INTRODUCTION

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Flagellated bacteria, such as Escherichia coli and Salmonella typhimurium, respond to a wide variety of positive or negative environmental stimuli through modulation of the direction of rotation of their flagella. An increase in the concentration of a chemical attractant (e.g., aspartate) or a decrease in the concentration of a chemical repellent (e.g., leucine) promotes counterclockwise rotation, or more persistent smooth swimming. The signal transduction chain includes a kinase, CheA, which phosphorylates a response regulator, CheY. CheY-phosphate (CheY-P), the active form, binds to the motor switch complex and promotes clockwise (CW) rotation, or more frequent tumbling. The turnover of CheY-P is relatively rapid: in vitro, CheY-P autodephosphorylates with a half time of about 14 s (27) and its dephosphorylation is accelerated by CheZ (11); in vivo, physiological measurements indicate that this acceleration should be by about a factor of 10 (26).

Several CheY mutants are active in the absence of phosphorylation. When overexpressed, the mutant protein CheY^13DK, for example, produces a tumbly phenotype in a host deleted for cheA (5, 6). However, it does not show enhanced binding to FliM or to CheZ, as does CheY-P (4, 35, 36). Another class of CheY mutants, including CheY^106YW and CheY^95IV, are hyperactive when expressed at normal levels in an otherwise wild-type background but are inactive in a cheA deletion background (25, 38). In vitro, the latter mutant exhibits enhanced binding to FliM (25), whereas the former shows no change in affinity for FliM (38). The double mutant CheY^13DK106YW (CheY**) is active at relatively low concentrations in a host deleted for cheA. In an earlier study, we used this mutant to establish quantitative relationships between intracellular concentrations and statistics of motor switching (24).

It is known from in vitro studies that the phosphatase CheZ does not bind to the switch component FliG, FliM, or FliN (8). However, it is not known whether CheZ has a direct effect on the switch in vivo, because it has not been possible to distinguish this action from inactivation of CheY-P. Early studies of intragenic suppression argued for direct interaction, because the suppression appeared to be allele specific (20, 21), but more recent work has made this less certain (13, 29). In the present study, we used the double mutant CheY** to settle the issue. We studied the effects of CheZ on CheY** and the switch, comparing these results with those obtained with wild-type CheY. CheZ did not affect the behavior of the flagellar motor, either directly or indirectly, e.g., by sequestration of CheY**. As judged in vitro by cross-linking, CheY** induced oligomerization of CheZ to the same extent as CheY (but much less so than CheY-P) and bound to FliM about as well as CheY-P.

	MATERIALS AND METHODS

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Bacteria and plasmids. The bacteria and plasmids used are listed in Table 1. Strains HCB900 and HCB915 contain the doubly mutated gene cheY^13DK106YW, under control of the IPTG (isopropyl--D-thiogalactopyranoside)-inducible promotor Ptrc, and a deletion in the adjacent gene, cheZ. The construction of HCB900, a derivative of RP9535 (cheA), has been described by Scharf et al. (24). HCB915, a derivative of RP437 (cheA⁺), was constructed in parallel. Both strains carry cat (chloramphenicol transacetylase).

Cell cultures. Cells were prepared in the same way for tethering assays and quantitative immunoblots, as described by Scharf et al. (24). They were grown in TB (1% tryptone, 0.5% NaCl) for 4 h at 33°C, and then (if required) inducers were added and incubation was continued for 2 h. For example, for tethering assays, IPTG was added to a final concentration of 15 µM, the culture was divided into two parts, and AHT was added to one part at a final concentration of 200 µg/liter.

Behavioral assays. Swarm assays were performed on soft agar plates (0.3% agar, 1% tryptone, 0.5% NaCl) inoculated with 2.5 µl of a 10-fold-concentrated culture that was grown in TB for 6 h at 30°C. The plates were incubated at temperatures and times indicated in the figure legends and text. Tethering, data acquisition, and analysis were done as described by Scharf et al. (24).

Immunoblots and cross-linking. Purified CheY^13DK was a gift from Phil Matsumura. Purification of CheY, CheY**, and CheZ was described previously (24). FliM was purified as described by Bren et al. (8), but the chromatography step was omitted.

Image enhancement. Photographs of swarms, immunoblots, and gels were scanned, and contrast was digitally enhanced.

