In vivo and in vitro characterization of Escherichia coli protein CheZ gain- and loss-of-function mutants.

Bacterial chemotaxis results from the ability of flagellated bacteria to control the frequency of switching between smooth-swimming and tumbling episodes in response to changes in concentration of extracellular substances. High levels of phosphorylated CheY protein are the intracellular signal for inducing the tumbling mode of swimming. The CheZ protein has been shown to control the level of phosphorylated CheY by regulating its rate of dephosphorylation. To identify functional domains in the CheZ protein, we made mutants by random mutagenesis of the cheZ gene and constructed a series of deletions. The map position and the in vivo and in vitro activity of the resulting gain- or loss-of-function mutant proteins define separate functional domains of the CheZ protein.     

Identification of a fourth cheY gene in Rhodobacter sphaeroides and interspecies interaction within the bacterial chemotaxis signal transduction pathway

The Escherichia coli chemotaxis signal transduction pathway has: CheA, a histidine protein kinase; CheW, a linker between CheA and sensory proteins; CheY, the effector; and CheZ, a signal terminator. Rhodobacter sphaeroides has multiple copies of these proteins (2 x CheA, 3 x CheW and 3 x CheY, but no CheZ). In this study, we found a fourth cheY and expressed these R. sphaeroides proteins in E. coli. CheA2 (but not CheA1) restored swarming to an E. coli cheA mutant (RP9535). CheW3 (but not CheW2) restored swarming to a cheW mutant of E. coli (RP4606). R. sphaeroides CheYs did not affect E. coli lacking CheY, but restored swarming to a cheZ strain (RP1616), indicating that they can act as signal terminators in E. coli. An E. coli CheY, which is phosphorylated but cannot bind the motor (CheY109KR), was expressed in RP1616 but had no effect. Overexpression of CheA2, CheW2, CheW3, CheY1, CheY3 and CheY4 inhibited chemotaxis of wild-type E. coli (RP437) by increasing its smooth-swimming bias. While some R. sphaeroides proteins restore tumbling to smooth-swimming E. coli mutants, their activity is not controlled by the chemosensory receptors. R. sphaeroides possesses a phosphorelay cascade compatible with that of E. coli, but has additional incompatible homologues.     

Reconstitution of signaling in bacterial chemotaxis.

Strains missing several genes required for chemotaxis toward amino acids, peptides, and certain sugars were tethered and their rotational behavior was analyzed. Null strains (called gutted) were deleted for genes that code for the transducers Tsr, Tar, Tap, and Trg and for the cytoplasmic proteins CheA, CheW, CheR, CheB, CheY, and CheZ. Motor switch components were wild type, flaAII(cheC), or flaBII(cheV). Gutted cells with wild-type motors spun exclusively counterclockwise, while those with mutant motors changed their directions of rotation. CheY reduced the bias (the fraction of time that cells spun counterclockwise) in either case. CheZ offset the effect of CheY to an extent that varied with switch allele but did not change the bias when tested alone. Transducers also increased the bias in the presence of CheY but not when tested alone. However, cells containing transducers and CheY failed to respond to attractants or repellents normally detected in the periplasm. This sensitivity was restored by addition of CheA and CheW. Thus, CheY both enhances clockwise rotation and couples the transducers to the flagella. CheZ acts, at the level of the motor, as a CheY antagonist. CheA or CheW or both are required to complete the signal pathway. A model is presented that explains these results and is consistent with other data found in the literature.     

Isolation and characterization of nonchemotactic CheZ mutants of Escherichia coli

