with a 82901M flexible disk drive, as described previously. (22). .... quired for complete recovery (the adaptation time) increased with the stronger stimulus ..... Public Health Service grant A106375 from the National Institutes of. Health to ...
JOURNAL OF BACTERIOLOGY, Sept. 1985, p. 983-990 0021-9193/85/090983-08$02.00/0 Copyright © 1985, American Society for Microbiology
Vol. 163, No. 3
Sensory Adaptation in Bacterial Chemotaxis: Regulation of Demethylation MARILYN R. KEHRY,t THOMAS G. DOAK, AND FREDERICK W. DAHLQUIST*
Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403 Received 19 February 1985/Accepted 17 May 1985
The behavioral responses of chemotactic bacteria to environmental stimuli are initiated by a family of membrane-bound transducer proteins that communicate excitatory signals to the flageliar apparatus. The adaptation process appears to turn off the excitatory signal and is mediated by the reversible methylation of multiple sites on the transducer proteins. The activities of two chemotaxis-specific enzymes, a methyltransferase and a methylesterase, are regulated during adaptation to maintain behavioral responsiveness. To monitor stimulus-induced changes in methylesterase activity in intact cells, we quantitated the continuous generation of methanol, the end product of the demethylation reaction, in a flow device. In this paper we describe studies of the regulation of the demethylation process. Changes in methylesterase activity after the simultaneous addition of opposing stimuli through two different transducer classes suggest that the sensory information detected by these transducers was integrated and that this integrated signal controlled demethylation.
Chemotactic bacteria such as Escherichia coli respond to environmental stimuli by regulation of the direction of flagellar rotation. Cells respond to attractait gradients by decreasing the frequency of tumbling (tumbling results from flagellar rotation in a clockwise direction). This decrease promotes a net migration towards increasing concentrations of attractaht. Conversely, negative stimuli produce an increase in tumbling frequency that results in the migration of cells away from increasing concentrations of repellent. Several minutes after responding to a stimulus, cells adapt to their environment by returning to their prestimulus pattern of behavior (1-3, 29). Sensory information is transduced in E. coli by a famnily of transmembrane proteins that are located in the inner membrane (36, 39). Each class of transducer molecules mediates the behavioral response to a specific subset of chemicals in the environment (27, 37, 38). The family of transducer proteins is composed of the products of four genes: tsr, tar, trg, and tap. The Tsr protein mediates responsiveness to the attractant serine and to repellents such as leucine and weak acids. Responses to the attractants aspartate and maltose and the heavy-netal repellents Ni(II) and Co(II) are mediated by the Tar protein (24, 35, 37, 38). Transducer proteins seem to act as the primary receptors for detecting changes in the concentrations of chemical stimuli. This interaction may occur by direct binding (serine, aspartate) (9, 19) or by indirect binding to the appropriate periplasmic binding protein (18, 26). The behavioral response is initiated by an excitatory signal which is communicated via the transducers to the flagella. In addition, transducer proteins undergo a reversible methylation reaction during adaptation to stimuli. The transducers have thus been designated the methyl-accepting chemotaxis proteins (MCP) (14). Methylation occurs at multiple sites in each transducer (four to six depending on the MCP class) on glutamate residues (5, 6, 10, 12, 20, 21, 25, 47). An increase in the level of methylation of the MCP class mediating a behavioral response correlates with the adapta-
