Response regulator output in bacterial chemotaxis
1998; Springer Nature; Volume: 17; Issue: 15 Linguagem: Inglês
10.1093/emboj/17.15.4238
ISSN1460-2075
Autores Tópico(s)Lipid Membrane Structure and Behavior
ResumoArticle3 August 1998free access Response regulator output in bacterial chemotaxis Uri Alon Uri Alon Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA Department of Physics, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Laura Camarena Laura Camarena Department of Molecular Biology, Instituto de Investigation Biomedicas, UNAM, Apartado Postal, 70-228 04510 Mexico D.F., Mexico Search for more papers by this author Michael G. Surette Michael G. Surette Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, AB, Canada, T2N 4N1 Search for more papers by this author Blaise Aguera y Arcas Blaise Aguera y Arcas Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Yi Liu Yi Liu Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Stanislas Leibler Stanislas Leibler Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA Department of Physics, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Jeffry B. Stock Jeffry B. Stock Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA Department of Chemistry, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Uri Alon Uri Alon Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA Department of Physics, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Laura Camarena Laura Camarena Department of Molecular Biology, Instituto de Investigation Biomedicas, UNAM, Apartado Postal, 70-228 04510 Mexico D.F., Mexico Search for more papers by this author Michael G. Surette Michael G. Surette Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, AB, Canada, T2N 4N1 Search for more papers by this author Blaise Aguera y Arcas Blaise Aguera y Arcas Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Yi Liu Yi Liu Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Stanislas Leibler Stanislas Leibler Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA Department of Physics, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Jeffry B. Stock Jeffry B. Stock Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA Department of Chemistry, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Author Information Uri Alon1,2, Laura Camarena3, Michael G. Surette4, Blaise Aguera y Arcas1, Yi Liu1, Stanislas Leibler1,2 and Jeffry B. Stock1,5 1Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA 2Department of Physics, Princeton University, Princeton, NJ, 08544 USA 3Department of Molecular Biology, Instituto de Investigation Biomedicas, UNAM, Apartado Postal, 70-228 04510 Mexico D.F., Mexico 4Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, AB, Canada, T2N 4N1 5Department of Chemistry, Princeton University, Princeton, NJ, 08544 USA The EMBO Journal (1998)17:4238-4248https://doi.org/10.1093/emboj/17.15.4238 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Chemotaxis responses in Escherichia coli are mediated by the phosphorylated response-regulator protein P-CheY. Biochemical and genetic studies have established the mechanisms by which the various components of the chemotaxis system, the membrane receptors and Che proteins function to modulate levels of CheY phosphorylation. Detailed models have been formulated to explain chemotaxis sensing in quantitative terms; however, the models cannot be adequately tested without knowledge of the quantitative relationship between P-CheY and bacterial swimming behavior. A computerized image analysis system was developed to collect extensive statistics on freeswimming and individual tethered cells. P-CheY levels were systematically varied by controlled expression of CheY in an E.coli strain lacking the CheY phosphatase, CheZ, and the receptor demethylating enzyme CheB. Tumbling frequency was found to vary with P-CheY concentration in a weakly sigmoidal fashion (apparent Hill coefficient ∼2.5). This indicates that the high sensitivity of the chemotaxis system is not derived from highly cooperative interactions between P-CheY and the flagellar motor, but rather depends on nonlinear effects within the chemotaxis signal transduction network. The complex relationship between single flagella rotation and free-swimming behavior was examined; our results indicate that there is an additional level of information processing associated with interactions between the individual flagella. An allosteric model of the motor switching process is proposed which gives a good fit to the observed switching induced by P-CheY. Thus the level of intracellular P-CheY can be estimated from behavior determinations: ∼30% of the intracellular pool of CheY appears to be phosphorylated in fully adapted wild-type cells. Introduction Bacteria such as E.coli swim by rotating several flagella (for a