The Escherichia coli CheZ protein stimulates dephosphorylation of CheY, a response regulator in the chemotaxis signal transduction pathway, by an unknown mechanism. Genetic analysis of CheZ has lagged behind biochemical and biophysical characterization. To identify putative regions of functional importance in CheZ, we subjected cheZ to random mutagenesis and isolated 107 nonchemotactic CheZ mutants. Missense mutations clustered in six regions of cheZ, whereas nonsense and frameshift mutations were scattered reasonably uniformly across the gene. Intragenic complementation experiments showed restoration of swarming activity when compatible plasmids containing genes for the truncated CheZ(1-189) peptide and either CheZA65V, CheZL90S, or CheZD143G were both present, implying the existence of at least two independent functional domains in each chain of the CheZ dimer. Six mutant CheZ proteins, one from each cluster of loss-of-function missense mutations, were purified and characterized biochemically. All of the tested mutant proteins were defective in their ability to dephosphorylate CheY-P, with activities ranging from 0.45 to 16% of that of wild-type CheZ. There was good correlation between the phosphatase activity of CheZ and the ability to form large chemically cross-linked complexes with CheY in the presence of the CheY phosphodonor acetyl phosphate. In consideration of both the genetic and biochemical data, the most severe functional impairments in this set of CheZ mutants seemed to be concentrated in regions which are located in a proposed large N-terminal domain of the CheZ protein.     

Characterization of the CheAS/CheZ complex: a specific interaction resulting in enhanced dephosphorylating activity on CheY-phosphate

The cheA gene encodes two overlapping polypeptides with a common carboxyl terminus: CheA_L and CheA_S. CheA_L plays a central role in the Escherichia coli chemotaxis signalling pathway by autophosphorylation and transferring the phosphate to both CheY and CheB. On the other hand, the physiological functions of CheA_S remain unknown.
We have observed that overproduction of CheA_S in wild-type cells increased counterclockwise-biased flagellar rotation, and this effect is dependent on the presence of CheZ. CheZ specifically facilitates CheY-phosphate (CheY-P) dephosphorylation and generates a smooth swimming signal. A physical interaction was detected between CheZ and CheA_S in wild-type cell lysates by immunoprecipitation. The CheA_S/CheZ interaction does not require other chemotaxis components, as we could form the complex using purified CheA_S and CheZ proteins. The ability of CheA_S to bind to CheZ depends on its being in the reduced state. We found that under non-reducing conditions, CheA_S appears to form intermolecular disulphide bonds and loses the ability to bind to CheZ. Finally, the CheA_S/CheZ complex formed in vitro shows a greater dephosphorylating activity on CheY-P than does free CheZ.     

Structure and function of an unusual family of protein phosphatases: the bacterial chemotaxis proteins CheC and CheX

In bacterial chemotaxis, phosphorylated CheY levels control the sense of flagella rotation and thereby determine swimming behavior. In E. coli, CheY dephosphorylation by CheZ extinguishes the switching signal. But, instead of CheZ, many chemotactic bacteria contain CheC, CheD, and/or CheX. The crystal structures of T. maritima CheC and CheX reveal a common fold unlike that of any other known protein. Unlike CheC, CheX dimerizes via a continuous beta sheet between subunits. T. maritima CheC, as well as CheX, dephosphorylate CheY, although CheC requires binding of CheD to achieve the activity of CheX. Structural analyses identified one conserved active site in CheX and two in CheC; mutations therein reduce CheY-phosphatase activity, but only mutants of two invariant asparagine residues are completely inactive even in the presence of CheD. Our structures indicate that the flagellar switch components FliY and FliM resemble CheC more closely than CheX, but attribute phosphatase activity only to FliY.     

Phosphorylation and binding interactions of CheY studied by use of Badan-labeled protein