tion of cells to a positive stimulus (14, 15, 33, 38). Thus, methylation of a transducer appears to turn off the excitatory signal that is being sent to the flagella. Methyl groups from S-adenosylmethionine are placed on MCPs by a protein methyltransferase, the product of the cheR gene, to form a -y-glutamyl methyl ester (11, 25, 40, 47). The removal of methyl groups during adaptation is mediated by a protein methylesterase, the product of the cheB gene, and generates methanol and the free glutamic acid residue (16, 41, 44). The only known substrates of the transferase and esterase enzymes are the sensory transducer proteins. Regulation of methylation and demethylation mnaintains behavioral responsiveness. In the absence of external stimuli, methyl groups on MCPs turn over continuously in a balance between transferase and esterase activities (28, 45). The mechanism for regulating these enzyme activities is not known, and several models for regulation have been proposed (13, 32, 42). We have used the production of methanol by chemotactically wild-type bacteria as the basis for a continuous assay for methylesterase activity in intact cells. Previous work has shown that after an attractant stimulus, methylesterase activity (as measured by methyl group turnover) decreases dramatically and then recovers over the course of several minutes to its prestimulus rate (22, 45). A negative stimulus produces a rapid and transient increase in methylesterase activity that corresponds to behavioral adaptation (22). It is known that the behavioral response of cells to several attractants is additive and that attractant and repellent stimuli applied simultaneously will cancel each other behaviorally (4). In this study we investigated the changes in methylesterase activity after stimuli were applied that reinforced or cancelled one another behaviorally and were directed through different transducer proteins. Modulation of methylesterase activity as a result of the simultaneous addition of parallel or opposing stimuli through the Tsr and Tar transducers suggested that the sensory information detected by these transducers was integrated and that this integrated signal conrolled demethylation. These data are discussed with regard to mechanisms for regulating methylesterase activity.
* Corresponding author. t Present address: DNAX Research Institute of Molecular and Cellular Biology, Inc., Palo Alto, CA 94304.
983
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MATERIALS AND METHODS
Strains. The E. coli strains used were gifts from J. S. Parkinson or M. I. Simon. Strain RP487(X ind) is chemotactically a wild type and requires threonine, leucine, histidine, and methionine for growth. Strain MS5228 is tar tap tsr and requires threonine, leucine, and histidine for growth. Strain RP2867 has the deletion A(tap-cheB) and is isogenic to RP487
(31). Growth of bacteria. Overnight cultures were grown from colonies at 30°C in minimal medium supplemented with 1% glycerol, threonine, leucine, histidine, and methionine as described previously (7, 20). Exponential cultures in tryptone broth supplemented with 1% glycerol were grown at 30°C from the overnight cultures. The cells were washed twice in 10 mM Tris hydrochloride (pH 7.4). Measurement of methanol production. Volatile 3H in cell supernatant fractions was measured by the method of Chelsky et al. (8) and Terwilliger et al. (43). Vapor phase transfer of radioactivity was performed in scintillation vials at room temperature for 14 to 18 h (22). Radioactivity was determined for preset 3H and 14C windows in a Beckman LS7000 liquid scintillation counter with automatic quench compensation. Data calculations that corrected for spillover and computed the ratio of the counts per minute of 3H/14C swarm
were
with
J. BACTERIOL.
KEHRY ET AL.
performed on a Hewlett Packard 87 terminal equipped 82901M flexible disk drive, as described previously
a
(22). Continuous-flow assay. The filter assembly (Gelman acrodisc, 0.2 p.m) used in all experiments was connected at the outlet end to a fraction collector. The inlet (luer fitting) was adapted to peristaltic-pump tubing (0.42 ml/min; Evergreen Scientific) as described in detail elsewhere (22). The filter was washed with 20 ml of 10 mM Tris hydrochloride (pH 7.4)-2 to 4 ml of medium before the addition of cells. Between 1.5 x 109 and 2 x 109 cells were pumped onto the filter and maintained by a continuous flow of medium at 23°C. Fractions of 0.8 min were collected from the time the cells were loaded. The actual flow rate with cells on the filter was 0.38 ml/min. A stimulus consisted of quickly switching the inlet tubing to medium lacking or containing L-aspartate or L-serine (ICN Pharmaceuticals) or NiSO4. The half time of mixing was 3.6 min (22). When all fractions were collected, samples of the medium that flowed over the cells (150 pu) were placed in 0.5-ml microcentrifuge tubes for vapor phase transfer. Two types of flow experiments were performed. In steadystate turnover experiments, cells