The switching mechanism of the bacterial rotary motor combines tight regulation with inherent flexibility
2021; Springer Nature; Volume: 40; Issue: 6 Linguagem: Inglês
10.15252/embj.2020104683
ISSN1460-2075
AutoresOshri Afanzar, Diana Di Paolo, Miriam Eisenstein, Kohava Levi, Anne Plochowietz, Achillefs N. Kapanidis, Richard M. Berry, Michael Eisenbach,
Tópico(s)Protist diversity and phylogeny
ResumoArticle23 February 2021Open Access Transparent process The switching mechanism of the bacterial rotary motor combines tight regulation with inherent flexibility Oshri Afanzar Oshri Afanzar orcid.org/0000-0001-9690-3180 Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Diana Di Paolo Diana Di Paolo Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, UK Search for more papers by this author Miriam Eisenstein Miriam Eisenstein Department of Chemical Research Support, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Kohava Levi Kohava Levi Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Anne Plochowietz Anne Plochowietz Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, UK Search for more papers by this author Achillefs N Kapanidis Achillefs N Kapanidis Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, UK Search for more papers by this author Richard Michael Berry Richard Michael Berry Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, UK Search for more papers by this author Michael Eisenbach Corresponding Author Michael Eisenbach [email protected] orcid.org/0000-0001-9048-4720 Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Oshri Afanzar Oshri Afanzar orcid.org/0000-0001-9690-3180 Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Diana Di Paolo Diana Di Paolo Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, UK Search for more papers by this author Miriam Eisenstein Miriam Eisenstein Department of Chemical Research Support, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Kohava Levi Kohava Levi Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Anne Plochowietz Anne Plochowietz Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, UK Search for more papers by this author Achillefs N Kapanidis Achillefs N Kapanidis Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, UK Search for more papers by this author Richard Michael Berry Richard Michael Berry Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, UK Search for more papers by this author Michael Eisenbach Corresponding Author Michael Eisenbach [email protected] orcid.org/0000-0001-9048-4720 Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Author Information Oshri Afanzar1,4, Diana Di Paolo2, Miriam Eisenstein3, Kohava Levi1, Anne Plochowietz2,5, Achillefs N Kapanidis2, Richard Michael Berry2 and Michael Eisenbach *,1 1Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel 2Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, UK 3Department of Chemical Research Support, The Weizmann Institute of Science, Rehovot, Israel 4Present address: Department of Chemical and Systems Biology, Stanford University, Stanford, CA, USA 5Present address: Hardware Research and Technology Laboratory, Palo Alto Research Center, Palo Alto, CA, USA *Corresponding author. Tel: +972 8 9343923; E-mail: [email protected] The EMBO Journal (2021)40:e104683https://doi.org/10.15252/embj.2020104683 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Regulatory switches are wide spread in many biological systems. Uniquely among them, the switch of the bacterial flagellar motor is not an on/off switch but rather controls the motor's direction of rotation in response to binding of the signaling protein CheY. Despite its extensive study, the molecular mechanism underlying this switch has remained largely unclear. Here, we resolved the functions of each of the three CheY-binding sites at the switch in E. coli, as well as their different dependencies on phosphorylation and acetylation of CheY. Based on this, we propose that CheY motor switching activity is potentiated upon binding to the first site. Binding of potentiated CheY to the second site produces unstable switching and at the same time enables CheY binding to the third site, an event that stabilizes the switched state. Thereby, this mechanism exemplifies a unique combination of tight motor regulation with inherent switching flexibility. SYNOPSIS Flagellated bacteria like E. coli change their behaviour from swimming to tumbling by altering the direction of flagellar motor rotation. Here, this directional change is shown to involve sequential interactions of the ligand, CheY, with its three binding sites at the motor, each with a different outcome. The motor employs one binding site to capture its ligand, phosphorylated CheY, and potentiate its motor switching activity. A second site binds potentiated CheY to bring