Identification of the binding interfaces on CheY for two of its targets, the phosphatase CheZ and the flagellar switch protein fliM.

CheY is the response regulator protein serving as a phosphorylation-dependent switch in the bacterial chemotaxis signal transduction pathway. CheY has a number of proteins with which it interacts during the course of the signal transduction pathway. In the phosphorylated state, it interacts strongly with the phosphatase CheZ, and also the components of the flagellar motor switch complex, specifically with FliM. Previous work has characterized peptides consisting of small regions of CheZ and FliM which interact specifically with CheY. We have quantitatively measured the binding of these peptides to both unphosphorylated and phosphorylated CheY using fluorescence spectroscopy. There is a significant enhancement of the binding of these peptides to the phosphorylated form of CheY, suggesting that these peptides share much of the binding specificity of the intact targets of the phosphorylated form of CheY. We also have used modern nuclear magnetic resonance methods to characterize the sites of interaction of these peptides on CheY. We have found that the binding sites are overlapping and primarily consist of residues in the C-terminal portion of CheY. Both peptides affect the resonances of residues at the active site, indicating that the peptides may either bind directly at the active site or exert conformational influences that reach to the active site. The binding sites for the CheZ and FliM peptides also overlap with the previously characterized CheA binding interface. These results suggest that interaction with these three proteins of the signal transduction pathway are mutually exclusive. In addition, since these three proteins are sensitive to the phosphorylation state of CheY, it may be that the C-terminal region of CheY is most sensitive for the conformational changes occurring upon phosphorylation.     

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.     

A mechanical role for the chemotaxis system in swarming motility

The chemotaxis system, but not chemotaxis, is essential for swarming motility in Salmonella enterica serovar Typhimurium. Mutants in the chemotaxis pathway exhibit fewer and shorter flagella, downregulate class 3 or 'late' motility genes, and appear to be less hydrated when propagated on a surface. We show here that the output of the chemotaxis system, CheY approximately P, modulates motor bias during swarming as it does during chemotaxis, but for a distinctly different end. A constitutively active form of CheY was found to promote swarming in the absence of several upstream chemotaxis components. Two point mutations that suppressed the swarming defect of a cheY null mutation mapped to FliM, a protein in the motor switch complex with which CheY approximately P interacts. A common property of these suppressors was their increased frequency of motor reversal. These and other data suggest that the ability to switch motor direction is important for promoting optimal surface wetness. If the surface is sufficiently wet, exclusively clockwise or counterclockwise directions of motor rotation will support swarming, suggesting also that the bacteria can move on a surface with flagellar bundles of either handedness.     

A Molecular Mechanism of Bacterial Flagellar Motor Switching

The high-resolution structures of nearly all the proteins that comprise the bacterial flagellar motor switch complex have been solved; yet a clear picture of the switching mechanism has not emerged. Here, we used NMR to characterize the interaction modes and solution properties of a number of these proteins, including several soluble fragments of the flagellar motor proteins FliM and FliG, and the response-regulator CheY. We find that activated CheY, the switch signal, binds to a previously unidentified region of FliM, adjacent to the FliM-FliM interface. We also find that activated CheY and FliG bind with mutual exclusivity to this site on FliM, because their respective binding surfaces partially overlap. These data support a model of CheY-driven motor switching wherein the binding of activated CheY to FliM displaces the carboxy-terminal domain of FliG (FliG_C) from FliM, modulating the FliG_C-MotA interaction, and causing the motor to switch rotational sense as required for chemotaxis.     

