Binding of the Chemotaxis Response Regulator CheY to the Isolated, Intact Switch Complex of the Bacterial Flagellar Motor
2003; Elsevier BV; Volume: 278; Issue: 28 Linguagem: Inglês
10.1074/jbc.m303201200
ISSN1083-351X
AutoresYael Sagi, Shahid Khan, Michael Eisenbach,
Tópico(s)Molecular Communication and Nanonetworks
ResumoIn 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∼P) was 4.0 ± 1.1 μm, well in line with the published range of CheY∼P concentrations to which the flagellar motor is responsive. Furthermore, the binding was not cooperative (Hill coefficient ≈ 1). This lack of CheY∼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∼P binding. 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∼P) was 4.0 ± 1.1 μm, well in line with the published range of CheY∼P concentrations to which the flagellar motor is responsive. Furthermore, the binding was not cooperative (Hill coefficient ≈ 1). This lack of CheY∼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∼P binding. The motility of bacteria such as Escherichia coli and Salmonella enterica serovar Typhimurium derives from the rotation of their flagella (for reviews, see Refs. 1Macnab R.M. Neidhardt F.C. Curtis III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 123-145Google Scholar and 2Eisenbach M. Mol. Microbiol. 1990; 4: 161-167Crossref PubMed Scopus (25) Google Scholar). Each flagellum is driven by a bidirectional rotary motor embedded in the cytoplasmic membrane. The direction of flagellar rotation determines the swimming behavior of the cells, enabling bacteria to approach beneficial environments and escape hostile ones. Thus, the essence of bacterial chemotaxis is modulation of the direction of flagellar rotation (3Larsen S.H. Reader R.W. Kort E.N. Tso W.-W. Adler J. Nature. 1974; 249: 74-77Crossref PubMed Scopus (275) Google Scholar). This modulation is carried out by a switch that responds to signals received from the chemotaxis receptors (for reviews, see Refs. 4Macnab R.M. Hoch J.A. Silhavy T.J. Two-component Signal Transduction. American Society for Microbiology, Washington D. C.1995: 181-199Google Scholar, 5Eisenbach M. Caplan S.R. Curr. Biol. 1998; 8: R444-R446Abstract Full Text Full Text PDF PubMed Google Scholar, 6Silversmith R.E. Bourret R.B. Trends Microbiol. 1999; 7: 16-22Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The switch extends from the base of the flagellar motor into the cytoplasm. It is composed of three proteins, FliG (∼35 molecules per switch (7Zhao R.B. Amsler C.D. Matsumura P. Khan S. J. Bacteriol. 1996; 178: 258-265Crossref PubMed Google Scholar, 8Thomas D.R. Morgan D.G. DeRosier D.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10134-10139Crossref PubMed Scopus (116) Google Scholar)), FliM (∼35 molecules per switch (7Zhao R.B. Amsler C.D. Matsumura P. Khan S. J. Bacteriol. 1996; 178: 258-265Crossref PubMed Google Scholar, 8Thomas D.R. Morgan D.G. DeRosier D.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10134-10139Crossref PubMed Scopus (116) Google Scholar)), and FliN (∼100 molecules per switch (9Zhao R.H. Pathak N. Jaffe H. Reese T.S. Khan S. J. Mol. Biol. 1996; 261: 195-208Crossref PubMed Scopus (90) Google Scholar)), and is mounted on the MS ring of the flagellar motor, formed from the protein FliF (10Ueno T. Oosawa K. Aizawa S.-I. J. Mol. Biol. 1992; 227: 672-677Crossref PubMed Scopus (134) Google Scholar, 11Oosawa K. Ueno T. Aizawa S. J. Bacteriol. 1994; 176: 3683-3691Crossref PubMed Google Scholar, 12Kubori T. Yamaguchi S. Aizawa S.-I. J. Bacteriol. 1997; 179: 813-817Crossref PubMed Google Scholar). The signals from the chemotaxis receptors to the switch are transduced by the response regulator CheY. The activity of this protein is modulated by phosphorylation. When phosphorylated in response to chemotactic stimulation, the protein is detached from the histidine kinase CheA, which is a part of the receptor supramolecular complex, and acquires an elevated affinity for the switch protein FliM (13Welch M. Oosawa K. Aizawa S.-I. Eisenbach M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8787-8791Crossref PubMed Scopus (349) Google Scholar, 14Schuster S.C. Swanson R.V. Alex L.A. Bourret R.B. Simon M.I. Nature. 1993; 365: 343-347Crossref PubMed Scopus (229) Google Scholar, 15Li J.Y. Swanson R.V. Simon M.I. Weis R.M. Biochemistry. 