	RESULTS AND DISCUSSION

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Expression of CheZ from pBES40. Plasmid pBES40 carries cheZ under tight control of TetR, the repressor of the tetracycline promoter. When this plasmid was introduced into the cheZ deletion strain RP1616 grown in the absence of inducer (AHT), only a slight increase in swarm drift velocities was seen (Fig. 1b). When CheZ was induced, swarm diameters increased substantially and chemotactic rings appeared (Fig. 1c). However, high levels of induction impaired chemotaxis (Fig. 1d). In Fig. 1a, the cells shown were tumbly; in Fig. 1d, they were smooth swimming.

View larger version (108K): [in this window] [in a new window]   FIG. 1.   Variations of swarm morphology upon expression of CheZ in the cheZ-deletion strain RP1616. Cells were transformed with pBES40 (cheZ inducible with AHT) or its parental plasmid pASK75 (no cheZ) and incubated on soft agar plates containing the following concentrations of AHT: pASK75 at 0 µg/liter (a), pBES40 at 0 µg/liter (b), pBES40 at 2 µg/liter (c), and pBES40 at 20 µg/liter (d). Plates were incubated for 14 h at 30°C. The outer diameter of the swarm in panel c is 46 mm.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Quantification of CheZ expressed from pBES40 in HCB917 (a cheZ-deletion strain) as a function of AHT induction. Cells were exposed to AHT for 2 h during exponential growth, harvested, and assayed on immunoblots with anti-CheZ MAb. CheZ levels were calculated as in Table 2. At least two immunoblots were performed for each concentration. Error bars are standard deviations of the means, with the result for each immunoblot weighted equally. The fit is of the form y = exp[C1  (C2/AHT)], where y is 103 molecules/cell, C1 and C2 are constants, and AHT is in micrograms per liter. The dashed line indicates the level of CheZ measured in strain RP437 (Table 2).

Effect of CheZ on swarming in wild-type cells. We also examined the effect of cheZ expression from pBES40 in wild-type strain RP437 and found swarm diameters to be remarkably sensitive to AHT (Fig. 3a). This sensitivity was not due to an unspecific antibiotic effect of AHT, which is known to be growth inhibiting at concentrations above 200 µg/liter (Fig. 3a). The swarms obtained in this experiment are shown in Fig. 3b. Chemotactic rings were not seen at concentrations of AHT of 12 µg/liter or higher. These results suggest that the ratio of CheZ to CheY (and hence the rate and/or extent of dephosphorylation of CheY) is important for effective chemotaxis. Huang and Stewart (12) found that swarming in a recA derivative of strain RP437 was impaired upon moderate induction of CheZ from E. coli but less so by induction from S. typhimurium. They concluded that "the chemotaxis system is quite sensitive to the CheY dephosphorylation activity of CheZ."

View larger version (44K): [in this window] [in a new window]   FIG. 3.   Variations of swarm size and morphology upon expression of CheZ in wild-type strain RP437. (a) Outer diameters of swarms measured as a function of AHT concentration for cells transformed with pBES40 (closed symbols) or its parental plasmid pASK75 (no cheZ) (open symbols), relative to the diameter observed without AHT. (b) Swarm morphologies for wild-type cells and cells transformed with pBES40. wt, wild-type strain. The numbers are AHT concentrations (in micrograms per liter). Plates were incubated for 9 h at 30°C.

Levels of CheY and CheZ in wild-type cells. Given this sensitivity to the ratio of CheZ to CheY, we measured the levels of these components in wild-type strains AW405 and RP437 (Table 2). Strain AW405 contained about three times as much of either component as strain RP437, but the CheY/CheZ molar ratios were about the same: 2.3:1. The swarming abilities of both strains were similar (data not shown), suggesting that the absolute values of CheY and CheZ are not as crucial for chemotaxis as their ratios. Kuo and Koshland (14) grew strain RP437 in a minimal medium and found a CheY/CheZ molar ratio of 8:1, with a CheZ concentration of 1 µM (about 900 molecules/cell using their cytoplasmic volume). Thus, their estimate of the number of molecules of CheZ per cell is close to ours, but their estimate of the number of CheY molecules is about 2.5 times higher. Matsumura et al. (17), working with the same strain, reported a 20-fold larger number of molecules of CheZ. The reason for this difference is not known.

