CheY-P Concentration Research Articles

The Chemoreceptor Sensory Adaptation System Produces Coordinated Reversals of the Flagellar Motors on an Escherichia coli Cell.

In isotropic environments, an Escherichia coli cell exhibits coordinated rotational switching of its flagellar motors, produced by fluctuations in the intracellular concentration of phosphorylated CheY (CheY-P) emanating from chemoreceptor signaling arrays. In this study, we show that these CheY-P fluctuations arise through modifications of chemoreceptors by two sensory adaptation enzymes: the methyltransferase CheR and the methylesterase CheB. A cell containing CheR, CheB, and the serine chemoreceptor Tsr exhibited motor synchrony, whereas a cell lacking CheR and CheB or containing enzymatically inactive forms did not. Tsr variants with different combinations of methylation-mimicking Q residues at the adaptation sites also failed to show coordinated motor switching in cells lacking CheR and CheB. Cells containing CheR, CheB, and Tsr [NDND], a variant in which the adaptation site residues are not substrates for CheR or CheB modifications, also lacked motor synchrony. TsrΔNWETF, which lacks a C-terminal pentapeptide-binding site for CheR and CheB, and the ribose-galactose receptor Trg, which natively lacks this motif, failed to produce coordinated motor switching, despite the presence of CheR and CheB. However, addition of the NWETF sequence to Trg enabled Trg-NWETF to produce motor synchrony, as the sole receptor type in cells containing CheR and CheB. Finally, CheBc, the catalytic domain of CheB, supported motor coordination in combination with CheR and Tsr. These results indicate that the coordination of motor switching requires CheR/CheB-mediated changes in receptor modification state. We conclude that the opposing receptor substrate-site preferences of CheR and CheB produce spontaneous blinking of the chemoreceptor array's output activity. IMPORTANCE Under steady-state conditions with no external stimuli, an Escherichia coli cell coordinately switches the rotational direction of its flagellar motors. Here, we demonstrate that the CheR and CheB enzymes of the chemoreceptor sensory adaptation system mediate this coordination. Stochastic fluctuations in receptor adaptation states trigger changes in signal output from the receptor array, and this array blinking generates fluctuations in CheY-P concentration that coordinate directional switching of the flagellar motors. Thus, in the absence of chemoeffector gradients, the sensory adaptation system controls run-tumble swimming of the cell, its optimal foraging strategy.

The Effect of the Second Messenger c-di-GMP on Bacterial Chemotaxis in Escherichia coli

c-di-GMP is a ubiquitous bacterial second messenger that plays a central regulatory role in diverse biological processes. c-di-GMP was known to regulate chemotaxis in multiple bacterial species, but its effect on Escherichia coli chemotaxis remained unclear. As an effector of c-di-GMP in E. coli, YcgR when bound with c-di-GMP interacts with the flagellar motor to reduce its speed and its probability of rotating clockwise (CW bias). Here, we found that a significant fraction of the c-di-GMP::YcgR dynamically exchange between the motor and the cytosol. Through fluorescent measurements, we found that there was no competitive binding between the chemotaxis response regulator CheY-P and c-di-GMP::YcgR to the motor. To test the influence of elevated c-di-GMP levels on the chemotaxis pathway, we measured the chemotactic responses of E. coli cells using a FRET assay, finding that elevated c-di-GMP levels had no effect on the upstream part of chemotaxis pathway down to the level of CheY-P concentration. This suggested that the possible effect of elevated c-di-GMP levels on chemotactic motion was through regulation of motor speed and CW bias. Using stochastic simulations of chemotactic swimming, we showed that the effects of reducing motor speed and decreasing CW bias on chemotactic drift velocity are compensating for each other, resulting in minimal effect of elevated c-di-GMP levels on E. coli chemotaxis. Therefore, elevated c-di-GMP levels promote the transition from motile to sedentary forms of bacterial life by reducing the bacterial swimming speed and CW bias, while still maintaining a nearly intact chemotaxis capability in E. coli. IMPORTANCE The ubiquitous bacterial second messenger c-di-GMP was known to regulate chemotaxis in many bacterial species, but its effect on E. coli chemotaxis was unclear. Here we studied the effect of elevated c-di-GMP levels on chemotaxis in E. coli. We found that the binding of c-di-GMP::YcgR (its effector) and the chemotaxis response regulator CheY-P to the flagellar motor are noncompetitive, and elevated c-di-GMP levels do not affect the upstream part of the chemotaxis pathway down to the level of CheY-P concentration. Elevated c-di-GMP levels exert direct effects on the flagellar motor by reducing its speed and CW bias, but the resulting effects on chemotaxis performance are compensating for each other. Our findings here showed that elevated c-di-GMP levels maintain a nearly intact chemotaxis capability when promoting the transition from motile to sedentary forms of bacterial life in E. coli.

