INTRODUCTION

Bacterial chemotaxis is a response to environmental changes in which cells swim toward chemical attractants and away from repellents (1). Extracellular stimuli are sensed by transmembrane receptors (2, 3). Signals initiated at the receptors are transduced through phosphotransfer reactions that regulate the extent of phosphorylation of CheY (3-7), the signal molecule of bacterial chemotaxis. Phosphorylated CheY (CheY-P)¹ interacts with the motor switch complex (8-11) to effect a reversal of flagellar rotation from counterclockwise to clockwise (12-14). Counterclockwise rotation causes a cell to swim in a straight path, whereas clockwise rotation causes cells to tumble and randomly change their direction of travel.

The concentration of CheY-P determines whether cells tumble or swim smoothly. The level of CheY-P in vivo is controlled by the autophosphorylation and phosphotransfer activities of the CheA kinase (15, 16) and by the dephosphorylation rate of CheY-P. The dephosphorylation rate is controlled by the intrinsic autophosphatase activity of CheY (15, 16) and by CheZ activity (15, 17). Although CheY-P is capable of autodephosphorylation, this reaction is relatively slow compared with the response time of the chemotaxis system (18). The dephosphorylation reaction is markedly accelerated by CheZ (15, 17, 19). The importance of CheZ activity is indicated by the extremely tumbly motility and loss of normal chemotactic ability of cheZ null mutants (20-23).

The interaction between CheZ and CheY has been investigated by several research groups (19, 24-30). The binding of CheZ to CheY-P is greater than its binding to apo-CheY (24, 30). The carboxyl-terminal domain in CheZ has been identified as the CheY-binding domain (27). The CheZ-binding region of CheY has not been completely elucidated, although two mutant CheY proteins with reduced ability to bind CheZ have been reported (29, 30). In this study, we characterize a number of mutant CheY proteins as substrates for CheZ. Nine mutant CheY proteins were more resistant to dephosphorylation by CheZ than was wild-type CheY. These nine mutant CheY proteins fell into two categories based on their affinity for CheZ: 1) mutant CheY proteins with reduced sensitivity but normal binding activity and 2) mutant CheY proteins with both reduced sensitivity and altered activity for CheZ.

MATERIALS AND METHODS

Bacterial strains and plasmids are listed in Table I. pRBB40Z(T87S) was a kind gift from Jeryl Appleby and Robert Bourret. pYM10, containing the mutation A88T, was derived from pRL22Z, in which pRL22Z was mutagenized in vitro with hydroxylamine and screened for Che (nonchemotactic) phenotypes in cheY mutant strain RP4079. pXYZM17W and pXYZV86M, containing the cheY mutations M17W and V86M, respectively, were constructed by the "megaprimer" polymerase chain reaction procedure as described (31). Primers containing the desired base changes were obtained from Operon Technologies, Inc. (Alameda, CA). pRL22Z DNA was used as a template. A HindIII-PvuII polymerase chain reaction fragment containing the cheY mutations was subcloned back to the HindIII and PvuII sites of pRL22Z by standard techniques (32). All of the mutations were confirmed by DNA sequencing.

Table I. Bacterial strains and plasmids Strain/plasmid Relevant genotype and description Source/Ref. Strains   RP437 Wild type (Che+) J. S. Parkinson   RP4079 cheY216 recA J. S. Parkinson   RP5135  tar-cheZ J. S. Parkinson   SG1 trpR(am)supDts cheY::Kanr; temperature-sensitive Lab collection   XYZ7 recD1903, cheYT871 Lab collection   XYZ9 recD1903, cheYT871/Y106W Lab collection Plasmids   pT7-7M penr, FliM expression Lab collection   pRL22 CheY, CheZ expression Lab collection   pRL22Z cheZ deletion, wild-type CheY expression Lab collection   pYM3 cheYE93K in pRL22Z Lab collection   pYM10 cheyYA88T in pRL22Z This study   pYM31 cheYT871 in PRL22Z Lab collection   pXYZ20 cheYY106W in pRL22Z Lab collection   pXYZ301 cheYT871/Y106W in pRL22Z Lab collection   pXYZM17W cheYM17W in pRL22Z This study   pXYZV86M cheYV86M in pRL22Z This study   pRBB40ZT87S cheYT87S in pRBB40Z J. Appleby and R. B. Bourret   pRYB0902Z cheYT112I in pRL22 Lab collection   pRYB0903Z cheYA90V in pRL22 Lab collection   pRYB0904Z cheYE117K in pRL22 Lab collection   pRYB0906Z cheYV108M in pRL22 Lab collection   pRYB1610Z cheYF111V in pRL22 Lab collection   pRYB2885Z cheYE27K in pRL22 Lab collection   pFZY low-copy penr vector Lab collection   pMM1 tar operon in pFZY Lab collection   pYB0902 cheYT112I in pMM1 Lab collection   pYB0903 cheYA90V in pMM1 Lab collection   pYB0904 cheYE117Kin pMM1 Lab collection   pYB1610 cheYF111V in pMM1 Lab collection   pYB2885 cheYE27K in pMM1 Lab collection