the motor into an unstable switched state. This interaction with the second site exposes a third binding site. Binding of acetylated CheY to the third site stabilizes the motor in the clockwise-rotation state. Introduction Switches are vastly known throughout the field of biology, from transcription and expression of genes to controlling processes of signal transduction, cell fate and cell cycle, to mention a few (Cross et al, 2002; Laslo et al, 2006; Pomerening, 2008). Most of these switches turn processes on and off. An exception is the switch of the bacterial flagellar motor, which controls the motor's direction of rotation rather than an on/off process (Eisenbach & Caplan, 1998). This dissimilarity combined with this switch's unique properties—controlling a mechanical rather than a chemical process and being exceptionally ultrasensitive with respect to the switching signal (see below) (Cluzel et al, 2000), made it a challenging system of investigation. Indeed, in spite of decades of studies, the molecular mechanism underlying switching of the bacterial flagellar motor has remained obscure. Switching of the motor enables bacterial cells to navigate. In bacteria like Escherichia coli, each cell contains multiple flagellar motors. When they rotate counterclockwise, the cell swims in a rather straight line, termed a "run". When a considerable fraction of flagella switch from the default direction of rotation, counterclockwise, to clockwise, the cell preforms a chaotic-like turning motion, termed a "tumble" (Berg & Brown, 1972; Turner et al, 2000) (Fig 1A), as a result of which the subsequent run (when the rotation switches back to counterclockwise) is in a randomly new direction. Conversely, very brief switching of some of the motors to clockwise generates slight changes in swimming direction without randomization rather than tumbles (Turner et al, 2000), thus maintaining directional swimming persistence (Vladimirov et al, 2010; Saragosti et al, 2011). This behavior was proposed to markedly improve the performance of collective migration (Saragosti et al, 2011), implying an evolutionary advantage. Maintenance of directional persistence requires extremely short intervals of clockwise rotation. However, it is unclear how the motor is regulated to produce both long clockwise intervals for tumbling and short intervals for directional persistence. Figure 1. Two modes of CheY binding to the switch A. Run-and-tumble swimming modes in E. coli are regulated by CheY activation by phosphorylation or acetylation. The electron density map of the switch was produced by Thomas et al (2006) and downloaded from http://dx.doi.org/10.1093/nar/gkv1126. B. Scheme demonstrating the internalization of Atto647-labeled CheY(I95V) to single cells by electroporation. C. Demonstration of single-molecule tracking in a single cell. Cell outline is shown as an ellipse. Left, Imaged FliM-YPet fluorescence. White dots indicate the estimated motor locations. White circles illustrate a 75 nm radius around these locations. Right, Contour map of the normalized sum of probabilities of the localization events of CheY(I95V)-Atto647 (from blue to red is low to high; black is zero probability). White circles show the motors' locations from the left panel. D. Survival probabilities of unmodified CheY(I95V)-Atto647 at the switch (ΔcheA background; strain EW668). 63 cells were recorded with a total of 1,316 trajectories in which CheY was found to interact with FliM. Note the logarithmic scale of the ordinate. Black line, a bi-exponential fit; dashed line, a fit of the fast decline process (see text for details). E. As in (D) for acetylated CheY(I95V)-Atto647 (presence of acetate, 50 mM, pH 7.0; strain EW668). 56 cells were recorded with a total of 1,904 trajectories. F. As in (D) for phosphorylated CheY(I95V)-Atto647 (ΔcheZ background; strain EW669). 82 cells were recorded with a total of 2,414 trajectories. G. As in (D) for phosphorylated and acetylated CheY(I95V)-Atto647 (presence of acetate, 50 mM, pH 7.0; ΔcheZ background, strain EW669). 40 cells were recorded with a total of 2660 trajectories. H. Experimental scheme of the FRET experiment. The attractant serine lowers the phosphorylation level of CheY. As a result, CheY dissociates from the switch and the energy transfer from YPet (conjugated to FliM) to mCherry (conjugated to CheY) is reduced. I. CheY interacts with FliM∆N and this interaction is sensitive to chemotactic stimuli. Each curve is the mean of two FRET measurements of cells in response to an attractant stimulus (0.1 mM serine; in the case of FliMwt, a single measurement was performed with serine; another measurement of FliMwt with 1mM aspartate produced similar results). See Appendix Fig S1 for the details of FRET analysis. FRET ratio is the ratio of