Effect of CheZ on swarming in cells expressing CheY**. We studied the effect of CheZ on cells expressing CheY**, a protein that induces CW flagellar rotation without phosphorylation (39). Earlier, we used variable expression of this protein to examine control of the direction of flagellar rotation in cells deleted for cheA and cheZ (24). In the present work, we also used cells deleted only for cheZ. Variable expression of CheZ in cells expressing a moderate amount of CheY** had no effect on swarm size, even when cells were grown for a relatively long period of time (Fig. 4). Nor did the swarm size of cells expressing variable amounts of CheY** depend on whether or not the cells expressed CheZ (Fig. 5). In the latter experiment, cells swam smoothly at low concentrations of IPTG and tumbled incessantly at high concentrations. Swarm size was maximal when cells both ran and tumbled (see Wolfe and Berg [37]). Similar results were obtained in a cheA cheZ deletion background with strain HCB900 (data not shown). These results indicate that CheZ has little, if any, direct effect on the behavior of the flagellar motor, nor does it sequester CheY**.

View larger version (156K): [in this window] [in a new window]   FIG. 4.   Similarities of swarm size and morphology with or without CheZ in strains HCB917 and HCB918, which also express CheY**. Both strains carry pMS421 (which provides LacI); in addition, HCB917 carries pBES40 (inducible cheZ) and HCB918 carries pASK75 (the parental plasmid). CheY was induced with 70 µM IPTG (see Fig. 5). Strains and AHT concentrations are as follows: HCB918, 0 µg/liter (a); HCB917, 0 µg/liter (b); HCB917, 2 µg/liter (c); HCB917, 20 µg/liter (d). Plates were incubated for 40 h at 33°C. Swarm diameters are about 30 mm.

View larger version (18K): [in this window] [in a new window]   FIG. 5.   Variations in swarm size as a function of the level of expression of CheY**, with (closed symbols, strain HCB917, AHT at 20 µg/liter) or without (open symbols, strain HCB918, AHT at 20 µg/liter) CheZ. Plates were incubated for 40 h at 33°C. (Inset) Level of expression of CheY** in strain HCB917 as measured by immunoblot analysis. Error bars are standard errors, with the result for each sample on an immunoblot weighted equally.

Effect of CheZ on switching behavior of cells expressing CheY**. The above conclusions were confirmed by measuring the rotational bias of tethered cells expressing small or large amounts of CheZ (Table 3). The strains examined were modified to take advantage of the self-tethering (sticky-filament) property of fliC(St) (24) by introducing a fliC mutation in the chromosome and adding a plasmid, pBES42, carrying both the fliC(St) allele and lacI^q. Strain HCB919 carries, in addition, the cheZ plasmid pBES40, and HCB920 carries, in addition, the parental plasmid pASK75. Shifts in CW bias were small: the effect of addition of AHT observed in the absence of CheZ (strain HCB920) was as large as or larger than that observed in the presence of small or large amounts of CheZ (strain HCB919). imilar results were found with the cheA-deleted strain HCB922 (data not shown). We found earlier (see Fig. 3a of reference 24) that the CW bias changes dramatically with CheY** concentration. So again, these results indicate that CheZ has little, if any, direct effect on the behavior of the flagellar motor, nor does it sequester CheY**.

Oligomerization of CheZ in the presence of CheY and CheY**. As just seen, CheZ has a great effect on the behavior of cells expressing wild-type CheY, but it has little, if any, effect on cells expressing CheY**, even though CheY** modulates rotational bias. Therefore, the effect of CheZ in wild-type cells must be due to an interaction with CheY itself and/or with an upstream component that controls the phosphorylation of CheY. To test the possibility that CheY** might interact with CheZ, for example, by promoting oligomerization of CheZ (3), we carried out the in vitro cross-linking procedure of Blat and Eisenbach (3) with purified proteins. Cross-linked products were detected on immunoblots of polyacrylamide gels by using anti-CheZ MAbs. Figure 6 shows a comparison of bandsformed in the presence of CheY or CheY** under nonphosphorylating and phosphorylating conditions. In the absence of CheY, CheZ formed a band with an apparent molecular mass of about 46 kDa, which is its dimeric form (Fig. 6, lane 2). In the presence of unphosphorylated CheY, the band shifted to a higher molecular mass, about 72 kDa (Fig. 6, lane 3). This complex did not contain CheY, because it did not appear on immunoblots probed with anti-CheY MAbs (data not shown). Therefore, it must be CheZ in its trimeric form. The addition of acetylphosphate promoted the phosphorylation of CheY, resulting in the formation of products in the 200-kDa range (Fig. 6, lane 4). The presence of CheY was evident in these products when the immunoblots were probed with anti-CheY MAbs (data not shown). These data are very similar to those obtained by Blat and Eisenbach (3), who used radioactively labeled proteins, except that the trimerization of CheZ promoted by CheY (Fig. 6, lane 3) is more pronounced (see also Fig. 5 of reference 2). The addition of CheY** in the absence of acetylphosphate (Fig. 6, lane 5) resulted in the formation of an oligomeric state of CheZ identical to that observed in the presence of unphosphorylated CheY. The result was the same on addition of CheY** in the presence of acetylphosphate (Fig. 6, lane 6); however, we do not know the extent of phosphorylation of CheY** by acetylphosphate at the concentration used (18 mM). The absence of CheY** in these 72 kDa-complexes was confirmed by using anti-CheY MAb as a probe. Thus, in vitro, CheY** has the same effect on CheZ as does unphosphorylated CheY, even though in vivo it also promotes CW flagellar rotation.