were equilibrated in medium containing L-[methyl-3H]methionine (80 Ci/mmol; New England Nuclear Corp.) that was dried under a stream of nitrogen before use and a trace of [14C]methanol (3.3 mCi/mmol; New England Nuclear Corp.). A stock solution of [14C]methanol was made (4,000 cpm/,ul) in glass-distilled methanol (Burdick and Jackson). The medium was the same as that used for maintaining cells on the filter and contained 10 mM Tris hydrochloride (pH 7.4), 50 mM sodium succinate (pH 7.4) (Sigma succinic acid), 0.25 ,uM methionine, 2.0 ,uCi of L-[methyl-3H]methionine per ml, and 5 RI of stock [14C]methanol per 9 ml of medium. Preparation of medium was as described previously (22). The second type of flow experiment was a chase turnover. Washed cells were suspended in 1.5 ml of 10 mM Tris hydrochloride-50 mM sodium succinate (pH 7.4) and incubated with 40 ,Ci of L-[methyl-3H]methionine (not dried) at 30°C for 40 to 50 min. The chase was initiated by suspending cells in medium containing nonradioactive methionine at
300-fold the concentration of L-[methyl-3H]methionine (50 mM sodium succinate, 10 mM Tris hydrochloride [pH 7.4], 0.1 mM methionine, 5 RI of stock [14C]methanol per 9 ml of medium). Cells were immediately pumped onto the filter and maintained with the chase medium described above. All other methods were the same as those used in steady-state turnover experiments (22). Rapid-flow chase experiments were the same as chase turnover experiments with minor changes (22). To increase resolution, the flow rate with cells on the filter was increased to 1.2 ml/min, and fractions of 0.3 min (-0.36 ml) were collected. Samples for vapor phase transfer were taken from every third fraction over the initial and final portions of the experiment and from every fraction through the stimulations. Preliminary experiments were performed to assess the toxicity of NiSO4 to cells. Nontoxic conditions were judged to be the concentrations of NiSO4 that allowed cell growth in minimal medium. Inhibition of cell growth was observed at 0.1 mM NiSO4; 0.05 mM NiSO4 was the highest concentration tested that did not inhibit cell growth and was used in all experiments. For several flow experiments the cells were adapted to aspartate at the start of the experiment. In steady-state turnover experiments, 1 mM aspartate was included in the medium used for equilibration. For chase turnover experiments, aspartate was added (to 1 mM) 15 min after the addition of L-[methyl-3H]methionine and increased to 2 mM after an additional 15 min. The chase medium in which the cells were suspended for the start of the experiment contained 1 mM aspartate. This decrease in aspartate concentration (still at saturation) was used to avoid an inadvertent, positive attractant stimulus at the beginning of the chase. RESULTS Chemotaxis-specific changes in the rate of methanol production. The steady-state production of methanol by intact
18 15-
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FIG. 1. Modulation of methylesterase activity after application of serine stimuli in wild-type and nonchemotactic bacteria. The steady-state turnover of methyl groups was measured as described in the text. A stimulus of 75 ,uM serine was applied at the point indicated by the first arrow and was removed at the point indicated by the second arrow along each curve. Symbols: 0, 2 x 109 RP487(A ind) cells (wild type); +, 2 x 109 MS5228 cells (tar tap tsr). The first peak at 2.4 min represents the accumulation of [3H]methanol during suspension and pumping of the cells onto the filter.
VOL. 163, 1985
CONTROL OF ADAPTATION IN CHEMOTAXIS 7
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, wild
type
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Minutes FIG. 2. Modulation of methylesterase activity after combined stimuli were applied through the Tsr and Tar transducers. Multiple stimuli were applied in a steady-state turnover experiment. Symbols: 0, 1.6 x 109 RP487(A ind) cells (wild type); +, 1.6 x 109 MS5228 cells (tar tap tsr). Stimuli were applied to wild-type cells as described in the text. Aspartate (1 mM) and serine (50 ,uM) were applied at 55.2 min to the tar tap tsr mutant and removed at 79.2 min. Results of the experiment with the mutant have been shifted on the abscissa to facilitate a comparison of the effects of the same stimuli on the two strains; the duration of the experiment on the tar tap tsr strain was 48 min.