Bacterial chemotactic ability was assayed on motility plates (1% Tryptone, 0.5% NaCl, and 0.3% agar) as described (33). Cells were grown in Tryptone broth at 30 °C to early post-exponential phase for cell tethering assays as described (33).

The wild-type and mutant CheY proteins were overexpressed by temperature-shift induction in strain SG1 (devoid of wild-type cheY) and purified as described previously (34, 35). The purified mutant proteins were concentrated to ~1.4 mg/ml with a Centriplus concentrator (Amicon, Inc.). The cheZ gene of plasmid pRL22 was induced in strain RP5135 for CheZ purification. CheZ was purified as described by Wang and Matsumura (36).

Phosphotransfer reactions from CheA to CheY were carried out as described (37, 38). The stability of phosphorylated CheY was analyzed as described previously (38, 39). Briefly, the autophosphorylating CheA kinase was coupled to Sepharose beads and phosphorylated with [-³²P]ATP. CheY was then added to the CheA beads to allow the phosphoryl group transfer reaction to occur. The phosphorylated CheA reaction mixture contained 3 µl of 10 × phosphorylation buffer, 3 µl of [-³²P]ATP, and 0.5 µl of unlabeled ATP (15 mM) in a total volume of 30 µl, with ~1 µg of CheA/µl of bead. The reaction was carried out at room temperature by rotating the beads for 30 min and was stopped by washing the beads with excess phosphorylation buffer. The phosphorylated CheA beads were stored on ice. 100 µl of purified CheY (1 µmol in 50 mM Tris, pH 7.5) was added to the beads attached to phosphorylated CheA, and phosphate transfer was allowed to progress for 30 s while rotating at 10-12 °C. Phosphorylated CheY was removed with a Hamilton syringe and immediately transferred at 10-12 °C into a buffer containing 0.2 mM Mg²⁺, 50 mM Tris, pH 7.5, and different amounts of CheZ. Samples of phosphorylated CheY were removed at various times, and the autodephosphorylation reaction or the CheZ-enhanced dephosphorylation reaction was quenched with 2 × SDS-PAGE sample buffer. Samples were loaded directly onto gels for SDS-PAGE (15% polyacrylamide). Radiolabeled proteins were visualized by autoradiography, and the radioactivity of the protein bands was determined with an AMBIS -scanning system.

Acetyl [³²P]phosphate was synthesized according to Welch et al. (10). CheY phosphorylation by acetyl [³²P]phosphate was performed as previously described (38). Identical amounts of wild-type or mutant CheY proteins were mixed with 20 mM acetyl [³²P]phosphate in a buffer containing 5 mM MgCl₂, 2 mM dithiothreitol, and 50 mM Tris-HCl, pH 7.9, in a total volume of 20 µl. The reactions were allowed to stand at room temperature (22 °C) for various times (1-10 min) before being quenched by the addition of 2 × SDS-PAGE loading buffer. 10 µl of reaction products was analyzed on 15% SDS-polyacrylamide gels. Labeled proteins were visualized by autoradiography.

The assays for binding of CheY to CheZ were carried out as described previously (26), with some modifications. CheY beads were suspended to homogeneity in 50 mM Tris-HCl, pH 7.9, and dispensed in aliquots of 50 µl in microcentrifuge tubes (~3 nmol of CheY in each aliquot). 100 µl of reaction buffer (50 mM Tris and 5 mM MgCl₂) and 35 µl of stabilizer buffer (3.4 M glycerol and and 12.5 mM MgCl₂) were added. Acetyl phosphate was added from a 1 M stock solution to 20 mM, as needed, and the phosphorylation reactions proceeded for 2 min at room temperature. Then, 3 nmol of purified CheZ was added to each tube. The final volume of each reaction mixture was adjusted to 200 µl with 50 mM Tris-HCl. The reactions were incubated at room temperature for 10 min. The beads were washed twice with 1 ml of 50 mM cold Tris buffer containing 5 mM MgCl₂ with acetyl phosphate added to the appropriate wash buffer. 60 µl of 2 × SDS-PAGE loading buffer was added, and the suspension was mixed at room temperature for 5 min. The beads were boiled for 2 min to remove the bound CheZ from the CheY beads. 10 µl of supernatant was loaded onto gels for 15% SDS-PAGE. Gels were stained with Coomassie Brilliant Blue. CheZ bands were scanned by Sigma Gel Gel Analysis software. Bovine serum albumin beads served as controls.