In the chemotaxis signal transduction pathway of Escherichia coli, the response regulator protein CheY is phosphorylated by the receptor-coupled protein kinase CheA. Previous studies of CheY phosphorylation and CheY interactions with other proteins in the chemotaxis pathway have exploited the fluorescence properties of Trp(58), located immediately adjacent to the phosphorylation site of CheY (Asp(57)). Such studies can be complicated by the intrinsic fluorescence and absorbance properties of CheA and other proteins of interest. To circumvent these difficulties, we generated a derivative of CheY carrying a covalently attached fluorescent label that serves as a sensitive reporter of phosphorylation and binding events and that absorbs and emits light at wavelengths well removed from potential interference by other proteins. This labeled version of CheY has the (dimethylamino)naphthalene fluorophore from Badan [6-bromoacetyl-2-(dimethylamino)naphthalene] attached to the thiol group of a cysteine introduced at position 17 of CheY by site-directed mutagenesis. Under phosphorylating conditions (or in the presence of beryllofluoride), the fluorescence emission of Badan-labeled CheY(M17C) exhibited an approximately 10 nm blue shift and an approximately 30% increase in signal intensity at 490 nm. The fluorescence of Badan-labeled CheY(M17C) also served as a sensitive reporter of CheY-CheA binding interactions, exhibiting an approximately 50% increase in emission intensity in the presence of saturating levels of CheA. Compared to wild-type CheY, Badan-labeled CheY exhibited reduced ability to autodephosphorylate and could not interact productively with the phosphatase CheZ. However, with respect to autophosphorylation and interactions with CheA, Badan-CheY performed identically to wild-type CheY, allowing us to explore CheA-CheY phosphotransfer kinetics and binding kinetics without interference from the fluorescence/absorbance properties of CheA and ATP. These results provide insights into CheY interactions with CheA, CheZ, and other components of the chemotaxis signaling pathway.     

Phosphorylation of three proteins in the signaling pathway of bacterial chemotaxis

Six cytoplasmic che gene products are required for signal transduction in bacterial chemotaxis, but the nature of their biochemical interactions is not known. We show that in vitro the CheA protein becomes autophosphorylated in the presence of ATP. In addition, the phosphate group on CheA can be rapidly transferred to CheB, a protein involved in adaptation to stimuli, or to CheY, a protein involved in the excitation response. The phosphorylation of CheB and CheY is transient; they readily dephosphorylate. We have also found that CheZ, a protein that appears to antagonize CheY function in vivo, accelerates the hydrolysis of the phosphate on CheY. These results suggest that signal transduction in bacterial chemotaxis may involve the flow of phosphate through a cascade of phosphorylated protein intermediates.     

Response regulation in bacterial chemotaxis

The signal transduction system that mediates bacterial chemotaxis allows cells to moduate their swimming behavior in response to fluctuations in chemical stimuli. Receptors at the cell surface receive information from the surroundings. Signals are then passed from the receptors to cytoplasmic chemotaxis components: CheA, CheW, CheZ, CheR, and CheB. These proteins function to regulate the level of phosphorylation of a response regulator designated CheY that interacts with the flagellar motor switch complex to control swimming behavior. The structure of CheY has been determined. Magnesium ion is essential for activity. The active site contains highly conserved Asp residues that are required for divalent metal ion binding and CheY phosphorylation. Another residue-at the active site, Lys109, is important in the phosphorylation-induced conformational change that facilitates communication with the switch complex and another chemotaxis component, CheZ. CheZ facilitates the dephosphorylation of phospho-CheY. Defects in CheY and CheZ can be suppressed by mutations in the flagellar switch complex. CheZ is thought to modulate the switch bias by varying the level of phospho-CheY. © 1993 Wiley-Liss, Inc.     

Sequence and characterization of Bacillus subtilis CheB, a homolog of Escherichia coli CheY, and its role in a different mechanism of chemotaxis

The Bacillus subtilis gene encoding CheB, which is homologous to Escherichia coli CheY, the regulator of flagellar rotation, has been cloned and sequenced. It has been verified, using a phage T7 expression system, by showing that a small protein, the same size as E. coli CheY, is actually made from this DNA. Despite the fact that the two proteins are 36% identical, with many highly conserved residues, they appear to play different roles. Unlike CheY null mutants, which swim smoothly, CheB null mutants tumble incessantly. However, a CheB point mutant swims smoothly, even in the presence of a plasmid bearing cheB, which restores the null mutants to wild type. Expression of CheB in wild type B. subtilis makes the cells exhibit more tumbling. Since both absence of CheB and presence of high levels of CheB cause tumbling, CheB appears to be required, in certain circumstances, for both smooth swimming and tumbling. Expression in wild type E. coli makes the cells smooth swimmers and strongly inhibits chemotaxis.     