1995; 34: 14626-14636Crossref PubMed Scopus (127) Google Scholar, 16McEvoy M. Bren A. Eisenbach M. Dahlquist F.W. J. Mol. Biol. 1999; 289: 1423-1433Crossref PubMed Scopus (88) Google Scholar). The outcome is an increased probability of shifting the direction of flagellar rotation from the default direction, counterclockwise, to clockwise (for recent reviews, see Refs. 17Bren A. Eisenbach M. J. Bacteriol. 2000; 182: 6865-6873Crossref PubMed Scopus (253) Google Scholar and 18Bourret R.B. Stock A.M. J. Biol. Chem. 2002; 277: 9625-9628Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Bacteria such as E. coli and Salmonella sense stimuli over a wide concentration range and, despite the wide range, do so with very high sensitivity, suggesting high amplification of the chemotactic signal (19Segall J.E. Block S.M. Berg H.C. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8987-8991Crossref PubMed Scopus (378) Google Scholar, 20Jasuja R. Yu-Lin T rentham D.R. Khan S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11346-11351Crossref PubMed Scopus (46) Google Scholar). One of the major questions in bacterial chemotaxis is which of the signal transduction steps amplify the chemotactic signal. One amplification step likely occurs at the receptor level, the amplification being provided by propagation of the excitation signal from the stimulated receptor molecule to neighboring receptor molecules within the receptor cluster (21Bray D. Levin M.D. Morton-Firth C.J. Nature. 1998; 393: 85-88Crossref PubMed Scopus (518) Google Scholar, 22Duke T.A.J. Bray D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10104-10108Crossref PubMed Scopus (230) Google Scholar, 23Ames P. Studdert C.A. Reiser R.H. Parkinson J.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7060-7065Crossref PubMed Scopus (280) Google Scholar, 24Kim S.-H. Wang W.R. Kim K.K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11611-11615Crossref PubMed Scopus (128) Google Scholar; for a review, see Ref. 17Bren A. Eisenbach M. J. Bacteriol. 2000; 182: 6865-6873Crossref PubMed Scopus (253) Google Scholar). In view of recent findings that switching from counterclockwise to clockwise upon an increase in the intracellular level of CheY∼P 1The abbreviations used are: CheY∼P, phosphorylated CheY; Ni-NTA, nickel-nitrilotriacetic acid.1The abbreviations used are: CheY∼P, phosphorylated CheY; Ni-NTA, nickel-nitrilotriacetic acid. is highly cooperative (a Hill coefficient of ∼10 (25Cluzel P. Surette M. Leibler S. Science. 2000; 287: 1652-1655Crossref PubMed Scopus (471) Google Scholar)), another amplification step probably occurs at the switch level (for a review, see Ref. 26Bray D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7-9Crossref PubMed Scopus (74) Google Scholar). However, it is not known whether this high amplification reflects cooperativity of CheY∼P binding to FliM (4Macnab R.M. Hoch J.A. Silhavy T.J. Two-component Signal Transduction. American Society for Microbiology, Washington D. C.1995: 181-199Google Scholar, 25Cluzel P. Surette M. Leibler S. Science. 2000; 287: 1652-1655Crossref PubMed Scopus (471) Google Scholar, 27Kuo S.C. Koshland D.E. J. Bacteriol. 1989; 171: 6279-6287Crossref PubMed Google Scholar, 28Spiro P.A. Parkinson J.S. Othmer H.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7263-7268Crossref PubMed Scopus (139) Google Scholar) or amplification of post-binding events at the switch (28Spiro P.A. Parkinson J.S. Othmer H.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7263-7268Crossref PubMed Scopus (139) Google Scholar). This question cannot be addressed by direct in vitro assays of CheY binding to purified FliM because it is not known whether purified FliM in solution truly represents the native protein within the switch complex and because the inter-FliM interactions, which normally occur within the switch (29Marykwas D.L. Schmidt S.A. Berg H.C. J. Mol. Biol. 1996; 256: 564-576Crossref PubMed Scopus (75) Google Scholar), probably do not occur with soluble FliM molecules. Recently, Lux et al. (30Lux R. Kar N. Khan S. J. Mol. Biol. 2000; 298: 577-583Crossref PubMed Scopus (18) Google Scholar) overexpressed, in a non-flagellated strain, all three switch proteins together with FliF and purified the resulting membrane-associated structure. These structures have now been identified on the basis of immunochemical (30Lux R. Kar N. Khan