchange kinetics of CheR and CheB, indicating that their association with clusters may depend on the ability to bind substrate sites on receptors and on the regulatory phosphorylation of CheB. In contrast, environmental temperature did not have a discernible effect on stability of the cluster core. Strain-specific loss of E. coli chemotaxis at high temperature could instead be explained by a heat-induced reduction in the chemotaxis protein levels. Nevertheless, high basal levels of chemotaxis and flagellar proteins in common wild type strains MG1655 and W3110 enabled these strains to maintain their chemotactic ability up to 42°C.ConclusionsOur results confirmed that clusters formed by less modified receptors are more dynamic, which can explain the previously observed adjustment of the chemotaxis response sensitivity according to the level of background stimulation. We further propose that the dependency of CheR exchange on the availability of unmethylated sites on receptors is important to improve the overall chemotaxis efficiency by suppressing molecular noise under conditions of high ligand concentrations. Moreover, the observed stability of the cluster core at high temperature is in line with the overall thermal robustness of the chemotaxis pathway and allows maintenance of chemotaxis up to 42°C in the common wild type strains of E. coli.

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