Alteration of a nonconserved active site residue in the chemotaxis response regulator CheY affects phosphorylation and interaction with CheZ

CheY is a response regulator in the well studied two-component system that mediates bacterial chemotaxis. Phosphorylation of CheY at Asp(57) enhances its interaction with the flagellar motor. Asn(59) is located near the phosphorylation site, and possible roles this residue may play in CheY function were explored by mutagenesis. Cells containing CheY59NR or CheY59NH exhibited hyperactive phenotypes (clockwise flagellar rotation), and CheY59NR was characterized biochemically. A continuous enzyme-linked spectroscopic assay that monitors P(i) concentration was the primary method for kinetic analysis of phosphorylation and dephosphorylation. CheY59NR autodephosphorylated at the same rate as wild-type CheY and phosphorylated similarly to wild type with acetyl phosphate and faster (4-14x) with phosphoramidate and monophosphoimidazole. CheY59NR was extremely resistant to CheZ, requiring at least 250 times more CheZ than wild-type CheY to achieve the same dephosphorylation rate enhancement, whereas CheY59NA was CheZ-sensitive. However, several independent approaches demonstrated that CheY59NR bound tightly to CheZ. A submicromolar K(d) for CheZ binding to CheY59NR-P or CheY.BeF(3)(-) was inferred from fluorescence anisotropy measurements of fluoresceinated-CheZ. A complex between CheY59NR-P and CheZ was isolated by analytical gel filtration, and the elution position from the column was indistinguishable from that of the CheZ dimer. Therefore, we were not able to detect large CheY-P.CheZ complexes that have been inferred using other methods. Possible structural explanations for the specific inhibition of CheZ activity as a result of the arginyl substitution at CheY position 59 are discussed.     

Mutations in the chemotactic response regulator, CheY, that confer resistance to the phosphatase activity of CheZ

CheY, a small cytoplasmic response regulator, plays an essential role in the chemotaxis pathway. The concentration of phospho-CheY is thought to determine the swimming behaviour of the cell: high levels of phospho-CheY cause bacteria to rotate their flagella clockwise and tumble, whereas low levels of the phos-phorylated form of the protein allow counter-ciockwise rotation of the flagella and smooth swimming. The phosphorylation state of CheY in vivo is determined by the activity of the phosphoryl donor CheA, and by the antagonistic effect of dephosphorylation of phospho-CheY. The dephosphorylation rate is controlled by the intrinsic autohydrolytic activity of phospho-CheY and by the CheZ protein, which accelerates dephosphorylation. We have analysed the effect of CheZ on the dephosphorylation rates of several mutant CheY proteins. Two point mutations were identified which were 50-fold and 5-fold less sensitive to the activity of CheZ than was the wild-type protein. Nonetheless, the phosphorylation and autodephos-phorylation rates of these mutants, CheY23ND and CheY26KE, were observed to be identical to those of wild-type CheY in the absence of CheZ. These are the first examples of CheY mutations that reduce sensitivity to the phosphatase activity of CheZ without being altered in terms of their intrinsic phosphorylation and autodephospborylation rates, interestingly, the residues Asn-23 and Lys-26 are located on a face of CheY far from the phosphorylation site (Asp-57), distinct from the previously described site of inter-action with the histidine kinase CheA, and partially overlapping with a region implicated in interaction with the flagellar switch.     

Elucidation of a PTS-carbohydrate chemotactic signal pathway in Escherichia coli using a time-resolved behavioral assay