RESULTS

The phosphorylation/dephosphorylation of CheY consists of a phosphotransfer from phospho-CheA and either the intrinsic autophosphatase activity of CheY or CheZ-enhanced dephosphorylation. To understand the effect of CheZ on the CheY dephosphorylation reaction, we screened a number of mutant CheY proteins for their ability to be dephosphorylated by CheZ. The mutant CheY proteins in this study were selected by one or more of four criteria. 1) They contain residue substitutions close to the phosphorylation site of Asp⁵⁷ (V86M, T87I, T87S, T87I/Y106W, and A88T). 2) They contain residue substitutions located close to those in previously described CheZ-binding mutants (29, 30). 3) They had previously been shown to have altered CheA binding (D93K, A90V, Y106W, V108M, F111V, and T112I) (35). 4) They had been identified as suppressors of mutations affecting the flagellar switch (A90V, V108M, F111V, T112I, E117K, and E27K) (9). To test the dephosphorylation rates of mutant CheY proteins, we first needed to know whether the mutant CheY proteins could be phosphorylated by CheA. Fourteen mutant CheY proteins were characterized by in vitro phosphotransfer from CheA (37, 38). All 14 mutant CheY proteins were found to be phosphorylated by CheA in vitro. Thirteen mutant CheY proteins had CheY-P levels similar to that of wild-type CheY under our conditions. The A88T mutant CheY protein had 40% phosphorylation activity relative to wild-type CheY (data not shown).

The dephosphorylation rates of these mutant CheY proteins in the presence of CheZ were assayed using purified CheY-P (36, 40). Different amounts of CheZ were added, and the dephosphorylation rates were measured. Examples of the results obtained are presented in Fig. 1, and Table II summarizes the results for all of the CheY proteins assayed. Nine of the phosphorylated mutant CheY proteins were 5- to >1000-fold more resistant to CheZ activity than was wild-type CheY-P. The others had sensitivity to CheZ similar to that of wild-type CheY. The autodephosphorylation rates of the nine mutant CheY proteins exhibiting decreased rates were also assayed using purified CheY-P in the absence of CheZ. Two mutant CheY proteins (T87I and T87I/Y106W) had 5-fold lower autodephosphorylation rates than did wild-type CheY. The other seven mutant CheY proteins had normal dephosphorylation.

Table II. Sensitivity of CheY-P to enhanced dephosphorylation in the presence of CheZ (wild type versus mutants) Mutant Half-lifea of CheY-P normalized to WTb CheZ required to reduce half-lifea of CheY-P by one-half pmol WT CheY 1 1 M17W 2 2 E27K 1 50 V86M 1.5-2 2 T87S 1.5-2 0.5-1 T87I 5 >1000 T87I/Y106W 5 >1000 A88T 1 >50 A90V 1 5 E93K 1 5 Y106W 2 1-2 V108M 1 2 F111V 1 100 T112I 1 >50 E117K 1 50 a The half-lives of CheY-P calculated as ln 2/slope. b WT, wild type.

One explanation for the phenotypes of these cheY mutations is that they disrupt CheY-CheZ interaction. To explore this possibility, the binding activity of wild-type and mutant CheY proteins with CheZ was measured. CheZ binds to the phosphorylated form of CheY with higher affinity than it does to the unphosphorylated form of CheY (24, 26, 30). Therefore, binding of apo-CheY and CheY-P to CheZ proteins was measured. Mutant CheY proteins were phosphorylated with excess acetyl [³²P]phosphate. Most mutant CheY proteins were phosphorylated to the same extent as wild-type CheY (data not shown); however, proteins with the T87I and T87I/Y106W substitutions were not phosphorylated by acetyl [³²P]phosphate, and the A88T protein could be phosphorylated to only 40% of the wild type.