Chemotactic response regulator mutant CheY95IV exhibits enhanced binding to the flagellar switch and phosphorylation-dependent constitutive signalling

CheY, a response regulator protein in bacterial chemotaxis, mediates swimming behaviour through interaction with the flagellar switch protein, FliM. In its active, phosphorylated state, CheY binds to the motor switch complex and induces a change from counterclockwise (CCW) to clockwise (CW) flagellar rotation. The conformation of a conserved aromatic residue, tyrosine 106, has been proposed to play an important role in this signalling process. Here, we show that an isoleucine to valine substitution in CheY at position 95 — in close proximity to residue 106 — results in an extremely CW, hyperactive phenotype that is dependent on phosphorylation. Further biochemical characterization of this mutant protein revealed phosphorylation and dephosphorylation rates that were indistinguishable from those of wild-type CheY. CheY95IV, however, exhibited an increased binding affinity to FliM. Taken together, these results show for the first time a correlation between enhanced switch binding and constitutive signalling in bacterial chemotaxis. Considering present structural information, we also propose possible models for the role of residue 95 in the mechanism of CheY signal transduction.     

Interactions of the chemotaxis signal protein CheY with bacterial flagellar motors visualized by evanescent wave microscopy

The chemotaxis signal protein CheY of enteric bacteria shuttles between transmembrane methyl-accepting chemotaxis protein (MCP) receptor complexes and flagellar basal bodies [1]. The basal body C-rings, composed of the FliM, FliG and FliN proteins, form the rotor of the flagellar motor [2]. Phosphorylated CheY binds to isolated FliM [3] and may also interact with FliG [4], but its binding to basal bodies has not been measured. Using the chemorepellent acetate to phosphorylate and acetylate CheY [5], we have measured the covalent-modification-dependent binding of a green fluorescent protein-CheY fusion (GFP-CheY) to motor assemblies in bacteria lacking MCP complexes by evanescent wave microscopy [6]. At acetate concentrations that cause solely clockwise rotation, GFP-CheY molecules bound to native basal bodies or to overproduced rotor complexes with a stoichiometry comparable to the number of C-ring subunits. GFP-CheY did not bind to rotors lacking FIiM/FliN, showing that these subunits are essential for the association. This assay provides a new means of monitoring protein-protein interactions in signal transduction pathways in living cells.     

Tyrosine 106 of CheY plays an important role in chemotaxis signal transduction in Escherichia coli.

CheY is the response regulator in the signal transduction pathway of bacterial chemotaxis. Position 106 of CheY is occupied by a conserved aromatic residue (tyrosine or phenylalanine) in the response regulator superfamily. A number of substitutions at position 106 have been made and characterized by both behavioral and biochemical studies. On the basis of the behavioral studies, the phenotypes of the mutants at position 106 can be divided into three categories: (i) hyperactivity, with a tyrosine-to-tryptophan mutation (Y106W) causing increased tumble signaling but impairing chemotaxis; (ii) low-level activity, with a tyrosine-to-phenylalanine change (Y106F) resulting in decreased tumble signaling and chemotaxis; and (iii) no activity, with substitutions such as Y106L, Y106I, Y106V, Y106G, and Y106C resulting in no chemotaxis and a smooth-swimming phenotype. All three types of mutants can be phosphorylated by CheA-phosphate in vitro to a level similar to that of wild-type CheY. Autodephosphorylation rates are similar for all categories of mutants. All mutant proteins displayed less than twofold increased rates compared with wild-type CheY. Binding of the mutant proteins to FliM was similar to that of the wild-type CheY in the CheY-FliM binding assays. The combined results from in vivo behavioral and in vitro biochemical studies suggest that the diverse phenotypes of the Y106 mutants are not due to a variation in phosphorylation or dephosphorylation ability nor in affinity for the switch. With reference to the structures of wild-type CheY and the T871 CheY mutant, our results suggest that rearrangements of the orientation of the tyrosine side chain at position 106 are involved in the signal transduction of CheY. These data also suggest that the binding of phosphoryl-CheY to the flagellar motor is a necessary, but not sufficient, event for signal transduction.     

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.     

Identification of a site of ATP requirement for signal processing in bacterial chemotaxis.