mCherry to YPet fluorescence. CheY-mCherry and mCherry concentrations were ~170 µM in the case of FliM∆N and ~15 µM in the case of FliMwt (Appendix Fig S2 for calibration of CheY concentration). Strains used: EW677 (FliMwt, red), EW659 (negative control of FliM∆N without CheY, purple), EW637 (FliM∆N, blue), and EW636 (FliM∆N ΔcheA, yellow). Download figure Download PowerPoint While the mechanism of switching is not resolved, much is known about the components of the switching machinery and the interactions between them. The switch of the flagellar motor is a large complex at the motor's base, consisting of multiple copies of the proteins FliM, FliN, and FliG (Fig 1A). Since chemotaxis of bacteria is achieved by modulating the direction of flagellar rotation, the main control target in chemotaxis is the switch. The switch shifts the direction of rotation from counterclockwise to clockwise in response to binding the signaling protein CheY, which shuttles back and forth between the chemotaxis receptor complex and the flagellar switch. Earlier studies of the corresponding author's group revealed that phosphorylated CheY (CheY~P) mainly binds to the switch at the N terminus of FliM (FliMN) (Welch et al, 1993; Bren & Eisenbach, 1998). Subsequently, Blair's group reported on two additional sites with weaker binding of CheY (termed hereafter "low-affinity sites"), one at FliN, to which the binding is FliMN-dependent and requires that CheY would be phosphorylated (Sarkar et al, 2010), and one at FliM at other location than FliMN (Mathews et al, 1998). On the basis of this evidence combined with mutational analysis and a structural model, this group further suggested that the interaction of CheY~P with FliMN serves to capture CheY~P and that switching to clockwise rotation involves the subsequent interaction of CheY~P with FliN (Sarkar et al, 2010). NMR analysis in Thermotoga maritima by Dahlquist's group identified the middle domain of FliM (FliMM) as a low-affinity binding site for CheY (Dyer et al, 2009). In view of this information, it is reasonable to assume that, also in E. coli, the other binding site is FliMM. (For simplicity, we will term hereafter this other site in E. coli FliMM even though its exact location is obscure.) It is not yet known how CheY binding to each of these sites affects the process of clockwise generation. CheY is bound to receptor clusters at the cell's poles. It is well established that it has to be activated by phosphorylation for switching the motor to clockwise. This activation results in CheY~P dissociation from the poles and, as mentioned above, in binding to FliMN. The level of CheY phosphorylation is regulated by CheA and CheZ as specific kinase and phosphatase, respectively (Fig 1A). A receptor-mediated attractant response (or removal of a repellent) inhibits CheA activity; stimulation by repellents (or attractant removal) enhances its activity [for reviews—(Berg, 2003; Eisenbach, 2004; Terashima et al, 2008; Porter et al, 2011)]. Another covalent modification that activates CheY to generate clockwise rotation is lysine acetylation (Wolfe et al, 1988; Barak et al, 1992, 1998). The regulation of acetylation is known to involve Acs and CobB as acetyl-transferase and deacetylase, respectively (Fig 1A) (Barak et al, 2004; Li et al, 2010). It has been shown that CheY acetylation is involved in bacterial chemotaxis (Barak & Eisenbach, 2001) and that it is inversely affected by CheA and CheZ (Barak & Eisenbach, 2004). Yet, the role that acetylated CheY (CheY~Ac) plays in chemotaxis is still obscure. While the dependence of clockwise generation on the intracellular concentration of active CheY is highly cooperative, meaning that the motor is ultrasensitive (Cluzel et al, 2000), binding assays between CheY and the switch, carried out both in vivo and in vitro, found that the binding is non-cooperative (Sourjik & Berg, 2002; Sagi et al, 2003). Subsequent studies, which employed a constitutively active CheY mutant protein, found that it binds better to clockwise-rotating motors than to counterclockwise-rotating motors (Fukuoka et al, 2014). This difference in binding may well result in cooperativity of binding (Duke et al, 2001). Here, we addressed the question of the mechanism underlying the switch function. We demonstrate that delicate, hitherto unknown, steps of the switching mechanism are resolved when FliMN is truncated from the switch. We bring evidence for three sequential steps of CheY binding to distinct sites at the switch, each with a different outcome. Binding to the first site (FliMN) potentiates CheY at the switch. Binding of potentiated CheY to the second site (FliN) is short-lived and generates transient motor switching. This seems to enable firm CheY binding to the third site (FliMM), with a