S. J. Mol. Biol. 2000; 298: 577-583Crossref PubMed Scopus (18) Google Scholar), morphological (31Young H.S. Dang H. Lai Y. DeRosier D.J. Khan S. Biophys. J. 2003; 84: 571-577Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), and in vivo CheY-binding criteria (32Khan S. Pierce D. Vale R.D. Curr. Biol. 2000; 10: 927-930Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) as intact functional switch complexes mounted on the transmembrane MS ring. These advances open up the possibility of detailed mechanistic analysis of the cooperative motor response upon binding CheY. Here, we have used this preparation to characterize the phosphorylation-dependent binding of CheY to the intact switch complex in vitro. Preparation of Switch Complex—For the preparation of switch complexes, we overproduced FliG and FliF from the overproducing plasmid pKOT107 and overproduced FliM and FliN from the overproducing plasmid pKLR2 in the nonflagellated E. coli strain BL21 (λDE3) (33Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (5998) Google Scholar). The overproduced switch complex, found at the cytoplasmic membrane, was purified according to the procedure of Francis et al. (34Francis N.R. Sosinsky G.E. Thomas D. DeRosier D.J. J. Mol. Biol. 1994; 235: 1261-1270Crossref PubMed Scopus (282) Google Scholar) for isolating extended basal bodies with several modifications. Cells (21 liters) of strain BL21 (λDE3) containing the plasmids pKOT107 and pKLR2 were grown in Luria broth supplemented with ampicillin (100 μg/ml) and chloramphenicol (30 μg/ml). The overproduction of the proteins was induced at OD600 = 0.4 by isopropyl-1-thio-β-d-galactopyranoside (1 mm). The cells were grown for an additional 3 h and harvested by centrifugation. The pellet was resuspended in 250 ml of ice-cold sucrose solution (0.5 m sucrose, 0.1 m Tris-HCl, pH 7.9), and then lysozyme (final concentration, 1 mg/ml, from a freshly prepared stock solution) and EDTA (final concentration, 10 mm) were added. The mixture was gently stirred at 4 °C for about 11 h. The resulting spheroplasts were lysed by two-step addition of Triton X-100: first to a final concentration of 1% in the presence of MgSO4 (10 mm); then, following gentle stirring at 4 °C for 3 h, to a final concentration of 2% in the presence of MgSO4 (10 mm). The suspension was further gently stirred at 4 °C for 0.5 h. EDTA (20 mm) was added, and the suspension was stirred for 60 min. Unlysed cells and inclusion bodies were removed by centrifugation at 23,000 × g for 30 min, and the pH of the supernatant was raised with NaOH to pH 10 to disintegrate the outer membrane structures. The lysate was spun at 123,000 × g for 60 min, and the pellet was resuspended in a solution containing Tris-HCl (10 mm, pH 7.9), KCl (100 mm), sucrose (10% w/v), and Triton X-100 (0.1%). The suspension was recentrifuged at 23,000 × g for 30 min, and then the supernatant was spun at 200,000 × g for 40 min. The pellet was resuspended in a solution containing Tris-HCl (50 mm, pH 7.9) and Triton X-100 (0.1%). To get rid of residual inclusion bodies that potentially remained in the suspension, the suspension was centrifuged again at 23,000 × g for 10 min. The purified switch complex was stored at 4 °C with Complete protease inhibitor (Roche Applied Science). Preparation of Switchless Basal Body—For the preparation of a switchless basal body, we used the same procedure used for the preparation of the switch complex, with the exception that the cells contained only one plasmid, the plasmid pKOT107 for the overproduction of FliG and FliF alone. Labeling of CheY—Initially we radiolabeled CheY in vitro by methylating the ∈-amine of its lysine residues and the N terminus with formaldehyde and NaB[3H]4, as described previously (35Blat Y. Eisenbach M. J. Biol. Chem. 1996; 271: 1232-1236Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The experiments shown in Fig. 2 were carried out with CheY labeled in this way. However, subsequently we found that high levels of labeling (higher than those used in Fig. 2) inhibited the ability of CheY to undergo in vitro phosphorylation by the phosphodonor acetyl phosphate. We, therefore, switched to in vivo labeling, which does not involve chemical modification of CheY. E. coli strain M15 (36Zamenhof P.J. Villarejo M. J. Bacteriol. 