bacteria was used as an assay for methylesterase activity. We have previously described a simple apparatus with which changes in methylesterase activity produced by attractant and repellent stimulation of cells are measured (22). Briefly, cells were incubated with [methyl-3H]methionine and maintained on a filter (pore size, 0.2 Rxm) assembly by a continuous flow of medium that contained [methyl-3H]methionine and a trace amount of [14C]methanol as an internal standard. Medium that flowed over the cells was collected in fractions, and a sample of each fraction was subjected to vapor phase transfer to measure volatile radioactivity. This measure of the relative rate of [3H]methanol production is a direct measure of the methylesterase activity for that time interval (22). A stimulus was given by switching the flow inlet to a medium that was identical except for the presence or absence of attractant or repellent. Changes in the rate of [3H]methanol production by wildtype cells given the attractant serine are shown in Fig. 1. A positive stimulus of 75 ,uM serine was applied at 15 min. Methylesterase activity was modulated (after a short delay caused by the dead volume of the tubing used) as a transient inhibition in the rate of [3H]methanol production; a gradual recovery to the prestimulus level occurred in 12 to 14 min. This recovery appeared to parallel the period required for behavioral adaptation to the stimulus (22, 45). Removal of serine (a repellent stimulus) at 39 min resulted in a rapid increase in methylesterase activity (peak of [3H]methanol release) that returned to the basal rate in 5 to 6 min. This long recovery time was a consequence of the prolonged negative gradient produced by the mixing time of the filter assembly (22). Behavioral adaptation to a temporal negative stimulus is usually complete in 1 to 2 min. The rate of [3H]methanol production by a mutant lacking the products of the two major transducer genes, tar and tsr, was very low relative to that by the wild type and was unaffected by the serine stimuli (Fig. 1). Inhibition of methylesterase activity by multiple attractant stimuli. The effects of multiple stimuli, given simultaneously or successively, on changes in methylesterase activity were examined in an experiment similar to that which provided the data for Fig. 1. Wild-type cells were stimulated through the Tar transducer with aspartate, through the Tsr transducer with serine, and through both transducers simulta-
neously. The addition of 1 mM aspartate at 15.2 min and its removal produced the usual inhibition and increase, respectively, in the rate of [3H]methanol production (Fig. 2). For aspartate stimuli, recovery from the decrease in methylesterase activity was faster, and the peak of [3H]methanol was always much less than the corresponding responses to serine stimuli. The addition and removal of 50 ,uM senine at 99.2 and 123.2 min, respectively, gave results similar to those in Fig. 1, even though the cells had been maintained on the filter for a much longer time (see Fig. 2). Stimulation of the cells with a combination of aspartate and serine (added at 55.2 min) resulted in an inhibition of [3H]methanol production that was more extended than that produced by the addition of either amino acid alone. Similarly, the removal of the two amino acids at 79.2 min resulted in a peak of methylesterase activity that was greater than the peak produced by either aspartate or serine. In the tar tap tsr mutant, the combined stimuli produced no effect on the rate of [3H]methanol formation (Fig. 2). Although the time required for complete recovery (the adaptation time) increased with the stronger stimulus (compare the second stimulation with the other two stimulations shown in Fig. 2), the overall pattern of inhibition of methylesterase activity with the combined addition of aspartate and serine was similar to the inhibition observed with the individual stimuli. In particular the three positive stimuli produced very similar initial decreases in the rate of [3H]methanol production; all decreases were to a level that was close to the base-line rate. Since the production of [3H]methanol was inhibited to similar extents by a stimulus through either or both major MCP classes, this suggested that the inhibition of methylesterase activity with a Tsr attractant stimulus might also inhibit the turnover of methyl groups on the Tar transducer and vice versa. We therefore peformed more sensitive measurements of the levels to which methanol production was inhibited by various positive stimuli. These measurements were made in rapid-flow chase turnover experiments. In such experiments medium containing nonradioactive methionine was washed over the cells as a chase, and the release of radioactive methanol was assayed by vapor phase transfer. The relative rate of formation of [3H]methanol was observed to continuously decrease as a multiple exponential decay during the chase (Fig. 3A). Positive and negative stimuli were applied
986
KEHRY ET AL.