As shown in Fig. 2, among seven CheZ-resistant mutant CheY proteins assayed for CheZ binding, four (E27K, F111V, T112I, and E117K) showed at least 50% reduction in CheZ binding, whereas three other mutant CheY proteins (A88T, A90V, and E93K) had a similar affinity for CheZ compared with wild-type CheY. The combined results from the dephosphorylation and CheY-CheZ binding assays indicate that there are two classes of CheY mutants that affect CheZ activity. One class is resistant to CheZ activity, but does not affect CheZ binding. The other decreases CheZ activity by reducing CheZ binding. We were not able to test CheZ binding with the phosphorylated T87I and T87I/Y106W proteins since they could not be phosphorylated by acetyl phosphate.

The black residues in Fig. 3 depict the positions on CheY where CheY residue substitutions reduce CheZ binding. Sites at which substitutions conferring CheZ resistance allow wild-type levels of CheZ binding are shown in gray. These latter residues are located near the active site of CheY, which is identified by the label for Asp⁵⁷ (D57). The residues associated with the two phenotypes cluster in distinctly different regions, suggesting that there is a CheZ-binding face consisting of the solvent-accessible surfaces of the ₁-helix (N23D, K26E, and E27K), part of the ₅-helix (E117K), and the ₅-₅ loop (F111V and T112I). These residues are located on the surface of CheY. The gray residues are in the ₄-₄ loop (A88T and A90V) and at the top of the ₄-helix (E93K), suggesting that this region is involved in CheZ catalytic activity.

A Structural Shift in the ₄-₄ (90's) Loop Affects CheY Autophosphatase Activity and Sensitivity to CheZ

Of these 14 mutant CheY proteins, four structures have been solved: T87I (39), T87I/Y106W (40), Y106W (40), and T87S.² The overall structure of all four mutant CheY proteins is the same as that of wild-type CheY. Both the T87I (39) and T87I/Y106W (40) proteins showed distinct backbone conformational changes in the 90's loop (41). This shift was directly attributable to the substitution of isoleucine for threonine at position 87 since no such backbone changes were found in the Y106W (40) or T87S ² protein (Fig. 4). The 90's loop consists of residues 88-92 and is near (7-14 Å) the Asp⁵⁷ phosphorylation site of CheY. The T87I and T87I/Y106W mutant proteins were completely resistant to CheZ activity and had five times lower autodephosphorylation rates than wild-type CheY (Fig. 1 and Table II). Furthermore, they were not able to be phosphorylated by acetyl phosphate, although they can be phosphorylated by CheA. On the other hand, the Y106W and T87S proteins, which lack the shift in the 90's loop, are not resistant to CheZ and have only slightly altered autodephosphorylation rates compared with wild-type CheY. Both of these proteins could be phosphorylated by either CheA or acetyl phosphate. These results indicate that the shift in the 90's loop of CheY is highly correlated with changes in CheY autodephosphorylation and enhanced dephosphorylation by CheZ.

The effects of these CheZ-resistant mutants on chemotaxis were analyzed in vivo using motility plates, the cell tethering assay, and direct microscopic observation of the liquid bacterial culture. Mutant cheY alleles, except those encoding the A88T and E93K proteins, were either subcloned into a low-copy-number plasmid (9) or introduced into the chromosome DNA (40) to ensure single-copy cheY expression. As shown in Table III, all these mutants, except A90V, were incapable of swarming on motility plates.

Table III. Behavioral and biochemical consequences of selected mutant CheY proteins CheY mutant Swarm Flagellar rotation CheA bindinga FliM bindingb CheZ binding Wild type Che+ CCW-CW Normal Normal Normal In 4-helix   E93K Che CCW Weak Normal Normal In 4-4 loop   T87I Che CCW Strong ND ND   A88T Che CCW ND Normal Normal   A90V Che CCW Weak Weak Normal In 5-sheet   V108M Che CCW Weak Weak Normal In 5-5 loop   F111V Che CCW Weak Weak Weak   T112I Che CCW Weak Weak Weak In 5-helix   E117K Che CCW Weak Weak Weak In 1-helix   E27K Che CCW Normal Weak Weak   K26Ed Che CW ND ND Weak   N23Dd Che CW ND ND Weak a CheY-CheA binding data are from Shukla, et al. (D. Shukla, X. Zhu, and P. Matsumura, in revision). b X. Zhu and P. Matsumura, unpublished data. c CCW, counterclockwise; CW, clockwise; ND, not determined. d Data for these two mutants are from Sanna et al. (29, 30).