1972; 110: 171-178Crossref PubMed Google Scholar), carrying the plasmid pQE12-CheY-His-tag for the overproduction of His6-CheY (received from A. Wolfe, Loyola University), was grown to OD590 = 0.5 in H1 minimal medium of Kaiser and Hogness (37Kaiser A.D. Hogness D.S. J. Mol. Biol. 1960; 2: 392-415Crossref PubMed Scopus (171) Google Scholar) supplemented with histidine, methionine, and threonine (1 mm each), as well as with leucine (200 μm), thiamin (5 μg/ml), ampicillin (100 μg/ml), and glucose (0.3%, w/v). The overproduction of CheY was induced at OD590 = 0.5 by isopropyl-1-thio-β-d-galactopyranoside (0.5 mm). For labeling, isopropyl-1-thio-β-d-galactopyranoside was supplemented with [14C]leucine (final concentration, 0.17 μCi/ml). The cells were grown for an additional 4 h, harvested by centrifugation, and sonicated. Non-soluble material (inclusion bodies and cell debris) was removed by centrifugation at 123,000 × g for 30 min. The His6-tagged CheY protein was purified from the supernatant by Ni-NTA affinity chromatography (Qiagen) according to the manufacturer's instructions, with some modifications. We followed protocol 11 (batch purification of His6-tagged proteins under native conditions) with the exception that, following the collection of the flow-through, the washing of the lysate-Ni-NTA column was with 80–90 ml of washing buffer (until no protein was eluted) rather than 4 ml, and the elution of CheY from the Ni-NTA column was with 20–30 ml of elution buffer instead of 2 ml. We concentrated CheY by ultrafiltration through a 10 kDa cut-off membrane in an Amicon chamber (model 52). CheY was stored at –80 °C. We verified that the His6-tag at the C terminus of CheY did not interfere with the function of the protein according to two criteria: (a) we expressed His6-tagged CheY in the non-chemotactic strain AW546 eda + cheY201 (received from J. Adler) and found that His6-tagged CheY restored chemotactic responsiveness to this strain, judged by its ability to form typical chemotactic rings on a semisolid agar (0.3% agar) plate containing Tryptone (38Adler J. Science. 1966; 153: 708-716Crossref PubMed Scopus (818) Google Scholar); and (b) we measured the ability of His6-tagged CheY to undergo acetyl phosphate-mediated in vitro phosphorylation by measuring the changes in the fluorescence of Trp58, known to be strongly reduced upon CheY phosphorylation (39Lukat G.S. McCleary W.R. Stock A.M. Stock J.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 718-722Crossref PubMed Scopus (409) Google Scholar). The fluorescence of CheY was determined using a fluorimeter with excitation and emission wavelengths set at 295 and 345 nm, respectively. Tag-free CheY or His6-tagged CheY was diluted to a final concentration of 10 μm into 50 mm Tris-HCl buffer (pH 7.9) containing 5 mm MgSO4, and its fluorescence was recorded. Subsequently, acetyl phosphate (final concentration, 20 mm) was added, and the fluorescence was again recorded. Both His6-tagged CheY and tag-free CheY were similarly quenched (by ∼60%) upon the addition of acetyl phosphate. Binding of CheY to the Switch Complex and to the Switchless Basal Body—The switch complex or the switchless basal body, at the amount indicated in the figure legends, was incubated with [3H]CheY (2000–7000 dpm/pmol) and [14C]glucose (final concentration, 3500 dpm/μl) or [14C]CheY (His6-tagged; 13–17 dpm/pmol) and [3H]glycine (600 dpm/pmol) in a solution of Tris-HCl (50 mm; pH 7.9), MgSO4 (5 mm), and where indicated, bovine serum albumin (10 mg/ml). For phosphorylating conditions, acetyl phosphate (25 mm) was added to the reaction mixture. After a 5-min incubation at room temperature (24 °C), the switch complex or the switchless basal body was pelleted by centrifugation at 200,000 × g for 10 min at 4 °C. The pellet was resuspended in 100 μl of Tris-HCl (50 mm, pH 7.9). Aliquots of the reaction mixture prior to centrifugation and of the supernatant and pellet subsequent to centrifugation were counted for 14C and 3H by a β counter (Tri-Carb liquid scintillation analyzers). Isolation of the Switch Complex for Biochemical Studies—To produce an intact switch complex in quantities sufficient for biochemical work, we overproduced the three switch proteins (FliG, FliM, and FliN) along with FliF from two plasmids as done by Lux et al. (30Lux R. Kar N. Khan S. J. Mol. Biol. 2000; 298: 577-583Crossref PubMed Scopus (18) Google