recent review of E.coli motility see Macnab, 1996). Each flagellum is attached to the cell at a complex basal body apparatus that functions as a bi-directional rotary motor. Rotating flagella filaments coalesce into a bundle that pushes the cell body in a curvilinear trajectory termed a 'run'. Runs are interrupted when the flagella fly apart and the cell tumbles in place, randomizing the direction of the subsequent run. By modulating the frequency of tumbles, the bacteria are able to perform chemotaxis, achieving a net motion towards attractants and away from repellants (Berg and Brown, 1972). The motion of individual motors can be visualized by tethering a flagellum to a coverslip and viewing the rotation of the cell body. From such experiments it has been determined that there is a general correlation of running behavior with counterclockwise (CCW) motor rotation, and a correlation of tumbling behavior with clockwise (CW) motor rotation (Larsen et al., 1974). Escherichia coli regulate their tumbling frequency through the activity of the Che proteins, which form a signal transduction network (for recent reviews see Eisenbach, 1996; Stock and Surette, 1996; Falke et al., 1997). This network interacts with the motor via the response regulator protein CheY. CheY is phosphorylated by the kinase CheA, and CheA kinase activity is regulated by the transmembrane chemotaxis receptors. P-CheY is dephosphorylated by the phosphatase CheZ. Genetic and biochemical studies indicate that P-CheY binds to the motor and promotes CW rotation. Thus, for instance, mutant strains that are deficient in CheA or CheY tend to run continually whereas mutants deficient in CheZ are highly tumbly. Increases in attractant concentration inhibit kinase activity, leading to reduced levels of P-CheY and suppression of tumbling. Thus, when a cell runs up an attractant gradient it tends to continue in that direction. The chemotaxis signal transduction system has been extensively studied under defined conditions with purified components, and models based on these results have been formulated to explain in quantitative terms the effects of various mutations and stimuli on the levels of P-CheY (Segel et al., 1986; Bray et al., 1993; Bray and Bourret, 1995; Hauri and Ross, 1995; Barkai and Leibler, 1997; Spiro et al., 1997). The strength of this approach would be significantly augmented if a direct quantitative relationship between the in vivo level of P-CheY and swimming behavior were known. In this report we have attempted to determine this relationship. Because of the inherent chemical instability of the aspartyl phosphate in P-CheY, it is not feasible to measure directly the fraction of CheY phosphorylated in the cell. Previous studies of the effect of CheY on cell behavior were performed using strains with undefined kinase activity (Kuo and Koshland, 1987, 1989), so the equivalent level of wild-type P-CheY could not be estimated. To bypass these problems, we have constructed a mutant strain that lacks both the P-CheY phosphatase, CheZ, and the downregulator of kinase activity, CheB. Several lines of evidence indicate that in this strain essentially all the CheY present is phosphorylated. Variable levels of CheY were expressed in this background from a low-copy plasmid with a regulated lac promoter, and CheY was quantitated by standard immunological techniques. Tethered-cell and swimming-cell assays were employed in parallel to establish a quantitative relation between behavior and P-CheY levels. Computerized image analysis (Sager et al., 1988; Khan et al., 1992; Amsler, 1996) was used to acquire extensive statistics in both assays. Both the tumbling frequency of swimming cells and the CW bias of tethered motors increase with increasing P-CheY concentration. However, the relationship between tethered-motor rotation and swimming behavior was not simple. Most of the differences could be ascribed to interactions between flagella that can be viewed as an additional level of information processing in the chemotaxis system. Comparing the behavior of wild-type cells with that induced by known amounts of P-CheY indicates that ∼30% of CheY is phosphorylated in adapted wild-type cells. The relation between P-CheY and tumbling frequency also allowed us to compare the in vivo function of several CheY mutants with that of wild-type P-CheY. Our results suggest that in vivo, the constitutively active mutant CheYD13K is quantitatively equivalent to P-CheY. This complements a recent study in which motor rotation was related to the intracellular concentration of a similar activated CheY mutant (Scharf et al., 1998). The tumbling frequency of swimming cells exhibited only a weakly sigmoidal dependence on P-CheY (apparent Hill coefficient ∼2.5). This result has important implications concerning