Suppression of cell-cell variation by cooperative interaction of phosphatase and response regulator

In bacterial chemotaxis, the output of chemosensing, the concentration of the response regulator CheY-P that was constantly adjusted by the opposing action of the kinase CheA and the phosphatase CheZ, serves as the input of the ultrasensitive flagellar motor that drives bacterial motility. The steady-state kinase activity exhibits large cell-to-cell variation that may result in similar variation in CheY-P concentration. Here, we found that the in vivo phosphatase activity is highly cooperative with respect to CheY-P concentration, and this suppresses the cell-to-cell variation of CheY-P concentration so that it falls within the operational range of the flagellar motor. Therefore, the cooperativity of the CheZ and CheY-P interaction we identified here provided a mechanism of robust coupling between the output of chemosensing and the input of the flagellar motor. Suppression of cell heterogeneity by cooperativity of protein-protein interaction is likely a common feature in many biological signaling systems.

Direct Mapping from Intracellular Chemotaxis Signaling to Single-Cell Swimming Behavior

Bacterial chemotaxis allows bacteria to sense the chemical environment and modulate their swimming behavior accordingly. Although the intracellular chemotaxis signaling pathway has been studied extensively, experimental studies are still lacking that could provide direct link from the pathway output (the intracellular concentration of the phosphorylated form of the response regulator phosphorylated CheY (CheY-P)) to single-cell swimming behavior. Here, we measured the swimming behavior of individual Escherichia coli cells while simultaneously detecting the intracellular CheY-P concentration, thereby providing a direct relationship between the intracellular CheY-P concentration and the single-cell run-and-tumble behavior. The measured relationship is consistent with the ultrasensitivity of the motor switch and a “veto model” that describes the interaction among individual flagella, although contribution from the voting mechanism could not be ruled out.

Fluctuations in Intracellular CheY-P Concentration Coordinate Reversals of Flagellar Motors in E. coli.

Signal transduction utilizing membrane-spanning receptors and cytoplasmic regulator proteins is a fundamental process for all living organisms, but quantitative studies of the behavior of signaling proteins, such as their diffusion within a cell, are limited. In this study, we show that fluctuations in the concentration of the signaling molecule, phosphorylated CheY, constitute the basis of chemotaxis signaling. To analyze the propagation of the CheY-P signal quantitatively, we measured the coordination of directional switching between flagellar motors on the same cell. We analyzed the time lags of the switching of two motors in both CCW-to-CW and CW-to-CCW switching (∆τCCW-CW and ∆τCW-CCW). In wild-type cells, both time lags increased as a function of the relative distance of two motors from the polar receptor array. The apparent diffusion coefficient estimated for ∆τ values was ~9 µm2/s. The distance-dependency of ∆τCW-CCW disappeared upon loss of polar localization of the CheY-P phosphatase, CheZ. The distance-dependency of the response time for an instantaneously applied serine attractant signal also disappeared with the loss of polar localization of CheZ. These results were modeled by calculating the diffusion of CheY and CheY-P in cells in which phosphorylation and dephosphorylation occur in different subcellular regions. We conclude that diffusion of signaling molecules and their production and destruction through spontaneous activity of the receptor array generates fluctuations in CheY-P concentration over timescales of several hundred milliseconds. Signal fluctuation coordinates rotation among flagella and regulates steady-state run-and-tumble swimming of cells to facilitate efficient responses to environmental chemical signals.