Scholar) and then purified the overproduced switch complex from the cytoplasmic membrane as described under "Experimental Procedures." In electron micrographs, the purified switch complex (Fig. 1) appeared similar to that obtained by Lux et al. (30Lux R. Kar N. Khan S. J. Mol. Biol. 2000; 298: 577-583Crossref PubMed Scopus (18) Google Scholar). As a negative control, we used the host BL21 cells, lacking the overproducing plasmids. These cells, following the same isolation procedure, did not yield the structures shown in Fig. 1. Demonstration of Specific CheY∼P Binding to the Switch Complex—To measure the binding between CheY and the purified switch complex, we equilibrated the complex with [3H]CheY and then separated switch-bound CheY from free CheY by centrifugation. To be able to measure the volume of medium entrapped within the pellet, we also included in the medium an inert radiolabeled compound, [14C]glucose. The 3H/14C ratio in the pellet was higher in the presence of the phosphodonor acetyl phosphate than in its absence (Fig. 2A), suggesting the occurrence of phosphorylation-dependent binding of CheY to the switch complex. Because CheY binds to FliM, we used, as a negative control, a switchless basal body. We isolated it from cells overexpressing FliF and FliG only, using the same isolation procedure as for the intact switch complex. Unlike the case of the intact switch complex, we did not observe a phosphorylation-dependent increase in CheY binding to the switchless basal body (Fig. 2A). These results suggest that the phosphorylation-dependent binding to the intact switch complex is specific and that the isolated switch complex is functional with respect to CheY binding. A potential source of concern in the above experiments was that traces of FliM-containing inclusion bodies might have remained in the preparation of the switch complex and might have contributed to the observed binding of CheY∼P. We verified that this was not the case by the following measures: (a) we did not observe a white fluffy precipitate, typical of inclusion bodies, on top of the precipitate of the switch complex; (b) before precipitating the switch complex at 200,000 × g, we got rid of FliM-containing inclusion bodies by spinning the preparation at 23,000 × g, which is above the force required for their full precipitation (12,500 × g (40Zhao R.B. Schuster S.C. Khan S. J. Mol. Biol. 1995; 251: 400-412Crossref PubMed Scopus (35) Google Scholar)); and (c) we carried out control experiments in which we equilibrated the switch complex with [3H]CheY and then separated switch-bound CheY from free CheY by centrifugation either at 23,000 × g or at 200,000 × g. We observed only a phosphorylation-dependent increase in the 3H/14C ratio in the pellet at 200,000 × g (Fig. 2B), suggesting that the preparation of isolated switch complex did not contain inclusion bodies and that the observed binding of CheY∼P was to the switch complex alone. Quantification of the CheY-Switch Binding—To quantify the binding between CheY and the switch complex, we titrated the complex with increasing amounts of radiolabeled CheY under phosphorylating and nonphosphorylating conditions (presence and absence of acetyl phosphate, respectively). As a control for nonspecific binding, we used the switchless basal body at equal total-protein concentrations. Under nonphosphorylating conditions, CheY bound to the switch complex only slightly better than to the negative control of switchless basal body (Fig. 3). Under phosphorylating conditions, CheY selectively bound to the switch complex. To quantify the binding, we had to estimate the total amount of FliM within the switch complex. We did this by two independent means: according to the number of CheY molecules bound to the switch complex at saturation and according to a Western blot analysis of the switch complex with anti-FliM antibodies (Fig. 4). Both methods yielded a similar estimate of the FliM amount, 0.95 and 1.1 nmol FliM/mg of total protein, respectively. These values were measured with the same preparation of switch complex used for the results shown in Figs. 3 and 4. The average ratio between these estimations for three different preparations of the switch complex was 0.9 ± 0.2 (mean ± S.D.), suggesting that the stoichiometry between CheY and FliM is 1:1. Quantitative analysis of the data