In Escherichia coli and Salmonella typhimurium, ATP is required for chemotaxis and for a normal probability of clockwise rotation of the flagellar motors, in addition to the requirement for S-adenosylmethionine (J. Shioi, R. J. Galloway, M. Niwano, R. E. Chinnock, and B. L. Taylor, J. Biol. Chem. 257:7969-7975, 1982). The site of the ATP requirement was investigated. The times required for S. typhimurium ST23 (hisF) to adapt to a step increase in serine, phenol, or benzoate were similar in cells depleted of ATP and in cells with normal levels of ATP. This established that ATP was not required for the chemotactic signal to cross the inner membrane or for adaptation to the transmembrane signal to occur. Depletion of ATP did not affect the probability of clockwise rotation in E. coli cheYZ scy strains that were defective in the cheY and cheZ genes and had a partially compensating mutation in the motor switch. Strain HCB326 (cheAWRBYZ tar tap tsr trg::Tn10), which was deficient in all chemotaxis components except the switch and motor, was transformed with the pCK63 plasmid (ptac-cheY+). Induction of cheY in the transformant increased the frequency of clockwise rotation, but except at the highest levels of CheY overproduction, clockwise rotation was abolished by depleting ATP. It is proposed that the CheY protein is normally in an inactive form and that ATP is required for formation of an active CheY* protein that binds to the switch on the flagellar motors and initiates clockwise rotation. Depletion of ATP partially inhibits feedback regulation of the cheB product, protein methylesterase, but this may reflect a second site of ATP action in chemotaxis.     

In situ structure of the Caulobacter crescentus flagellar motor and visualization of binding of a CheY-homolog

Bacterial flagellar motility is controlled by the binding of CheY proteins to the cytoplasmic switch complex of the flagellar motor, resulting in changes in swimming speed or direction. Despite its importance for motor function, structural information about the interaction between effector proteins and the motor are scarce. To address this gap in knowledge, we used electron cryotomography and subtomogram averaging to visualize such interactions inside Caulobacter crescentus cells. In C. crescentus, several CheY homologs regulate motor function for different aspects of the bacterial lifestyle. We used subtomogram averaging to image binding of the CheY family protein CleD to the cytoplasmic Cring switch complex, the control center of the flagellar motor. This unambiguously confirmed the orientation of the motor switch protein FliM and the binding of a member of the CheY protein family to the outside rim of the C ring. We also uncovered previously unknown structural elaborations of the alphaproteobacterial flagellar motor, including two novel periplasmic ring structures, and the stator ring harboring eleven stator units, adding to our growing catalog of bacterial flagellar diversity.     

Helicobacter pylori CheZHP and ChePep form a novel chemotaxis‐regulatory complex distinct from the core chemotaxis signaling proteins and the flagellar motor

Chemotaxis is important for Helicobacter pylori to colonize the stomach. Like other bacteria, H. pylori uses chemoreceptors and conserved chemotaxis proteins to phosphorylate the flagellar rotational response regulator, CheY, and modulate the flagellar rotational direction. Phosphorylated CheY is returned to its non‐phosphorylated state by phosphatases such as CheZ. In previously studied cases, chemotaxis phosphatases localize to the cellular poles by interactions with either the CheA chemotaxis kinase or flagellar motor proteins. We report here that the H. pylori CheZ, CheZ_HP, localizes to the poles independently of the flagellar motor, CheA, and all typical chemotaxis proteins. Instead, CheZ_HP localization depends on the chemotaxis regulatory protein ChePep, and reciprocally, ChePep requires CheZ_HP for its polar localization. We furthermore show that these proteins interact directly. Functional domain mapping of CheZ_HP determined the polar localization motif lies within the central domain of the protein and that the protein has regions outside of the active site that participate in chemotaxis. Our results suggest that CheZ_HP and ChePep form a distinct complex. These results therefore suggest the intriguing idea that some phosphatases localize independently of the other chemotaxis and motility proteins, possibly to confer unique regulation on these proteins' activities.     