CW and CCW Conformations of the E. coli Flagellar Motor C-Ring Evaluated by Fluorescence Anisotropy

The molecular cascade that controls switching of the direction of rotation of Escherichia coli flagellar motors is well known, but the conformational changes that allow the rotor to switch are still unclear. The signaling molecule CheY, when phosphorylated, binds to the C-ring at the base of the rotor, raising the probability that the motor spins clockwise. When the concentration of CheY-P is so low that the motor rotates exclusively counterclockwise (CCW), the C-ring recruits more monomers of FliM and tetramers of FliN, the proteins to which CheY-P binds, thus increasing the motor's sensitivity to CheY-P and allowing it to switch once again. Motors that rotate exclusively CCW have more FliM and FliN subunits in their C-rings than motors that rotate exclusively clockwise. How are the new subunits accommodated? Does the diameter of the C-ring increase, or do FliM and FliN get packed in a different pattern, keeping the overall diameter of the C-ring constant? Here, by measuring fluorescence anisotropy of yellow fluorescent protein-labeled motors, we show that the CCW C-rings accommodate more FliM monomers without changing the spacing between them, and more FliN monomers at the same time as increasing their effective spacing and/or changing their orientation within the tetrameric structure.

Marine Bacterial Chemoresponse to a Stepwise Chemoattractant Stimulus

We found recently that polar flagellated marine bacterium Vibrio alginolyticus is capable of exhibiting taxis toward a chemical source in both forward and backward swimming directions. How the microorganism coordinates these two swimming intervals, however, is not known. The work presented herein is aimed at determining the response functions of the bacterium by applying a stepwise chemoattractant stimulus while it is swimming forward or backward. The important finding of our experiment is that the bacterium responds to an identical chemical signal similarly during the two swimming intervals. For weak stimuli, the difference is mainly in the amplitudes of the response functions while the reaction and adaptation times remain unchanged. In this linear-response regime, the amplitude in the forward swimming interval is approximately a factor of two greater than in the backward direction. Our observation suggests that the cell processes chemical signals identically in both swimming intervals, but the responses of the flagellar motor to the output of the chemotaxis network, the regulator CheY-P concentration, are different. The biological significance of this asymmetrical response in polar flagellated marine bacteria is discussed.

Single-Cell E. coli Response to an Instantaneously Applied Chemotactic Signal

In response to an attractant or repellant, an Escherichia coli cell controls the rotational direction of its flagellar motor by a chemotaxis system. When an E. coli cell senses an attractant, a reduction in the intracellular concentration of a chemotaxis protein, phosphorylated CheY (CheY-P), induces counterclockwise (CCW) rotation of the flagellar motor, and this cellular response is thought to occur in several hundred milliseconds. Here, to measure the signaling process occurring inside a single E. coli cell, including the recognition of an attractant by a receptor cluster, the inactivation of histidine kinase CheA, and the diffusion of CheY and CheY-P molecules, we applied a serine stimulus by instantaneous photorelease from a caged compound and examined the cellular response at a temporal resolution of several hundred microseconds. We quantified the clockwise (CW) and CCW durations immediately after the photorelease of serine as the response time and the duration of the response, respectively. The results showed that the response time depended on the distance between the receptor and motor, indicating that the decreased CheY-P concentration induced by serine propagates through the cytoplasm from the receptor-kinase cluster toward the motor with a timing that is explained by the diffusion of CheY and CheY-P molecules. The response time included 240 ms for enzymatic reactions in addition to the time required for diffusion of the signaling molecule. The measured response time and duration of the response also revealed that the E. coli cell senses a similar serine concentration regardless of whether the serine concentration is increasing or decreasing. These detailed quantitative findings increase our understanding of the signal transduction process that occurs inside cells during bacterial chemotaxis.

Adaptation at the output of the chemotaxis signalling pathway

In the bacterial chemotaxis network, receptor clusters process input1–3, and flagellar motors generate output4. Receptor and motor complexes are coupled by the diffusible protein CheY-P. Receptor output (the steady-state concentration of CheY-P) varies from cell to cell5. However, the motor is ultrasensitive, with a narrow [CheY-P] operating range6. How might the match between receptor output and motor input be optimized? Here we show that the motor can shift its operating range by changing its composition. The number of FliM subunits in the C-ring increases in response to a decrement in the concentration of CheY-P, increasing motor sensitivity. This shift in sensitivity explains the slow partial adaptation observed in mutants that lack the receptor methyltransferase and methylesterase7–8 and why motors exhibit signal-dependent FliM turnover9. Adaptive remodelling is likely to be a common feature in the operation of many molecular machines.