of this representative experiment revealed a dissociation constant of 3.6 and 120 μm for phosphorylated and nonphosphorylated CheY, respectively (Fig. 3, insets). Correcting the CheY∼P concentration in the experiment according to the estimate that CheY is 85.5% phosphorylated in vitro under these conditions (41Blat Y. Gillespie B. Bren A. Dahlquist F.W. Eisenbach M. J. Mol. Biol. 1998; 284: 1191-1199Crossref PubMed Scopus (44) Google Scholar), we calculated that the dissociation constant for the CheY∼P-switch complex is 2.9 μm. A Hill fit to the binding data yielded a Hill coefficient of 1.0 (r 2 = 0.99), suggesting that, within the uncertainties of the experimental error, the binding of CheY∼P to the isolated switch complex is not cooperative. This apparent lack of cooperativity was observed in each of the three batches of isolated switch complex assayed for cooperativity (average Hill coefficient = 1.3 ± 0.2 (± S.D.)). The dissociation constant varied in the range of 2.9–5.6 μm between different batches of the isolated switch complex, yielding a mean of 4 ± 1 μm (± S.D.).Fig. 4FliM quantification by Western blots. Aliquots of purified switch complex and of purified FliM (15 ng/μl) were mixed with sodium dodecyl sulfate sample buffer, boiled for 10 min, resolved on sodium dodecyl sulfate-12% (w/v) polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes as described previously (46Bren A. Eisenbach M. J. Mol. Biol. 2001; 312: 699-709Crossref PubMed Scopus (25) Google Scholar). The blots were probed with anti-FliM antibody (a gift from S. I. Aizawa) and peroxidase-conjugated anti-rabbit antibody (Sigma). Films were scanned with an imaging densitometer (model GS-690; Bio-Rad). The intensities of the bands were quantified by Multi-Analyst software (Bio-Rad). Calculation of the amount of FliM in each preparation of the purified switch complex was made on the basis of a calibration curve prepared according to the band intensities of increasing concentrations of FliM.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In this study we isolated, in quantities sufficient for biochemical work, the flagellar switch complex of E. coli and demonstrated that it is functional, at least with respect to CheY binding. We found that this binding (K d = 4 ± 1 μm) is phosphorylation-dependent, is specific, has a CheY:FliM stoichiometry of 1:1, and is not cooperative. The significance of these findings is discussed below. Earlier in vivo studies found a steep dependence of the probability of clockwise rotation on the intracellular concentration of active CheY (25Cluzel P. Surette M. Leibler S. Science. 2000; 287: 1652-1655Crossref PubMed Scopus (471) Google Scholar, 27Kuo S.C. Koshland D.E. J. Bacteriol. 1989; 171: 6279-6287Crossref PubMed Google Scholar, 42Scharf B.E. Fahrner K.A. Turner L. Berg H.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 201-206Crossref PubMed Scopus (164) Google Scholar, 43Alon U. Camarena L. Surette M.G. Arcas B.A.Y. Liu Y. Leibler S. Stock J.B. EMBO J. 1998; 17: 4238-4248Crossref PubMed Scopus (187) Google Scholar). Carrying out the measurements in single cells, Cluzel et al. (25Cluzel P. Surette M. Leibler S. Science. 2000; 287: 1652-1655Crossref PubMed Scopus (471) Google Scholar) found that the increase was over a narrow range of CheY∼P concentrations and highly cooperative (Hill coefficient ≈ 10). These observations suggested that the switch acts as some sort of amplifier, translating small changes in the concentration of CheY∼P into large changes in clockwise probability. However, these observations could not distinguish between amplification caused by cooperativity of CheY binding to the switch (4Macnab R.M. Hoch J.A. Silhavy T.J. Two-component Signal Transduction. American Society for Microbiology, Washington D. C.1995: 181-199Google Scholar, 25Cluzel P. Surette M. Leibler S. Science. 2000; 287: 1652-1655Crossref PubMed Scopus (471) Google Scholar, 27Kuo S.C. Koshland D.E. J. Bacteriol. 1989; 171: 6279-6287Crossref PubMed Google Scholar, 28Spiro P.A. Parkinson J.S. Othmer H.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7263-7268Crossref PubMed Scopus (139) Google Scholar) and amplification evolved within the switch (28Spiro P.A. Parkinson J.S. Othmer H.