our current understanding of the chemotaxis signal transduction system. Simulations based on the known biochemistry of the chemotaxis system indicate that the high sensitivity that cells exhibit to attractant stimuli (Berg and Brown, 1972; Segall et al., 1986; Khan et al., 1993) implies a highly cooperative interaction between P-CheY and the motor [with apparent Hill cofficients ranging from 8 to 11 (Ninfa et al., 1991; Spiro et al., 1997)]. The observation that this interaction is not highly cooperative suggests that the sensitivity of the chemotaxis system is due to some hitherto undefined molecular interaction within the signal transduction network that controls the level of CheY phosphorylation. Results Strategy for measuring behavior as a function of P-CheY Escherichia coli strain PS2001, a derivative of the chemotaxis wild-type strain RP437 with a deletion from the beginning of cheB through cheY to the end of cheZ, was transformed with the low-copy-number plasmid pLC576 in which cheY is under control of the lac promoter (Table I). Production of CheY was controlled by varying the concentration of isopropyl-β-D-thiogalactoside (IPTG). In this strain, lack of CheB leads to full methylation of the chemoreceptors, and thus to a high activity of the kinase, CheA, which phosphorylates CheY. In addition, CheZ is missing, greatly reducing the rate of CheY dephosphorylation. Thus, in this strain virtually all of the CheY expressed is expected to be phosphorylated. This is supported by three lines of evidence. (i) From in vitro measurements of CheY phosphorylation by highly methylated receptor-CheA complexes, and of P-CheY dephosphorylation in the absence of CheZ, one would predict that >99.9% of CheY is maintained in a phosphorylated state in PS2001–pLC576 (Table II). From these calculations, even if the in vivo rate of CheY phosphorylation were 100-fold lower than the in vitro estimates, >90% of CheY would still be phosphorylated in this strain. (ii) The tumbling frequency of RP1616, a strain deleted for CheZ, is the same as the tumbling frequency of PS2001–pLC576 with CheY induced to wild-type level. (iii) The tumbling behavior of PS2001–pLC576 was not affected by addition of repellents (50 mM L-leucine or 100 μM indole, at various levels of CheY induction; data not shown). Thus, repellents can not further increase the P-CheY levels in this strain, consistent with phosphorylation of essentially all of CheY. Table 1. Strains and plasmids Comment Source Strain RP437 wild-type for chemotaxis, lacY−, ara− J.S.Parkinson PS2001 ΔcheBcheYcheZ this study PS2002 ΔcheA–cheZ, 'gutted strain' this study RP1616 ΔcheZ J.S.Parkinson Plasmid pHSG576 low copy, CmR Takeshita et al. (1987) pLC576 low copy, cheY under lac promoter, CmR this study pMS164 low copy, cheYD13K under lac promoter, CmR this study pMS168 medium copy, cheYD57A under araBAD promoter, AmpR this study Table 2. Estimates of fraction of CheY phosphorylated in various strains, based on in vitro rate constants Strain Kinase CheA autophosphorylation activity, kA (s−1) Fraction of CheY phosphorylated PS2001–pLC576 20 0.9995 RP1616 (ΔcheZ) 13 0.9990 Kuo and Koshland (1987, 1989) 0.1 0.5–0.9 The relevant reactions are: CheA + MgATP − kA → P-CheA + MgADP; P-CheA + CheY ← KD → P-CheA–CheY − ktrans → CheA + P-CheY; and P-CheY − kY → CheY + Pi. The fraction of CheY phosphorylated was calculated from the steady state equation: kY×[P-CheY] = kA[CheA] ([CheY]total − [P-CheY])/([CheY]total − [P-CheY] + KD). Measurements with purified components indicate that the equilibrium dissociation constant for CheY binding to P-CheA, KD, is ∼2 μM (Li et al., 1995; Shukla and Matsumura, 1995), the phosphotransfer reaction from P-CheA to CheY is not rate-limiting (ktrans >600/s; Stewart, 1997), and the rate of P-CheY dephosphorylation in the absence of CheZ is relatively slow, kY ∼0.04/s (Hess et al., 1988; Lukat et al., 1991). Estimated rate constants for CheA autophosphorylation at saturating MgATP, kA, in PS2001–pLC576 and in RP1616 correspond to values measured for CheA in complexes with CheW and the fully modified (all Q) or half modified (QEQE) receptor signaling domains, respectively (Borkovich et al., 1992; Liu et al., 1997). The strain used by Kuo and Koshland (1989) expressed a CheA–CheZ fusion that is not subject to receptor activation. The value of kA in the Kuo and Koshland experiments is thus assumed to correspond to that of pure CheA in the absence of receptor (Tawa and Stewart, 1994; Surette and Stock, 1996). The level of CheA in E.coli has been estimated to be ∼5 μM (Wang and Matsumura, 1997). The calculated values for fraction of CheY phosphorylated are essentially constant over the range of total CheY concentrations used in these studies (<100 μM) in the case of PS2001–pLC576 and RP1616, but