Adaptation by target remodelling

Bacteria direct their movement in response to certain chemicals by controlling the rotation of whip-like appendages called flagella. The sensitivity of the response can be adjusted at the signal's target, the flagellar motor. See Letter p.233 Chemotaxis, the process used by bacteria to move in response to a chemical stimulus, is driven by a flagellar rotary motor that is exquisitely sensitive to intracellular levels of the signalling protein CheY-P within a narrow operating range. However, there is a large cell-to-cell variation in steady-state CheY-P concentration, prompting speculation as to whether Escherichia coli could possibly control its steady-state CheY-P concentration with sufficient precision to engage the flagellar motor when required. Howard Berg and colleagues have found an answer to this paradox. The bacterium seems to avoid the problem in a counter-intuitive way, by constantly adjusting the number of CheY-P target proteins in the motor. Such adaptive remodelling at the output end of a control process goes against the principles of good engineering practice, yet is likely to be common to many biological machines.

Action at a Distance: Amino Acid Substitutions That Affect Binding of the Phosphorylated CheY Response Regulator and Catalysis of Dephosphorylation Can Be Far from the CheZ Phosphatase Active Site

Two-component regulatory systems, in which phosphorylation controls the activity of a response regulator protein, provide signal transduction in bacteria. For example, the phosphorylated CheY response regulator (CheYp) controls swimming behavior. In Escherichia coli, the chemotaxis phosphatase CheZ stimulates the dephosphorylation of CheYp. CheYp apparently binds first to the C terminus of CheZ and then binds to the active site where dephosphorylation occurs. The phosphatase activity of the CheZ(2) dimer exhibits a positively cooperative dependence on CheYp concentration, apparently because the binding of the first CheYp to CheZ(2) is inhibited compared to the binding of the second CheYp. Thus, CheZ phosphatase activity is reduced at low CheYp concentrations. The CheZ21IT gain-of-function substitution, located far from either the CheZ active site or C-terminal CheY binding site, enhances CheYp binding and abolishes cooperativity. To further explore mechanisms regulating CheZ activity, we isolated 10 intragenic suppressor mutations of cheZ21IT that restored chemotaxis. The suppressor substitutions were located along the central portion of CheZ and were not allele specific. Five suppressor mutants tested biochemically diminished the binding of CheYp and/or the catalysis of dephosphorylation, even when the suppressor substitutions were distant from the active site. One suppressor mutant also restored cooperativity to CheZ21IT. Consideration of results from this and previous studies suggests that the binding of CheYp to the CheZ active site (not to the C terminus) is rate limiting and leads to cooperative phosphatase activity. Furthermore, amino acid substitutions distant from the active site can affect CheZ catalytic activity and CheYp binding, perhaps via the propagation of structural or dynamic perturbations through a helical bundle.

Coordinated Reversal of Flagellar Motors on a Single Escherichia coli Cell

An Escherichia coli cell transduces extracellular stimuli sensed by chemoreceptors to the state of an intracellular signal molecule, which regulates the switching of the rotational direction of the flagellar motors from counterclockwise (CCW) to clockwise (CW) and from CW back to CCW. Here, we performed high-speed imaging of flagellar motor rotation and show that the switching of two different motors on a cell is controlled coordinatedly by an intracellular signal protein, phosphorylated CheY (CheY-P). The switching is highly coordinated with a subsecond delay between motors in clear correlation with the distance of each motor from the chemoreceptor patch localized at a cell pole, which would be explained by the diffusive motion of CheY-P molecules in the cell. The coordinated switching becomes disordered by the expression of a constitutively active CheY mutant that mimics the CW-rotation stimulating function. The coordinated switching requires CheZ, which is the phosphatase for CheY-P. Our results suggest that a transient increase and decrease in the concentration of CheY-P caused by a spontaneous burst of its production by the chemoreceptor patch followed by its dephosphorylation by CheZ, which is probably a wavelike propagation in a subsecond timescale, triggers and regulates the coordinated switching of flagellar motors.

Switching of the Bacterial Flagellar Motor Near Zero Load

Flagellated bacteria, such as Escherichia coli, are able to swim up gradients of chemical attractants by modulating the direction of rotation of their flagellar motors, which spin alternately clockwise (CW) and counterclockwise (CCW). Chemotactic behavior has been studied under a variety of conditions, mostly at high loads (at large motor torques). Here, we examine motor switching at low loads. Nano-gold spheres of various sizes were attached to hooks (the flexible coupling at the base of the flagellar filament) of cells lacking flagellar filaments in media containing different concentrations of the viscous agent Ficoll. The speeds and directions of rotation of the spheres were measured. Contrary to the case at high loads, motor switching rates increased appreciably with load. Both the CW → CCW and CCW → CW switching rates increased linearly with motor torque. Evidently, the switch senses stator–rotor interactions as well as the CheY-P concentration.