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7263-7268Crossref PubMed Scopus (139) Google Scholar). Earlier studies, which used in vitro binding assays with purified soluble proteins (or peptides thereof), could not address this question either (13Welch M. Oosawa K. Aizawa S.-I. Eisenbach M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8787-8791Crossref PubMed Scopus (349) Google Scholar, 16McEvoy M. Bren A. Eisenbach M. Dahlquist F.W. J. Mol. Biol. 1999; 289: 1423-1433Crossref PubMed Scopus (88) Google Scholar, 44Welch M. Oosawa K. Aizawa S.-I. Eisenbach M. Biochemistry. 1994; 33: 10470-10476Crossref PubMed Scopus (79) Google Scholar, 45Bren A. Eisenbach M. J. Mol. Biol. 1998; 278: 507-514Crossref PubMed Scopus (109) Google Scholar). A distinction between both possibilities, although indirect, was later made by Bren and Eisenbach (46Bren A. Eisenbach M. J. Mol. Biol. 2001; 312: 699-709Crossref PubMed Scopus (25) Google Scholar), who used FliM proteins almost locked in their clockwise or counterclockwise states as representatives of CheY-bound and CheY-free FliM, respectively. By expressing these FliM proteins to different levels, they found that the dependence of the clockwise probability on the relative level of FliM in the clockwise state is very steep. Because that study bypassed the CheY∼P-FliM binding step, it suggested that at least part of the signal amplification occurs at a post-binding step. In the current study, we found no cooperativity of CheY∼P binding to the switch, endorsing the notion that the chemotactic signal is amplified within the switch rather than at the preceding binding step of CheY∼P to the switch. While preparing this manuscript for publication, a study by Sourjik and Berg (47Sourjik V. Berg H.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12669-12674Crossref PubMed Scopus (207) Google Scholar) was published in which resonance energy transfer was used for in vivo measurements of CheY binding to the switch. Sourjik and Berg (47Sourjik V. Berg H.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12669-12674Crossref PubMed Scopus (207) Google Scholar) found essentially no cooperativity of binding and a dissociation constant of ∼3.7 μm, very similar to the value measured by us in this study. The similarity between the in vivo results of Sourjik and Berg (47Sourjik V. Berg H.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12669-12674Crossref PubMed Scopus (207) Google Scholar) and the in vitro results with purified intact switch complex obtained in our current study suggests that the isolated switch complex is functional and reliably represents the function of the switch within the cell. Furthermore, our measured dissociation constant for CheY∼P (4 ± 1 μm) is well in line with the range of CheY∼P concentrations over which the motor changes its bias from counterclockwise to clockwise (2–6 μm) (25Cluzel P. Surette M. Leibler S. Science. 2000; 287: 1652-1655Crossref PubMed Scopus (471) Google Scholar). As might be expected, it is lower than the dissociation constant measured for the binding of an N terminus-containing peptide of FliM (45Bren A. Eisenbach M. J. Mol. Biol. 1998; 278: 507-514Crossref PubMed Scopus (109) Google Scholar) to CheY∼P (∼27 μm) (16McEvoy M. Bren A. Eisenbach M. Dahlquist F.W. J. Mol. Biol. 1999; 289: 1423-1433Crossref PubMed Scopus (88) Google Scholar). The current study, taken together with the studies of Sourjik and Berg (47Sourjik V. Berg H.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12669-12674Crossref PubMed Scopus (207) Google Scholar), Cluzel et al. (25Cluzel P. Surette M. Leibler S. Science. 2000; 287: 1652-1655Crossref PubMed Scopus (471) Google Scholar), and Bren and Eisenbach (46Bren A. Eisenbach M. J. Mol. Biol. 2001; 312: 699-709Crossref PubMed Scopus (25) Google Scholar), strongly argues that the chemotactic signal is amplified within the switch, subsequent to the CheY∼P binding. Such amplification suggests that the switching process can be described as an allosteric transition between the rotational states of the switch, with CheY∼P as an allosteric activator. A model for an allosteric transition of switching was recently proposed by Duke et al. (48Duke T.A.J. Le Novère N. Bray D. J. Mol. Biol. 2001; 308: 541-553Crossref PubMed Scopus (185) Google Scholar). We thank Dr. S. R. Caplan for insightful discussions and critical reading of the manuscript.
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