resultant stabilization of the switched state. Results Two dwelling modes of CheY at the switch; phosphorylation affects one mode, acetylation affects both Following the findings of Fukuoka et al (2014), mentioned just above, that constitutively active CheY binds differently to counterclockwise- and clockwise-rotating motors, we investigated whether two modes of binding can also be observed with CheY activated by phosphorylation and acetylation. To this end, we measured in vivo the dwell time of single CheY molecules at motors whose FliM molecules were labeled with YPet. We compared between phosphorylating conditions, acetylating conditions, and conditions under which CheY was both phosphorylated and acetylated. We electroporated CheY(I95V) molecules labeled with a maleimide modification of the photo-stable organic dye Atto647 into FliM-YPet expressing cells (Fig 1B) (Di Paolo et al, 2016). The experiments were carried out in the following settings: in a ΔcheZ background to make CheY fully phosphorylated, in a ΔcheA background to make CheY non-phosphorylated, and, in each of these settings, also in the presence of the acetyl donor acetate to make CheY acetylated (Barak et al, 1992, 2004). The cheY(I95V) mutation was designed to increase CheY affinity for FliMN (Schuster et al, 2000), thus enhancing sampling of otherwise rare binding events. Custom-written software tracked CheY(I95V)-Atto647 molecules and estimated switch locations in each cell using FliM-YPet fluorescence images. We interpreted CheY(I95V)-Atto647 molecules dwelling within 75 nm of a switch for >30 ms as binding to the motor (Fig 1C; see Materials and Methods). We observed CheY(I95V)-Atto647 molecules dwelling at the motor both in the absence and presence of acetate. However, in the latter case, the dwell time was markedly longer (Fig 1D vs. E and Fig 1F vs. G for the survival probability, i.e., for the probability to remain bound to the switch; Movie EV1). Notably, the very long dwell events in Movie EV1 were only detected in the presence of acetate. The survival distributions in all experiments appeared to be biphasic, i.e., each of them comprised two exponentially decaying distributions, fast and slow (note the logarithmic scale of the ordinates in Fig 1D–G). A biphasic distribution is indicative of two modes of CheY dissociation from the switch, fast and slow. These two modes can either reflect CheY binding to two different sites at the switch or to two different states of the same site. To quantify the effect of phosphorylation and acetylation on each mode, we fitted each of the distributions in Fig 1D–G with a bi-exponential expression [ A 1 e - k 1 t + A 2 e - k 2 t , with pre-exponential factors A1 > A2, marking the fraction of each mode in the overall distribution, and k1, k2 being the rate constants of the modes; t is time]. From the fitted rate constants (and their inverse, the average dwell time of CheY at the switch), we could learn how phosphorylation, acetylation, or both modifications combined, affected each mode of CheY binding. Unmodified CheY exhibited the fastest decline rate in both modes (k1 and k2 being 99.5 and 9.7 s−1, and expected average dwell times being about 10 and 100 ms, respectively; Fig 1D). Phosphorylation markedly decreased the rate of the fast mode, but did not affect the slow mode (k1 and k2 being 45.3 and 11.1 s−1, and expected average dwell times being about 22 and 90 ms, respectively; Fig 1F). Acetylation alone decreased the decline rate of both modes (k1 and k2 being 32.8 and 5.6 s−1, and typical dwell times being about 30 and 180 ms, respectively; Fig 1E). Phosphorylation and acetylation combined yielded values comparable to acetylation alone (k1 and k2 being 28.7 and 4.9 s−1, and typical dwell times being about 35 and 200 ms, respectively; Fig 1G). Hence, it seems that phosphorylation mostly affects the fast mode, i.e., the mode with relatively short dwell times at the motor, whereas acetylation likely affects both modes. CheY binds to the switch even in the absence of FliMN To study the association of both binding modes with clockwise generation, we sought to read the direct output of these modes. We suspected that the high-affinity binding of CheY to FliMN might mask fine outputs related to these binding modes. Therefore, we studied the binding and functional interaction of CheY with motors in which FliM had been truncated to remove FliMN (termed hereafter FliM∆N), i.e., with motors that only contained the low-affinity binding sites of CheY. To examine whether CheY can at all bind to such motors, we employed in vivo Förster resonance energy transfer (FRET), measuring the interaction between overexpressed CheY-mCherry and FliM∆N-YPet-labeled motors (Fig 1H for an explanatory scheme). First, to validate our FRET approach, we