with the much lower kinase activity in the strain used by Kuo and Koshland the fraction of CheY phosphorylated is expected to vary with total CheY as indicated. Cultures of PS2001–pLC576 grown in the presence of varying concentrations of the inducer IPTG were harvested at mid-log phase and the rotation of single tethered cells as well as the behavior of free-swimming cells was analyzed. Cells from each culture were also pelleted and frozen for subsequent quantitation of CheY by Western blot determinations. CheY expression increased with IPTG concentration from a basal level of ∼700 CheY/cell in the absence of IPTG to ∼35 000 CheY/cell in cells grown in 50 μM IPTG (Figure 1, inset). The total protein per cell, as well as the number of flagella per cell (6.7 ± 0.5, mean and SE for 25 cells), did not measurably vary with CheY expression. Figure 1.Fraction of time tethered cells spend turning CW (fCW). Cells induced with various amounts of IPTG were tethered and their motion was analyzed using video tracking. Each point is an average of data from 15–40 different tethered cells, analyzed for 100 s each. Open circles, strain PS2001–pLC576; solid circle, wild-type strain RP437–pHSG576. Error bars correspond to standard error of the mean. Solid line, fit to the allosteric model (see Discussion). The model parameters are: N = 30 binding sites for P-CheY, allosteric constant of L0 = 107, dissociation constant for P-CheY binding K = 7.6 μM and ratio of binding affinity to CW and CCW states Ψ = 2.0. Dashed line, allosteric model with K = 12.5 μM and Ψ = 2.3, a best fit to the data of Scharf et al. (1998), where tethered-cell CW bias was measured as a function of the concentration of the activated CheY mutant CheYD13KY106W. Inset, intracellular CheY concentrations as a function of IPTG induction of strain PS2001–pLC576. The number of CheY molecules/cell were determined using Western blots. CheY intracellular concentration was calculated assuming that 1000 molecules/cell correspond to 2.8 μM (Scharf et al., 1998). The line is a smooth fit. Download figure Download PowerPoint Tethered cells To measure the rotation of single flagellar motors, cells were tethered to coverslips coated with anti-flagella antiserum and analyzed by computerized image-processing. The analysis included all rotating cells in a field of view according to uniform criteria, eliminating any bias introduced by manual cell selection procedures. From the angular velocity trace for each cell, switches between CCW and CW motion were determined. Each cell typically displayed approximately the same absolute angular velocity in both CW and CCW states. The average fraction of time spent turning CW, fCW, increased with increasing CheY concentration with an apparent Hill coefficient of 3.5 ± 1.0 (Figure 1). The switching process was further characterized by the mean duration of CW and CCW intervals (Figure 2). As expected, the mean CW duration increased and the mean CCW duration decreased with increasing P-CheY concentration. These results are similar to those of Kuo and Koshland (1989) who measured the effects of CheY expression in a strain that contained an incomplete deletion from cheA to cheZ that produces a fragment of CheA that is not subject to receptor activation. Because of the kinase deficiency, one might assume that relatively low levels of P-CheY were present in these experiments. Our results indicate that, in fact, a large fraction of the CheY must have been phosphorylated in these cells. A kinetic analysis based on in vitro rate estimates is consistent with this interpretation (Table II). A recent study showed a similar dependence of motor rotation on the level of an activated CheY mutant (Scharf et al., 1998; Figure 1). This indicates that the mutant has approximately the same activity as P-CheY. Figure 2.Mean duration of CCW (A) and CW (B) intervals of tethered-cell rotation in PS2001–pLC576, as a function of intracellular CheY concentration. Download figure Download PowerPoint Motor behavior displays significant variations from cell to cell (Berg and Tedesco, 1975; Spudich and Koshland, 1976; Ishihara et al., 1983; Eisenbach et al., 1990; Levin et al., 1998). We found that in PS2001–pLC576, at levels of CheY induction that caused approximately wild-type motor bias, a distribution of behaviors similar to wild-type was observed (Figure 3). This suggests that the plasmid system used in the present study does not introduce fluctuations between cells larger than those found naturally. Figure 3.Distribution of CW bias between different individual cells. Plotted is the fraction of time spent CW, fCW, divided by the mean of fCW over all cells with the same CheY induction level, . White bars, cumulative data from PS2001–pLC576 cells induced to a level of ∼10, 14 and 20 μM CheY. Shaded