View larger version (62K): [in this window] [in a new window]   FIG. 6.   Immunoblot of cross-linked products of CheZ recorded on film by enhanced chemiluminescence. The molecular mass scale is shown on the right. Symbols on the bottom: +, present; , absent; wt, wild-type CheY; **, CheY**; AcP, acetylphosphate (which phosphorylates wild-type CheY); EDC-NHS, cross-linking reagents (see Materials and Methods). Each lane contained 100 ng of CheZ.

Cross-linking of wild-type and mutant CheY to FliM. CheY-P is known to bind to the switch component FliM (36). In our earlier work (24), we showed that CheY** promotes switching without being phosphorylated. Here, we demonstrated the affinity of CheY** for FliM by using the in vitro cross-linking assay of Bren et al. (8). Figure 7 shows a polyacrylamide gel after Coomassie staining of the cross-linked products of FliM with wild-type CheY, CheY**, and CheY^13DK in the absence or presence of acetylphosphate. The presence of CheY in the bands at 52 kDa was confirmed by immunoblot analysis, and the immunoblot band intensities agreed with those observed with Coomassie staining (data not shown). As expected, increased cross-linking of CheY to FliM occurred when CheY was phosphorylated (Fig. 7, lanes 1 and 2) (see also reference 8). On the other hand, the cross-linked products between CheY** or CheY^13DK and FliM were the same under both nonphosphorylating and phosphorylating conditions (Fig. 7, lanes 3 and 4 and lanes 5 and 6). But once again, we do not know the extent of phosphorylation of either mutant protein by acetylphosphate at the concentration used (22 mM). However, the affinity of the double mutant to FliM was about four times higher than that of the single mutant, i.e., about 80% of the affinity of CheY-P to FliM. By this assay, CheY** has nearly the same affinity to FliM as CheY-P.

View larger version (41K): [in this window] [in a new window]   FIG. 7.   Coomassie-stained polyacrylamide gel of cross-linked products of FliM and CheY. Molecular mass markers appear on the left and right. FliM appears at 38 kDa, and the cross-linked product CheY-FliM appears at 52 kDa. Monomeric CheY (14.3 kDa) is not shown. Symbol not identified in the legend to Fig. 6: *, CheY13DK. The cross-linking reagent was DMS. Each lane contained 7 µg of FliM.

Summary. We have shown that overexpression of CheZ in wild-type cells abolishes chemotaxis (see also reference 12). However, it has no effect on the behavior of cells whose motors are activated for switching by the double mutant CheY**. This is true in both cheA⁺ and cheA mutant backgrounds. We conclude that CheZ does not affect the motor directly. This is consistent with in vitro studies showing that CheZ does not bind to the switch component FliG, FliM, or FliN (8) and supports the contention that direct interaction between CheZ and the motor has not been established by studies of intragenic suppression (13, 29). We have shown previously, in a cheA mutant background, that rotational bias is a sensitive function of the level of expression of CheY** (24). Therefore, our data also indicate that CheZ does not sequester CheY**. It is possible, a priori, that some CheY** is bound to CheZ. If so, this binding does not interfere with the interaction of CheY** with the switch. We were not able to detect any cross-linking between CheY** and CheZ in vitro. It also is possible that some CheY** is phosphorylated in a cheA⁺ background, since both CheY^13DK and CheY^106YW can be phosphorylated by CheA, although only to a limited extent (5, 38). If so, dephosphorylation or sequestration of this form of CheY** by CheZ also has a negligible effect. Our measurements do not address the possibility that CheZ interacts with CheA_short or CheA_long (34), because those interactions would modulate the activity of the kinase, to which our assays are insensitive.