J. BACTERIOL.
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FIG. 3. Measurement of the degree of inhibition of methylesterase activity by added stimuli in a rapid-flow chase. Cells were labeled with [methyl'3H]methionine for a rapid-flow chase turnover experiment. At 0 min, [methyl-3H]methionine was removed and replaced with medium conitaining a 300-fold excess of nonradioactive methionine. Volatile radioactivity in every third fraction from 0 to 10 min and 35 to 65 min was determined. For this reason most of the peaks of [3H]methanol release produced by attractant removal at 45 min in panels B through D were not observed. Data are expressed as the natural logarithm of the ratio of volatile 3H counts per minute/'4C counts per minute. RP487(X ind) cells (wild type; 2.5 x 109) were used in the experiment shown in each panel. (A) Unstimulated; (B) stimulated with 10 mM aspartate at 15 min (arrow) (attractant was removed at 45 min [arrow]); (C) stimulated with 100 mM AIBU at the points indicated by the arrows as in panel B; (D) stimulated with 10 mM aspartate and 100 mM AIBU as in panel B. as in the steady-state experinients and produced similar results (see Fig. 3). The rapid-flow chase method provided increased resolution and greater sensitivity for observing the stimulus-induced changes in methylesterase activity (22). To quantitate the extent to which methyl turnover was inhibited by positive stimuli during a chase, the change in the rate of [3H]methanol production from the maximum inhibition in the trough to the base-line value that would have occurred at this point if no stimulus were applied (obtained by interpolation across the top of the trough with the unstimulated control [Fig. 3A], which was superimposable) was measured on a semilogarithmic scale (Fig. 3). The antilogarithm of this number indicated the factor by which turnover of the original, labeled (basal) methyl groups had been inhibited by the attractant stimulus. The addition of 10 mM aspartate (Tar stimulus) to wildtype cells in Fig. 3B resulted in a 4.1-fold (or 76%) decrease in the rate of [3H]methanol production from the prestimulus rate. Stimulation with 100 mM aminoisobutyric acid (AIBU; a nonmetabolizable analog of serine and a Tsr attractant) produced a 2.5-fold (60%) inhibition (Fig. 3C). AIBU was used as a Tsr stimulus instead of serine because cells do not adapt behaviorally to high serine concentrations (>0.1 mM)
(3, 23). Stimulation with the
same
concentrations of
aspartate and AIBU together gave a 7.9-fold (87%) inhibition in the rate of [3H]methanol production (Fig. 3D). Thus, a
stimulus through either the Tar protein (aspartate) or the Tsr protein (AIBU) inhibited the rate of [3H]methanol production by significantly more than 50% of the base-line rate. The observation that inhibition of [3H]methanol production by the combined stimuli was only 87% suggested that stimulation of one MCP class may elicit a general tnodulation of methylesterase activity resulting in inhibition of methyl turnover on the other MCP class. Changes in methylesterase activity produced by opposing stimuli. The additivity of opposing stimuli through different transducers was examined in experiments analogous to those described above. Experimentally, this involved a simultaneous treatment of wild-type cells with a positive stimulus through one transducer (e.g., Tsr) and a negative stimulus through the other transducer (e.g., Tar) that resulted in little or no modulation of methylesterase activity. The positive and negative stimuli appeared to cancel or balance one another. Appropriate concentrations of aspartate, NiSO4, and serine were determined empirically in chase turnover experiments (as in Fig. 3) because of the increased sensitivity relative to behavioral experiments. The
CONTROL OF ADAPTATION IN CHEMOTAXIS
VOL. 163, 1985