Dependence of Bacterial Chemotaxis on Gradient Shape and Adaptation Rate

Simulation of cellular behavior on multiple scales requires models that are sufficiently detailed to capture central intracellular processes but at the same time enable the simulation of entire cell populations in a computationally cheap way. In this paper we present RapidCell, a hybrid model of chemotactic Escherichia coli that combines the Monod-Wyman-Changeux signal processing by mixed chemoreceptor clusters, the adaptation dynamics described by ordinary differential equations, and a detailed model of cell tumbling. Our model dramatically reduces computational costs and allows the highly efficient simulation of E. coli chemotaxis. We use the model to investigate chemotaxis in different gradients, and suggest a new, constant-activity type of gradient to systematically study chemotactic behavior of virtual bacteria. Using the unique properties of this gradient, we show that optimal chemotaxis is observed in a narrow range of CheA kinase activity, where concentration of the response regulator CheY-P falls into the operating range of flagellar motors. Our simulations also confirm that the CheB phosphorylation feedback improves chemotactic efficiency by shifting the average CheY-P concentration to fit the motor operating range. Our results suggest that in liquid media the variability in adaptation times among cells may be evolutionary favorable to ensure coexistence of subpopulations that will be optimally tactic in different gradients. However, in a porous medium (agar) such variability appears to be less important, because agar structure poses mainly negative selection against subpopulations with low levels of adaptation enzymes. RapidCell is available from the authors upon request.

Kinetic Characterization of Catalysis by the Chemotaxis Phosphatase CheZ: MODULATION OF ACTIVITY BY THE PHOSPHORYLATED CheY SUBSTRATE

CheZ catalyzes the dephosphorylation of the response regulator CheY in the two-component regulatory system that mediates chemotaxis in Escherichia coli. CheZ is a homodimer with two active sites for dephosphorylation. To gain insight into cellular mechanisms for the precise regulation of intracellular phosphorylated CheY (CheYp) levels, we evaluated the kinetic properties of CheZ. The steady state rate of CheZ-mediated dephosphorylation of CheYp displayed marked sigmoidicity with respect to CheYp concentration and a k(cat) of 4.9 s(-1). In contrast, the gain of function mutant CheZ-I21T with an amino acid substitution far from the active site gave hyperbolic kinetics and required far lower CheYp for half-saturation but had a similar k(cat) value as the wild type enzyme. Stopped flow fluorescence measurements demonstrated a 6-fold faster CheZ/CheYp association rate for CheZ-I21T (k(assoc) = 3.4 x 10(7) M (-1) s(-1)) relative to wild type CheZ (k(assoc) = 5.6 x 10(6) M(-1) s(-1)). Dissociation of the CheZ.CheYBeF(3) complex was slow for both wild type CheZ (k(dissoc) = 0.040 s(-1)) and CheZ-I21T (k(dissoc) = 0.023 s(-1)) and, when taken with the k(assoc) values, implied K(d) values of 7.1 and 0.68 nm, respectively. However, comparison of the k(dissoc) and k(cat) values implied that CheZ and CheYp are not at binding equilibrium during catalysis and that once CheYp binds, it is almost always dephosphorylated. The rate constants were collated to formulate a kinetic model for CheZ-mediated dephosphorylation that includes autoregulation by CheYp and allowed prediction of CheZ activities at CheZ and CheYp concentrations likely to be present in cells.

Switching Statistics of a Flagellar Motor: First-Passage Time and Dynamic Binding

Bacterial chemotaxis relies on the clockwise-to-counterclockwise switching (and the reverse CCW-to-CW switching) of the flagellar motor’s rotational direction. Yet, despite its central role, the switch mechanism remains poorly understood. Here we study the switch statistics of the flagellar motor using computer simulations that combine first-passage time ideas and the property of dynamic binding of CheY-P to FliM. The simulated clockwise bias and switching frequency as a function of CheY-P concentration agree with the experimental results for E. coli. Moreover, the simulation results show a robust peak, which is present in some experimental reports, in the distribution of the time intervals of both clockwise and counterclockwise rotation.