Fundamental Constraints on the Abundances of Chemotaxis Proteins

Flagellated bacteria, such as Escherichia coli, perform directed motion in gradients of concentration of attractants and repellents in a process called chemotaxis. The E. coli chemotaxis signaling pathway is a model for signal transduction, but it has unique features. We demonstrate that the need for fast signaling necessitates high abundances of the proteins involved in this pathway. We show that further constraints on the abundances of chemotaxis proteins arise from the requirements of self-assembly both of flagellar motors and of chemoreceptor arrays. All these constraints are specific to chemotaxis, and published data confirm that chemotaxis proteins tend to be more highly expressed than their homologs in other pathways. Employing a chemotaxis pathway model, we show that the gain of the pathway at the level of the response regulator CheY increases with overall chemotaxis protein abundances. This may explain why, at least in one E. coli strain, the abundance of all chemotaxis proteins is higher in media with lower nutrient content. We also demonstrate that the E. coli chemotaxis pathway is particularly robust to abundance variations of the motor protein FliM.     

Acetyladenylate or its derivative acetylates the chemotaxis protein CheY in vitro and increases its activity at the flagellar switch.

CheY, a key protein in the mechanism of bacterial chemotaxis, is known to interact with the flagellar switch and thereby cause clockwise rotation. This activity of CheY was significantly increased by producing acetyladenylate (AcAMP) within cytoplasm-free bacterial envelopes containing purified CheY. This was achieved by including in the envelopes the enzyme acetyl-CoA synthetase (ACS) and ATP, and adding acetate externally. The fraction of clockwise-rotating envelopes, tethered to glass by their flagella, increased from 14% to 58% by the presence of AcAMP (or its derivative). In parallel experiments carried out with [14C]acetate under similar conditions, CheY became acetylated: [1-14C]acetate was as effective as [2-14C]acetate in labeling CheY, and ACS-dependent labeling of CheY by [alpha-32P]ATP was not detected. The switch proteins, FliG, FliM, and FliN, isolated to purity, were not acetylated. The acetylation was specific for CheY and dependent on its native conformation. The acetylated form the CheY was estimated to be more active than its nonacetylated form by 4-5 orders of magnitude. Acetylated CheY was stable in the presence of the strong nucleophiles hydroxylamine or ethanolamine, indicative of N-acetylation. There was a correlation between the activity of CheY in vivo and its ability to be acetylated in vitro. Thus, proteins with a single substitution at their active site, CheY57DE and CheY109KR, are not active in vivo and accordingly were not acetylated in vitro; in contrast, the protein CheY13DK is active in vivo and was normally acetylated in vitro. The possibility that CheY acetylation plays a role in bacterial chemotaxis is discussed.     

Binding of the chemotaxis response regulator CheY to the isolated,intact switch complex of the bacterial flagellar motor: lack of cooperativity

In bacteria, the chemotactic signal is greatly amplified between the chemotaxis receptors and the flagellar motor. In Escherichia coli, part of this amplification occurs at the flagellar switch. However, it is not known whether the amplification results from cooperativity of CheY binding to the switch or from a post-binding step. To address this question, we purified the intact switch complex (constituting the switch proteins FliG, FliM, and FliN and the scaffolding protein FliF) in quantities sufficient for biochemical work and used it to investigate whether the binding of CheY to the switch complex is cooperative. As a negative control, we used complexes of switchless basal bodies, formed from the proteins FliF and FliG and similarly isolated. Using double-labeling centrifugation assays for binding, we found that CheY binds to the isolated, intact switch complex in a phosphorylation-dependent manner. We observed no significant phosphorylation-dependent binding to the negative control of the switchless basal body. The dissociation constant for the binding between the switch complex and phosphorylated CheY (CheY approximately P) was 4.0 +/- 1.1 microm, well in line with the published range of CheY approximately P concentrations to which the flagellar motor is responsive. Furthermore, the binding was not cooperative (Hill coefficient approximately 1). This lack of CheY approximately P-switch complex binding cooperativity, taken together with earlier in vivo studies suggesting that the dependence of the rotational state of the motor on the fraction of occupied sites at the switch is sigmoidal and very steep (Bren, A., and Eisenbach, M. (2001) J. Mol. Biol. 312, 699-709), indicates that the chemotactic signal is amplified within the switch, subsequent to the CheY approximately P binding.     