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appropriately balanced concentrations and several unbalanced concentrations that resulted in either a net Tar or Tsr stimulation were subsequently tested for their effects on the behavior of cells, by visual inspection in the microscope. Similar behavioral studies have been performed by Berg and Tedesco (4) with the nonmetabolizable attractants amethylaspartate (Tar stimulus) and AIBU (Tsr stimulus). In all experiments presented below, we used the simultaneous removal of 1 mM aspartate and addition of 0.05 mM NiSO4 as a negative stimulus through the Tar protein (34, 38, 46) that produced a larger change in methylesterase activity than that observed with aspartate alone. This was balanced by a positive stimulus of 35 jxM serine through the Tsr transducer. Behaviorally, these combined stimuli produced no response that was observable under the microscope. A lower concentration of serine (20,uM) as the positive Tsr stimulus caused a brief period of tumbling when serine was added with NiSO4; increasing the serine to 80 jxM resulted in a net positive Tsr stimulus as indicated by a brief suppression of tumbling. Therefore, the opposing stimuli applied to the cells appeared to elicit little or no net behavioral response. The results of applying these opposing stimuli to wild-type cells in a steady-state turnover experiment are shown in Fig. 4. The simultaneous application of Ni(II) and removal of aspartate produced a peak of demethylation (Fig. 4A). Reversal of the stimuli resulted in the decrease in [3H]methanol production expected from the addition of attractant. Stimulation with serine produced the usual modulation (Fig. 4B). When the same stimuli applied in panels A and B were combined (Fig. 4C), they nearly canceled each other with respect to effects on methylesterase activity. Combining the stimuli resulted in a small net Tar stimulus in both directions. A confirming result was obtained by applying opposing stimuli in a chase turnover experiment. This experiment was more sensitive to small changes in the rate of [3H]methanol production. In addition, the difference in curve shape between the inhibition of [3H]methanol production and the peak of [3H]methanol release allowed us to easily monitor small changes in the rate of demethylation. The stimuli applied in the chase experiments which provided the data for Fig. 5 were the same as those for the experiments illustrated in Fig. 4. For the Tar stimuli, cells were incubated with aspartate and chased on the filter assembly with medium containing aspartate (Fig. 5A). The results of application of the Tsr stimuli are shown in Fig. SB. The modulation of methylesterase activity after these stimuli were applied was as expected from the data in Fig. 4. The simultaneous application to cells of the same stimuli which provided the results in Fig. SA and B produced the curve shown in Fig. SC. Again, the stimuli nearly canceled each other and produced only very small changes in methylesterase activity. The slight negative and positive Tar stimuli (also observed in Fig. 4C) were seen as a very small peak and a small trough, respectively. We observed no significant peaks. The experiment shown in Fig. 5 was repeated three times with identical results. The following experiments were also performed as controls. First, serine was used to stimulate wild-type cells in the continuous presence of 10 mM aspartate. The results were the same as those in the absence of aspartate (e.g., Fig. SB). Second, the combined stimuli used in Fig. SC were applied to a mixture of equal numbers of cells from two strains, RP5884 (tsr deletion) and RP3841 (tar deletion). The results were very different from those observed in Fig. SC and consisted of two peaks of
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Minutes FIG. 4. Modulation of methylesterase activity after the application of opposing stimuli through the Tsr and Tar transducers. Cells [RP487(X ind), wild type] were used for steady-state turnover experiments. (A) 2 x 109 cells were equilibrated for 45 min in 1 mM aspartate before being maintained on the filter. Aspartate was removed, and 0.05 mM NiSO4 was added at the point indicated by the first arrow. The stimulus was reversed at the point indicated by
F.M
the second arow. (B) 1.55 x 109 cells were stimulated with 35 serine at 16.8 min. Attractant was removed at 40 min (arrows). (C) 2 x 109 cells were stimulated with the combined stimuli shown for panels A and B (arrows).
[3H]methanol
shown;
see
release, nearly identical
to Fig.
5D
(data not
below).