measured the response of non-truncated FliM (termed hereafter FliMwt)-YPet motors in cells that expressed CheY-mCherry to approximately the endogenic CheY expression level (due to leaky promoter expression; no inducer added). With these cells, the addition and subsequent removal of the attractant serine caused reduction and enhancement of the FRET signal, respectively, thus validating the method (Fig 1I, red curve). To determine whether CheY binds to FliM∆N (within the switch or soluble in the cytoplasm), we repeated the experiment with FliM∆N-YPet in cells expressing CheY-mCherry, using ΔcheZ background to ensure that CheY-mCherry was mostly phosphorylated. We observed a very weak, hardly detectable FRET response when the intracellular CheY-mCherry concentration was comparable to the endogenic CheY expression level (Fig 1I, green). However, when we overexpressed CheY-mCherry (inducer present), we observed a response similar in amplitude to that of FliMwt (Fig 1I, blue). This difference between cells containing FliMwt motors and cells containing FliM∆N motors was expected due to the absence of the high-affinity binding site from FliM∆N motors. These results imply a low-affinity interaction of CheY~P with FliM∆N. When, as a negative control, we overexpressed mCherry instead of CheY-mCherry, we observed no response (Fig 1I, purple). This implies that the FRET responses, observed with CheY-mCherry, did not emerge from an interaction between the fluorescent proteins themselves. The response of FliM∆N cells overexpressing CheY-mCherry~P was slower than that of FliMwt cells (Fig 1I). This was likely because of the longer time required to phosphorylate and dephosphorylate such high CheY concentrations and because of the possible impairment of the on-rate of CheY~P binding to the switch by the absence of FliMN. The response of CheY-mCherry under conditions that do not allow its phosphorylation (ΔcheA background, i.e., the kinase is missing and the phosphatase is present) was minor (Fig 1I, yellow). This response possibly reflected the alternative pathway for chemotaxis, demonstrated in E. coli cells lacking most of the chemotaxis machinery but overexpressing CheY (Barak & Eisenbach, 1999). To distinguish between CheY-mCherry binding to the switch and binding to free FliM∆N-YPet molecules within the cytoplasm, we attempted to measure CheY binding to individual motors, employing FRET photobleaching (Appendix Fig S3A and B). As detailed in Appendix 1, we indeed found an increased CheY~P binding at FliM∆N-YPet spots (presumably spots of individual motors) when the CheY-mCherry expression level was elevated (Appendix Fig S3C). However, as the CheY-mCherry concentration increased, the fluorescence of the spots became diffusive. This avoided conclusive differentiation between switch-originated and cytoplasm-originated signals (Appendix 1). To verify that CheY~P can, indeed, bind to FliM∆N motors, we examined whether it can generate clockwise rotation of such motors, relying on the fact that, for generating clockwise rotation, CheY~P must first bind to the motor. We tethered cells containing FliM∆N motors (FliM∆N-YPet motors in some of the experiments) and~100 µM CheY in a ΔcheZ background (to ensure phosphorylation of CheY) to glass via their flagella (Fig 2A for experimental scheme and Fig 2B for a demonstration of a tethered cell and for a representative trace of the rotation rate) and analyzed their direction of rotation with an automated home-made software. The mere overexpression of CheY was enough to produce clockwise rotation (Fig 2C prior to stimulation), indicating that CheY can bind to FliM∆N motors to generate clockwise rotation. The cells responded to positive stimuli (attractant addition or repellent removal) with reduced clockwise rotation (Fig 2C). Because positive stimuli work by lowering the phosphorylation level of CheY (Borkovich et al, 1989), the observed response implies that CheY had to be phosphorylated for binding to these FliM∆N motors. It also suggests that, as in cells containing FliMwt motors (Sourjik & Berg, 2002), lowering the level of CheY~P results in lowering the clockwise level. Repellent stimulation, known to work by elevating the phosphorylation level of CheY (Sourjik & Berg, 2002), had hardly any effect (Fig 2D). This is because the absence of CheZ caused CheY to be fully phosphorylated already prior to the repellent stimulation. Thus, CheY~P functionally binds to FliM∆N motors. Figure 2. FliM∆N motors respond differently to various modifications of CheY and FliMN-CheY A. Experimental scheme of flagellar motor tethering. The stub of sheared flagellum is tethered to glass by an anti-flagellin antibody (Silverman & Simon, 1974) in a flow chamber (Berg & Block, 1984). B. Demonstration of rotation rate measured from a single cell; negative and positive values (below and above the blue bar) are counterclockwise and clockwise, respectively. The switch from negative to positive rotation rate at 3 s is shown in the montage to the right. C. The response of tethered fliM∆N ΔcheZ cells (strain EW635), induced for CheY expression from a plasmid (200 µM IPTG), to positive stimuli. Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The arrow marks the estimated time at which the stimulant arrived to, or left (as indicated), the flow chamber. N is the number of cells. α-Methyl-DL-aspartate (MeAsp) and leucine were used at 1 mM, benzoate (pH 7.0) at 50 mM. D. As in (C) for negative stimuli. E. Response of tethered fliM∆N ΔcheA cells (strain EW634) to acetate (50 mM, pH 7.0) at different CheY concentrations. Lines and shaded regions are mean ± SEM. F. Response of tethered fliM∆N ΔcheZ cells (strain EW635) to acetate. Details as in (E). G. Contribution of acetylation and phosphorylation of CheY as well as its D13K mutation to clockwise rotation. The points are the mean clockwise rotation calculated from panels E and F at time segments 0–20 s and 160–180 s. The variants shown are fliM∆N ΔcheZ cells expressing CheY in the absence and presence of acetate (10 mM, pH 7.0) (i.e., CheY~P and CheY~P~Ac; blue and green curves; strain EW635), fliM∆N ΔcheA cells expressing CheY, in the absence and presence of acetate (CheY and CheY~Ac; purple and red, respectively; EW634), and fliM∆N ΔcheA cells expressing CheY(D13K) (burgundy; EW737). Each data point is the average of all experiments at a given CheY concentration, weighted by the sample number of each experiment. For data, see Table EV1. The CheY concentrations shown are estimates based on the calibration curves in Appendix Fig S2, for which a similar CheY expression system was used. H. FliMN further activates CheY variants to produce clockwise rotation. The variants shown are fliM∆N ΔcheZ cells expressing FliMN-CheY (i.e., FliMN-CheY~P; blue curve; strain EW697), fliM∆N ΔcheA cells expressing FliMN-CheY, in the absence and presence of acetate (10 mM, pH 7.0) (FliMN-CheY and FliMN-CheY~Ac; purple and red, respectively; EW696), and fliM∆N ΔcheA cells expressing FliMN-CheY(D13K) (burgundy; EW739). See G for other details. I. Response of fliM∆N ΔcheA cells expressing FliMN-CheY (strain EW696) to acetate and benzoate (10 mM each; pH 7.0). FliMN-CheY expression was induced with 800 μM IPTG. Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The black arrow indicates the estimated time point at which the stimulus entered the flow chamber. Download figure Download PowerPoint FliM∆N motors respond differently to CheY~P and CheY~Ac The observed different effects of phosphorylation and acetylation on the modes of CheY binding to FliMwt motors (Fig 1D–G) raised the possibility that CheY~P and CheY~Ac generate clockwise rotation by somewhat different mechanisms. To examine this possibility, we compared clockwise generation by CheY, CheY~P, CheY~Ac, and CheY~P~Ac in cells containing FliM∆N motors. Studying FliM∆N motors is advantageous in this case because it avoids complications caused by differences in binding—CheY~P binds well to FliMN (McEvoy et al, 1999) whereas CheY~Ac does not (Liarzi et al, 2010; Li et al, 2010). To assess the contribution of non-phosphorylated CheY to clockwise generation, we measured the clockwise level of cells that lacked the kinase of CheY (ΔcheA cells); for CheY~P, we used cells that lacked the phosphatase of CheY (ΔcheZ cells); for CheY~Ac, we used ΔcheA cells supplied with the acetyl donor acetate (Appendix Fig S4 for a control of FliM∆N cells with intact chemotactic machinery, demonstrating that acetate acted as an acetyl donor rather than as a repellent); and for CheY~P~Ac, we employed ΔcheZ cells supplied with acetate. To ascertain that the effects are not limited by CheY availability, we measured each of these CheY forms at increasing CheY concentrations that were far beyond the endogenous concentration of chromosomally expressed CheY [estimated at ~10 μM (Li & Hazelbauer, 2004)]. While nonmodified CheY did not generate clockwise rotation (Fig 2E prior to acetate addition; Fig 2G, purple), CheY~P did (Fig 2F prior to acetate addition; Fig 2G, blue). Notably, the dependence of clockwise rotation on the CheY~P concentration had the shape of a saturation curve (Fig 2G, blue), unlike the ultrasensitive (sigmoidal, cooperative-like) dependence observed in FliMwt motors (Cluzel et al, 2000). This suggests that the truncation of FliMN resulted in loss of ultrasensitivity. Also, the concentration-dependence curve of clockwise rotation on CheY~P (Fig 2G, blue) further endorses the conclusion, made above, that the lack of a repell
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