Chemotaxis of Escherichia coli toward phosphotransferase systems (PTSs)-carbohydrates requires phosphoenolpyruvate-dependent PTSs as well as the chemotaxis response regulator CheY and its kinase, CheA. Responses initiated by flash photorelease of a PTS substrates D-glucose and its nonmetabolizable analog methyl alpha-D-glucopyranoside were measured with 33-ms time resolution using computer-assisted motion analysis. This, together with chemotactic mutants, has allowed us to map out and characterize the PTS chemotactic signal pathway. The responses were absent in mutants lacking the general PTS enzymes EI or HPr, elevated in PTS transport mutants, retarded in mutants lacking CheZ, a catalyst of CheY autodephosphorylation, and severely reduced in mutants with impaired methyl-accepting chemotaxis protein (MCP) signaling activity. Response kinetics were comparable to those triggered by MCP attractant ligands over most of the response range, the most rapid being 11.7 +/- 3.1 s-1. The response threshold was <10 nM for glucose. Responses to methyl alpha-D-glucopyranoside had a higher threshold, commensurate with a lower PTS affinity, but were otherwise kinetically indistinguishable. These facts provide evidence for a single pathway in which the PTS chemotactic signal is relayed rapidly to MCP-CheW-CheA signaling complexes that effect subsequent amplification and slower CheY dephosphorylation. The high sensitivity indicates that this signal is generated by transport-induced dephosphorylation of the PTS rather than phosphoenolpyruvate consumption.     

Control of bacterial chemotaxis

Bacterial chemotaxis, which has been extensively studied for three decades, is the most prominent model system for signal transduction in bacteria. Chemotaxis is achieved by regulating the direction of flagellar rotation. The regulation is carried out by the chemotaxis protein, CheY. This protein is activated by a stimulus-dependent phosphorylation mediated by an autophosphorylatable kinase (CheA) whose activity is controlled by chemoreceptors. Upon phosphorylation, CheY dissociates from its kinase, binds to the switch at the base of the flagellar motor, and changes the motor rotation from the default direction (counter-clockwise) to clockwise. Phosphorylation may also be involved in terminating the response. Phosphorylated CheY binds to the phosphatase CheZ and modulates its oligomeric state and thereby its dephosphorylating activity. Thus CheY phosphorylation appears to be involved in controlling both the excitation and adaptation mechanisms of bacterial chemotaxis. Additional control sites might be involved in bacterial chemotaxis, e.g. lateral control at the receptor level, control at the motor level, or control by metabolites that link central metabolism with chemotaxis.     

Co-regulation of acetylation and phosphorylation of CheY, a response regulator in chemotaxis of Escherichia coli

CheY, a response regulator of the chemotaxis system in Escherichia coli, can be activated by either phosphorylation or acetylation to generate clockwise rotation of the flagellar motor. Both covalent modifications are involved in chemotaxis, but the function of the latter remains obscure. To understand why two different modifications apparently activate the same function of CheY, we studied the effect that each modification exerts on the other. The phosphodonors of CheY, the histidine kinase CheA and acetyl phosphate, each strongly inhibited both the autoacetylation of the acetylating enzyme, acetyl-CoA synthetase (Acs), and the acetylation of CheY. CheZ, the enzyme that enhances CheY dephosphorylation, had the opposite effect and enhanced Acs autoacetylation and CheY acetylation. These effects of the phosphodonors and CheZ were not caused by their respective activities. Rather, they were caused by their interactions with Acs and, possibly, with CheY. In addition, the presence of Acs elevated the phosphorylation levels of both CheA and CheY, and acetate repressed this stimulation. These observations suggest that CheY phosphorylation and acetylation are linked and co-regulated. We propose that the physiological role of these mutual effects is at two levels: linking chemotaxis to the metabolic state of the cell, and serving as a tuning mechanism that compensates for cell-to-cell variations in the concentrations of CheA and CheZ.     

Interaction of CheY with the C-terminal peptide of CheZ

Chemotaxis, a means for motile bacteria to sense the environment and achieve directed swimming, is controlled by flagellar rotation. The primary output of the chemotaxis machinery is the phosphorylated form of the response regulator CheY (P~CheY). The steady-state level of P~CheY dictates the direction of rotation of the flagellar motor. The chemotaxis signal in the form of P~CheY is terminated by the phosphatase CheZ. Efficient dephosphorylation of CheY by CheZ requires two distinct protein-protein interfaces: one involving the strongly conserved C-terminal helix of CheZ (CheZ_C) tethering the two proteins together and the other constituting an active site for catalytic dephosphorylation. In a previous work (J. Guhaniyogi, V. L. Robinson, and A. M. Stock, J. Mol. Biol. 359:624-645, 2006), we presented high-resolution crystal structures of CheY in complex with the CheZ_C peptide that revealed alternate binding modes subject to the conformational state of CheY. In this study, we report biochemical and structural data that support the alternate-binding-mode hypothesis and identify key recognition elements in the CheY-CheZ_C interaction. In addition, we present kinetic studies of the CheZ_C-associated effect on CheY phosphorylation with its physiologically relevant phosphodonor, the histidine kinase CheA. Our results indicate mechanistic differences in phosphotransfer from the kinase CheA versus that from small-molecule phosphodonors, explaining a modest twofold increase of CheY phosphorylation with the former, observed in this study, relative to a 10-fold increase previously documented with the latter.     