The A90V mutant could swarm somewhat. All of the cheY mutant strains exhibited smooth-swimming behavior when observed under the microscope, and all showed counterclockwise-biased flagellar rotation in the cell tethering assay. These results were surprising because one would expect that the CheZ-resistant cheY mutants will cause the accumulation of CheY-P in vivo, resulting in a tumbly phenotype. Under "Discussion," we review the data for the interactions of CheY with CheA (35) and of CheY with FliM (9) that enable us to speculate why these mutant CheY proteins confer a smooth-swimming phenotype rather than a tumbly phenotype like the CheZ-resistant N23D and E26K CheY mutations described by Sanna et al. (29).

DISCUSSION

Nine CheZ-resistant cheY mutants were obtained in this study (Table II). Seven of these mutant CheY proteins were tested for their CheZ binding ability. They showed two different CheZ binding properties. Four mutant CheY proteins had reduced binding of CheZ (Fig. 2). The residues, altered in these proteins, cluster in the three-dimensional structure of CheY on the same face of CheY as the N23D and K26E CheY mutations that alter CheZ binding (29, 30). Fig. 3 indicates the positions of CheY altered residues that reduce CheZ binding. This surface consists of the ₁-helix (N23D, K26E, and E27K), the ₅-₅ loop (F111V and T112I), and part of the ₅-helix (E117K). The mutation M17W is also located in the ₁-helix, but it does not affect CheZ activity (Table II), and it has only slightly less CheZ binding ability than wild-type CheY (Fig. 2). Residue 17 is not on the solvent-accessible surface of CheY, and therefore, it is unlikely to interact directly with CheZ. CheY proteins with the substitutions A88T and A90V in the 90's loop and E93K in the ₄-helix next to the 90's loop bind CheZ almost as well as does wild-type CheY, but they are resistant to CheZ activity. This finding suggests that the 90's loop is involved in the catalytic activity of CheZ rather than in CheZ binding.

Unfortunately, we could not measure CheZ binding by the phosphorylated T87I and T87I/Y106W CheY proteins because of their inability to be phosphorylated by acetyl phosphate. Although both mutant CheY proteins can be phosphorylated by CheA, CheY-P from CheA phosphotransfer is not stable enough (with a half-life 40 s in our conditions) for use in the binding assay, which requires 10 min. Considering the structural shift that exists in the 90's loop of both these mutant CheY proteins and the fact that the T87S mutation does not change CheZ binding, we believe that the T87I and T87I/Y106W mutations affect CheZ activity in the same way as the mutations in the 90's loop. Our hypothesis for the mechanism of CheZ-enhanced CheY dephosphorylation is that CheZ binds to the CheZ-binding surface of CheY-P and that CheZ-induced changes in the conformation of the 90's loop lead to accelerated dephosphorylation.

The concentration of CheY-P in vivo determines the direction of the flagellar rotation, and CheZ helps control the cytoplasmic concentration of CheY-P. CheZ apparently forms oligomers upon interaction with CheY-P (25). This CheZ oligomerization is thought to be a mechanism for regulating CheZ activity. In our CheY-CheZ binding assay, in which CheZ was coupled to Sepharose beads, CheY did not exhibit increased binding upon phosphorylation (data not shown). This result could be viewed as further evidence that CheZ must be oligomerized to increase its affinity for CheY-P since the beads could prevent CheZ from oligomerizing. CheZ activity on CheY-P might also be regulated by a CheA_S-CheZ complex since this complex enhances dephosphorylation of CheY-P in vitro (36).

CheY is a single-domain protein that interacts with at least three other polypeptides: CheA (35, 42, 43), CheZ (24, 26, 30), and FliM (10, 11, 36). Previous studies (35) showed that the mutant CheY proteins A90V, E93K, Y106W, V108M, F111V, T112I, and E117K have altered CheA binding (Table III). These data suggest that the region containing ₄-₅-₅ is involved in CheY-CheA recognition. Similar results were reported in a two-dimensional NMR study of CheY-CheA interaction (44). Studies of cheY suppressors of flagellar switch mutants indicated that the CheY mutants V11M, E27K, A90V, V108M, F111V, T112I, and E117K are fliG suppressors (9), whereas E27K, A90V, V108M, F111V, T112I, and E117K are fliM suppressors³ (Table III). These data suggest that an area of CheY including the ₅-₅ loop and part of the ₁-helix might be involved in the interaction with the flagellar switch (Table III). The data reported here suggest that the CheZ-binding surface of CheY consists of the ₅-₅ loop, the top of the ₅-helix, and part of the ₁-helix (Fig. 3 and Table III). Therefore, the three proteins bind to surfaces of CheY that overlap but are not completely identical.