bars, wild-type cells RP437–pHSG576. Error bars represent standard errors of the mean. Download figure Download PowerPoint Swimming cells Swimming behavior of cells was determined using the same cultures that were used to analyze the rotation of tethered motors. Cells were placed between a glass slide and a cover slip in a thin fluid layer of ∼10 μm thickness. Their motion was observed by dark-field microscopy and recorded on videotape. The cells remained in the focal plane of the microscope at all times. The videotapes were analyzed by a computerized image analysis method developed to acquire extensive statistics on various aspects of swimming behavior. As expected, in the absence of CheY no tumbles were observed and cells tumbled with increasing frequency as P-CheY increased (Figure 4A–C). At very high levels of induction, however, the cells actually became less tumbly (Figure 4D). At low CheY concentrations, the tumble frequency increased in a weakly sigmoidal fashion with an apparent Hill coefficient of 2.5 ± 1 (Figure 5). The maximal degree of tumbling, 1.8 ± 0.1 tumbles/s, was observed at CheY concentrations of ∼50 μM. At higher CheY concentrations, cells tumbled less, dropping to ∼1.4 tumbles/s at 100 μM CheY. It seems probable that this decrease in tumbling frequency at CheY concentrations >50 μM is due to runs caused by the formation of CW rotating rather than CCW rotating flagella bundles (Wolf and Berg, 1989). The existence of such CW bundles and their ability to support runs has previously been demonstrated in strains with mutations that lock the motor in a CW state (Khan et al., 1978). The average run speed decreased slightly with increasing CheY levels (Figure 6), with a rather sharp drop to ∼50% of the maximal run speed at CheY concentrations >50 μM. The initial gentle decrease in run speed with increasing tumbling frequency is probably due to the fact that the cell speed takes a certain amount of time to recover from each tumble (Berg and Brown, 1972). Figure 4.Paths of swimming cells expressing varying amounts of P-CheY. Each path represents the trajectory of an individual bacterium over an average of ∼2 s. (A) Strain deleted for cheB, cheY and cheZ (PS2001), the bar is 100 μm long, (B) PS2001–pLC576 expressing 10 ± 2 μM CheY, (C) PS2001–pLC576 expressing 60 ± 10 μM CheY, (D) PS2001–pLC576 expressing 100 ± 20 μM CheY, (E) RP437–pHSG576 (wild-type) and (F) RP1616–pHSG576 (ΔcheZ). Download figure Download PowerPoint Figure 5.Tumbling frequency as a function of CheY concentration. Open circles, PS2001–pLC576 induced with various amounts of IPTG; solid circle, wild-type (RP437–pHSG576); solid triangle, ΔcheZ (RP1616–pHSG576) cells. The tumbling frequency is the total number of tumbles detected by the computerized algorithm, divided by the total duration of all paths. The error bars represent standard errors of the mean. Solid line, fit to a Hill equation, with Hill coefficient of 2.5 and half maximal effect at 18.8 μM. Dashed line, guide to the eye. Download figure Download PowerPoint Figure 6.Run speed as a function of CheY concentration in strain PS2001–pLC576 (open circles), wild-type (RP437–pHSG576) (solid circle) and ΔcheZ (RP1616–pHSG576) cells (solid triangle). Run speed is defined as the mean of the top 10% of the speeds along each bacterial trail, averaged over all trails. Shown are means ± SE of the mean. Download figure Download PowerPoint We also measured the duration of run and tumble events. The mean run duration decreased with increasing CheY concentration until a minimum was reached at a CheY concentration of ∼50 μM (Figure 7B). In contrast, the duration of tumbling intervals remained approximately constant at 0.20 ± 0.05 s over the entire range of CheY induction (Figure 7A). Figure 7.Mean duration of tumbles (A) and runs (B), as a function of CheY concentration in PS2001–pLC576 in the wild-type strain (RP437–pHSG576) (solid circle) and ΔcheZ (RP1616–pHSG576) cells (solid triangle). Download figure Download PowerPoint Estimates of P-CheY levels in strains that contain wild-type levels of CheY In order to estimate the fraction of CheY that is phosphorylated in wild-type cells, we employed the computerized tethering and swimming assays on strain RP437, the parental strain of all mutants used here, which is wild-type for chemotaxis. We determined the total amount of CheY in RP437 under the present experimental conditions to be 17 500 ± 1000 molecules/cell (average ± SE of eight independent measurements), corresponding to a cytoplasmic concentration of ∼49 ± 3 μM. This is close to the CheY levels in Salmonella typhimurium (15 000–20 000 CheY/cell; Stock et al., 1985; Zhao et al., 1996), but is ∼3-fold higher than the value previously determined for the copy number in E.coli (Kuo and Koshland, 1987), perhaps