Genetic and behavioral analysis of flagellar switch mutants of Salmonella typhimurium.

At the interface between the sensory transduction system and the flagellar motor system of Salmonella typhimurium, the switch complex plays an important role in both sensory transduction and energy transduction. To examine the function of the switch complex, we isolated from 10 cheY mutants 500 pseudorevertants with a suppressor mutation in one of the three genes (fliG, fliM, and fliN) encoding the switch complex. Detailed mapping revealed that these suppressor mutations were localized to several segments of each switch gene, suggesting localization of functional sites on the switch complex. These switch mutations were introduced into the wild-type background and into a chemotaxis deletion background. Behavior of the pseudorevertants and their derivatives (1,500 strains in all) was observed by light microscopy. In the chemotaxis deletion background, about 70% of the switch mutants showed smooth swimming and the rest showed more or less tumbly swimming. There was some correlation between the mutational sites and the swimming patterns in the chemotaxis deletion background, suggesting that there is segregation of functional sites on the switch complex. The interaction of the switch complex with the chemotaxis protein, CheY, and the stochastic nature of switching in the absence of CheY are discussed.     

Different evolutionary constraints on chemotaxis proteins CheW and CheY revealed by heterologous expression studies and protein sequence analysis

CheW and CheY are single-domain proteins from a signal transduction pathway that transmits information from transmembrane receptors to flagellar motors in bacterial chemotaxis. In various bacterial and archaeal species, the cheW and cheY genes are usually encoded within homologous chemotaxis operons. We examined evolutionary changes in these two proteins from distantly related proteobacterial species, Escherichia coli and Azospirillum brasilense. We analyzed the functions of divergent CheW and CheY proteins from A. brasilense by heterologous expression in E. coli wild-type and mutant strains. Both proteins were able to specifically inhibit chemotaxis of a wild-type E. coli strain; however, only CheW from A. brasilense was able to restore signal transduction in a corresponding mutant of E. coli. Detailed protein sequence analysis of CheW and CheY homologs from the two species revealed substantial differences in the types of amino acid substitutions in the two proteins. Multiple, but conservative, substitutions were found in CheW homologs. No severe mismatches were found between the CheW homologs in positions that are known to be structurally or functionally important. Substitutions in CheY homologs were found to be less conservative and occurred in positions that are critical for interactions with other components of the signal transduction pathway. Our findings suggest that proteins from the same cellular pathway encoded by genes from the same operon have different evolutionary constraints on their structures that reflect differences in their functions.     

Deletion analysis of the FliM flagellar switch protein of Salmonella typhimurium.

The flagellar switch of Salmonella typhimurium and Escherichia coli is composed of three proteins, FliG, FliM, and FliN. The switch complex modulates the direction of flagellar motor rotation in response to information about the environment received through the chemotaxis signal transduction pathway. In particular, chemotaxis protein CheY is believed to bind to switch protein FliM, inducing clockwise filament rotation and tumbling. To investigate the function of FliM and its interactions with FliG and FliN, we engineered a series of 34 FliM deletion mutant proteins, each lacking a different 10-amino-acid segment. We have determined the phenotype associated with each mutant protein, the ability of each mutant protein to interfere with the motility of wild-type cells, and the effect of additional FliG and FliN on the function of selected FliM mutant proteins. Overall, deletions at the N terminus produced a counterclockwise switch bias, deletions in the central region of the protein produced poorly motile or nonflagellate cells, and deletions near the C terminus produced only nonflagellate cells. On the basis of this evidence and the results of a previous study of spontaneous FliM mutants (H. Sockett, S. Yamaguchi, M. Kihara, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 174:793-806, 1992), we propose a division of the FliM protein into four functional regions: an N-terminal region primarily involved in switching, an extended N-terminal region involved in switching and assembly, a middle region involved in switching and motor rotation, and a C-terminal region primarily involved in flagellar assembly.     

Phosphorylation-independent bacterial chemoresponses correlate with changes in the cytoplasmic level of fumarate.