Localization of components of the chemotaxis machinery of Escherichia coli using fluorescent protein fusions

We prepared fusions of yellow fluorescent protein [the YFP variant of green fluorescent protein (GFP)] with the cytoplasmic chemotaxis proteins CheY, CheZ and CheA and the flagellar motor protein FliM, and studied their localization in wild-type and mutant cells of Escherichia coli. All but the CheA fusions were functional. The cytoplasmic proteins CheY, CheZ and CheA tended to cluster at the cell poles in a manner similar to that observed earlier for methyl-accepting chemotaxis proteins (MCPs), but only if MCPs were present. Co-localization of CheY and CheZ with MCPs was CheA dependent, and co-localization of CheA with MCPs was CheW dependent, as expected. Co-localization with MCPs was confirmed by immunofluorescence using an anti-MCP primary antibody. The motor protein FliM appeared as discrete spots on the sides of the cell. These were seen in wild-type cells and in a fliN mutant, but not in flhC or fliG mutants. Co-localization with flagellar structures was confirmed by immunofluorescence using an antihook primary antibody. Surprisingly, we did not observe co-localization of CheY with motors, even under conditions in which cells tumbled.     

Protein phosphorylation in the bacterial chemotaxis system

Bacterial chemotaxis involves the detection of changes in concentration of specific chemicals in the environment of the cell as a function of time. This process is mediated by a series of cell surface receptors that interact with and activate intracellular protein phosphorylation. Five cytoplasmic proteins essential for chemotaxis have been shown to be involved in a coupled system of protein phosphorylation. Ligand binding to cell surface receptors affects the rate of autophosphorylation of the CheA protein. In the absence of an attractant bound to receptor and in the presence of the CheW protein, the rate of CheA autophosphorylation is markedly increased. Phosphorylated CheA can transfer phosphate to the CheY or CheB proteins; phosphorylation of these "effector" proteins may increase their activity. The CheY protein is thought to regulate flagellar rotation and thus control swimming behavior. The CheB protein modifies the cell surface receptor and thus regulates receptor function. Finally, another chemotaxis protein, CheZ, acts to specifically dephosphorylate CheY-phosphate. This system shows marked similarity to the 2-component sensor-regulator systems found to control specific gene expression in a variety of bacteria.     

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 CheZ₂ dimer exhibits a positively cooperative dependence on CheYp concentration, apparently because the binding of the first CheYp to CheZ₂ 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.     

The dynamics of protein phosphorylation in bacterial chemotaxis

The chemotaxis signal transduction pathway allows bacteria to respond to changes in concentration of specific chemicals (ligands) by modulating their swimming behavior. The pathway includes ligand binding receptors, and the CheA, CheY, CheW, and CheZ proteins. We showed previously that phosphorylation of CheY is activated in reactions containing receptor, CheW, CheA, and CheY. Here we demonstrate that this activation signal results from accelerated autophosphorylation of the CheA kinase. Evidence for a second signal transmitted by a ligand-bound receptor, which corresponds to inhibition of CheA autophosphorylation, is also presented. We postulate that CheA can exist in three forms: a "closed" form in the absence of receptor and CheW; an "open" form that results from activation of CheA by receptor and CheW; and a "sequestered" form in reactions containing ligand-bound receptor and CheW. The system's dynamics depends on the relative distribution of CheA among these three forms at any time.