due to the different growth conditions used in that study. In tethering assays, RP437 spent 0.22 ± 0.05 of the time turning CW (Figure 1). PS2001–pLC576 cells exhibited an equivalent behavior at a CheY concentration of ∼13.5 ± 1 μM. Thus, in wild-type cells under these steady-state conditions ∼30% of the intracellular pool of CheY is phosphorylated. The same conclusion was obtained from an analysis of swimming behavior. Wild-type cells exhibited a tumbling frequency of 0.53 ± 0.05/s, and PS2001–pLC576 cells display the same tumbling frequency when the concentration of CheY is 13 ± 1 μM. Thus, the swimming assay also suggests that ∼30% of the CheY is phosphorylated in RP437. The tumbling behavior and level of CheY expression in RP437 were not affected by the presence of the plasmid pHSG576, nor by growth in the presence of 50 μM IPTG (data not shown). The swimming speed measured for wild-type cells (RP437) was 16.5 ± 1.0 μm/s (Figure 6). This is in good agreement with previous measurements where the cells were tracked near a glass slide (Amsler et al., 1993; Khan et al., 1993), though it is lower than some measurements of the run speed by three-dimensional tracking of cells swimming far from any surface (Lowe et al., 1987). In the present assay, the cells swim in a thin fluid layer at a distance of at most ∼5 μm from a surface. Evidently, this has an effect on cell motion as compared with motion in a free solution (Ramia et al., 1993; Frymier et al., 1995; Vigeant and Ford, 1997). This of course should not affect the use of the present method as a read-out of the intracellular P-CheY levels. The swimming behavior of RP1616–pHSG576 was also measured. This strain lacks the CheY phosphatase CheZ. Because of the CheZ defect, P-CheY levels are expected to be high in this strain (Table II), and in fact its tumbling frequency, 1.7 ± 0.1/s, is approximately equal to the maximal tumbling frequency of PS2001–pLC576 corresponding to wild-type levels of CheY (Figure 5). Thus, the observed RP1616 tumbling rate is consistent with the assumption that it contains a wild-type amount of CheY protein, essentially all in the phosphorylated state. In vivo function of cheY mutants The relation between P-CheY and tumbling frequency allowed us to compare quantitatively the in vivo activity of CheY mutants with that of wild-type P-CheY. Various amounts of the mutant proteins were expressed from inducible promoters, protein expression was quantified and swimming behavior was measured using the same procedure as for wild-type CheY. Two mutants were studied: CheYD13K, an activated mutant that causes tumbling in the absence of phosphorylation (Bourret et al., 1993), and CheYD57A, a mutant that cannot be phosphorylated. The corresponding mutant genes were cloned into expression plasmids (Table I): cheYD13K was cloned into the low-copy vector pHSG576 under lac control, and cheYD57A into a complementary multicopy plasmid under an arabinose inducible promoter. The plasmids were transformed separately and together into the 'gutted' strain PS2002 that contains no Che genes (deleted from the beginning of cheA to the end of cheZ). Whereas the activated mutant CheYD13K caused tumbling (Figure 8), CheYD57A induced no tumbles even at intracellular concentrations >200 μM (data not shown). Moreover, swimming behavior as a function of CheYD13K concentration was not affected by simultaneous expression of >200 μM CheYD57A (Figure 8). Thus, unphosphorylated CheY does not appear to compete with active CheY for binding to the motor, and does not appear to cause either tumbles or runs. This result is consistent with the in vitro studies of CheY binding to FliM where it has been shown that CheY binding to the motor switch protein is substantially enhanced by phosphorylation (Welch et al., 1993, 1994). Figure 8.Tumbling frequency as a function of mutant CheY proteins expressed in the gutted strain PS2002 which is deleted for cheA–cheZ. Open circles, PS2002–pMS164 induced with various amounts of IPTG expressing CheYD13K; solid circles, PS2002–pMS164–pMS168 induced with various amounts of IPTG expressing CheYD13K and CheYD57A, the latter induced with 0 μM arabinose; open squares, PS2002–pMS164–pMS168 induced with 100 μM arabinose and varying amounts of IPTG (CheYD13K coexpressed with CheYD57A, the latter at an intracellular concentration of 260 ± 40 μM). The solid and dashed lines represent the tumbling frequency induced by wild-type CheY in strain PS2001 (from Figure 5). Download figure Download PowerPoint The activated mutant, CheYD13K, had approximately the same quantitative effect in vivo as P-CheY (Figure 8). This finding was unexpected; previous in vitro studies have indicated that CheYD13K binds FliM only marginally more av
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