Bacterial chemotaxis is based on modulation of the probability to switch the direction of flagellar rotation. Responses to many stimuli are transduced by a two-component system via reversible phosphorylation of CheY, a small cytoplasmic protein that directly interacts with the switch complex at the flagellar motor. We found that the chemorepellents indole and benzoate induce motor switching in Escherichia coli cells with a disabled phosphorylation cascade. This phosphorylation-independent chemoresponse is explained by reversible inhibition of fumarase by indole or benzoate which leads to an increased level of cellular fumarate, a compound involved in motor switching for bacteria and archaea. Genetic deletion of fumarase increased the intracellular concentration of fumarate and enhanced the switching frequency of the flagellar motors irrespective of the presence or absence of the phosphorylation cascade. These correlations provide evidence for fumarate-dependent metabolic signal transduction in bacterial chemosensing.     

Only one of the five CheY homologs in Vibrio cholerae directly switches flagellar rotation

Vibrio cholerae has three sets of chemotaxis (Che) proteins, including three histidine kinases (CheA) and four response regulators (CheY) that are encoded by three che gene clusters. We deleted the cheY genes individually or in combination and found that only the cheY3 deletion impaired chemotaxis, reinforcing the previous conclusion that che cluster II is involved in chemotaxis. However, this does not exclude the involvement of the other clusters in chemotaxis. In other bacteria, phospho-CheY binds directly to the flagellar motor to modulate its rotation, and CheY overexpression, even without CheA, causes extremely biased swimming behavior. We reasoned that a V. cholerae CheY homolog, if it directly controls flagellar rotation, should also induce extreme swimming behavior when overproduced. This was the case for CheY3 (che cluster II). However, no other CheY homolog, including the putative CheY (CheY0) protein encoded outside the che clusters, affected swimming, demonstrating that these CheY homologs cannot act directly on the flagellar motor. CheY4 very slightly enhanced the spreading of an Escherichia coli cheZ mutant in semisolid agar, raising the possibility that it can affect chemotaxis by removing a phosphoryl group from CheY3. We also found that V. cholerae CheY3 and E. coli CheY are only partially exchangeable. Mutagenic analyses suggested that this may come from coevolution of the interacting pair of proteins, CheY and the motor protein FliM. Taken together, it is likely that the principal roles of che clusters I and III as well as cheY0 are to control functions other than chemotaxis.     

Proposed Signal Transduction Role for Conserved CheY Residue Thr87, a Member of the Response Regulator Active-Site Quintet

CheY serves as a structural prototype for the response regulator proteins of two-component regulatory systems. Functional roles have previously been defined for four of the five highly conserved residues that form the response regulator active site, the exception being the hydroxy amino acid which corresponds to Thr87 in CheY. To investigate the contribution of Thr87 to signaling, we characterized, genetically and biochemically, several cheY mutants with amino acid substitutions at this position. The hydroxyl group appears to be necessary for effective chemotaxis, as a Thr→Ser substitution was the only one of six tested which retained a Che⁺ swarm phenotype. Although nonchemotactic, cheY mutants with amino acid substitutions T87A and T87C could generate clockwise flagellar rotation either in the absence of CheZ, a protein that stimulates dephosphorylation of CheY, or when paired with a second site-activating mutation, Asp13→Lys, demonstrating that a hydroxy amino acid at position 87 is not essential for activation of the flagellar switch. All purified mutant proteins examined phosphorylated efficiently from the CheA kinase in vitro but were impaired in autodephosphorylation. Thus, the mutant CheY proteins are phosphorylated to a greater degree than wild-type CheY yet support less clockwise flagellar rotation. The data imply that Thr87 is important for generating and/or stabilizing the phosphorylation-induced conformational change in CheY. Furthermore, the various position 87 substitutions differentially affected several properties of the mutant proteins. The chemotaxis and autodephosphorylation defects were tightly linked, suggesting common structural elements, whereas the effects on self-catalyzed and CheZ-mediated dephosphorylation of CheY were uncorrelated, suggesting different structural requirements for the two dephosphorylation reactions.