All the residues shown by genetic studies to be involved in interactions with CheA, CheZ, and FliM were mapped on the CheY surface (Fig. 5). The red residues (E93K and Y106W) represent residues that are specifically required for CheA binding. The pink residues (A90V and V108M) are involved in both CheA and FliM binding. The white residues (F111V, T112I, and E117K) indicate positions at which substitutions affect binding of all three proteins. The green residue (E27K) is involved in both FliM and CheZ binding. The blue residues (N23D and K26E) represent positions at which substitutions reduce CheZ binding (29, 30) but presumably allow normal binding of CheA and FliM since they must bind both CheA and the motor to generate a tumbly phenotype (29, 30). As shown in Fig. 5, the CheA-binding surface extends from the ₄- to ₅-helix of CheY; the CheZ-binding surface is mainly located on the ₁-helix; and the FliM surface overlaps both the CheA- and CheZ-binding surfaces. The Venn diagram depicts this pattern.

CheA binds to apo-CheY and dissociates from CheY-P when it is phosphorylated (43). In contrast, CheZ and FliM bind to CheY-P with a higher affinity than to apo-CheY (10, 11, 24, 30). It is possible that the overlap region in the unphosphorylated state contributes to the CheA-binding surface (35) and that CheY phosphorylation alters the topology of this region, causing the release of CheY from CheA and increasing the affinity of CheY for the switch proteins or CheZ. Our unpublished data⁴ show that when an equal molar solution of FliM and CheZ is allowed to bind to immobilized CheY, they bind with a 1:1 ratio. When this solution is allowed to bind to immobilized CheY-P, the overall binding is increased, but the ratio of FliM to CheZ remains 1:1. These results suggest that CheY-P has no preference between FliM and CheZ.

CheY is phosphorylated by CheA through a phosphotransfer reaction. It has been suggested that this reaction is catalyzed by CheY rather than CheA (45, 46). The claim that CheY possesses kinase activity is supported by the observation that small molecule phosphodonors such as acetyl phosphate can act in place of CheA-P to donate the phosphoryl group to CheY (47, 48). The T87I and T87I/Y106W mutant CheY proteins, which have a structural shift in their 90's loop, cause severe defects in autodephosphorylation and then are completely resistant to CheZ activity (Table II). Furthermore, they cannot be phosphorylated by acetyl phosphate, although they can be phosphorylated by CheA. These results suggest not only that the activity of CheZ on CheY might be through enhancement of CheY's own autodephosphorylation reaction, but also that the mechanism of CheY phosphorylation by acetyl phosphate may involve the reverse dephosphorylation reaction rather than the forward phosphorylation by CheA. Alternatively, the mutant CheY proteins may alter binding to acetyl phosphate.

CheY is phosphorylated by CheA, and CheY-P binds to the motor switch, resulting in tumbles. A CheZ-resistant mutant CheY protein dephosphorylates more slowly, resulting in an elevated level of CheY-P. An increased level of CheY-P should generate clockwise rotation of the flagella and a tumbly phenotype. Indeed, the N23D and K26E mutant cells are more tumbly than wild-type cells (29, 30). However, all nine of our CheZ-resistant CheY mutants have smooth-swimming phenotypes and counterclockwise-biased rotation of the flagella.

Any cheY mutation causing a defect in CheY phosphorylation would affect signal transduction. An example is a mutation at Asp⁵⁷, which cannot be phosphorylated by CheA and results in a smooth-swimming phenotype (49). One of our nine CheZ-resistant mutants (A88T) could be phosphorylated to only 40% of wild-type CheY levels. Any CheY mutation with a defect in binding to the switch will also block signal transfer. Five of our nine CheZ-resistant mutant CheY proteins (E27K, A90V, F111V, T112I, and E117K) show reduced binding to FliM. Furthermore, CheY phosphorylation and binding of CheY-P to the switch are necessary (but not sufficient) events in generating the tumble signal since a mutant CheY protein (Y106L) exhibits normal phosphorylation and dephosphorylation properties and normal binding to FliM, yet it fails to generate a tumble signal (38). It is possible that some of these CheZ-resistant mutant CheY proteins affect signal transduction at a step after CheY phosphorylation, like the Y106L mutant. The CheY mutants T87I and T87I/Y106W restrict the rotation of residue 106 (40), a limitation that may block signal propagation from CheY-P to the switch.