DISCUSSION Models for methylesterase regulation. Measurements of stimulus-induced changes in the rate of methyl group turn-
used to study the effects on methylesterase activity of behaviorally reinforcing and canceling stimuli. over were
988
J. BACTERIOL.
KEHRY ET AL. l
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Several mechanisms for regulating the methylation level are discussed below with respect to our findings. In this study we did examine the activity of the methyltransferase enzyme, and our discussion is limited to the methylesterase activity. One proposal for regulation of the activities of methyltransferase and methylesterase has been described in detail (13, 42). This model suggests that methylation and demethylation are controlled by interactions of the enzymes with the transducer proteins. Changes induced in the transducers as a result of chemoreception directly alter their reactivity to the transferase and esterase, which are bound to separate sites on the MCP. Stimulus-induced changes in transducer structure control the degree to which the glutamate residues are exposed to either methyltransferase or methylesterase (42). An alternative model for regulating the methylation level is general or global in nature and proposes that for all transducers in a stimulated cell, the regulation occurs at a point further along the sensory processing pathway than the transducer proteins. Chemoreception by the transducers ultimately results in the generation of an excitatory signal that controls behavior. A behavioral regulator that is generally modulated may feed back from subsequent steps in the sensory processing pathway (for example, via the flagella or various che gene products) to regulate methyltransferase or methylesterase (23). Communication between MCP classes. Studies of the addition of positive stimuli suggested that stimulation through one major MCP class produced a general inhibition of methyl turnover on the other MCP class (Fig. 3). It is likely that Tsr and Tar molecules are not present in equal numbers in an E. coli cell (9, 17). Thus, one class would contribute more to the overall observed rate of methanol production. Nonetheless, we observed that a positive stimulus through either the Tar or the Tsr transducer inhibited methyl turnover by significantly more than 50% of the base-line rate. An 87% inhibition in methylesterase activity was actually produced by a combined stimulation through both MCPs (Fig. 3; see above). Thus, somehow a stimulus through Tsr molecules was communicated to Tar molecules. Both models above are consistent with this observation. However, regulation of demethylation via direct transducerenzyme interactions seems likely only if an interactive complex composed of different MCP classes exists. A stimulus received by one MCP class would be communicated to other MCP classes within the complex. The existence of signaling mutants in the tsr gene that are dominant and alter methylation levels of both Tsr and Tar proteins in the cell
2-
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FIG. 5. Changes in methylesterase activity produced by the application of opposing stimuli through the Tar and Tsr transducers in a chase. Cells [1.4 x 109 RP487(X ind), wild type, used for each experiment in panels A through C] were labeled and maintained for
chase turnover experiments. The data in all panels are expressed in logarithmic form. (A) Aspartate was removed and 0.05 mM NiSO4 was added at the first arrow (16 min). NiSO4 was removed and replaced by 1 mM aspartate at the second arrow (36 min). (B) Serine (35 ,uM) was applied at 16 min and removed at 40 min (arrows). (C) Cells received the combined stimuli used in panels A and B (arrows). (D) The data represented by the curves in panels A and B were added together as described in the text. The second stimulus was offset in panels A and B by 4 min; corresponding portions were therefore matched by adding the data from panel A from 36 to 56 min to the data from panel B from 40 to 60 min. The experiment was repeated three times without the difference in stimulation times between panels A and B. All repetitions gave similar results for the observed and predicted added stimuli. Thus, the duplication of the 4-min unstimulated period in panel A between 32 and 36 min did not affect any features of the predicted result in panel D.
VOL. 163, 1985
(30) is consistent with the notion that signaling properties are communicated between MCP classes. It is not clear, though, that such communication is mediated by direct transducerto-transducer interactions. In particular, since tar and tsr mutants exhibit normal chemotactic responses and changes in methylesterase activity after the appropriate attractant and repellent stimuli are applied (22, 37, 38), a complex of different MCP classes is not a requirement for functional chemotaxis. The alternative possibility that demethylation is regulated in a global fashion throughout the cell is also consistent with the results. In a global mechanism the pathway that regulates demethylation is common to all MCP classes and is modulated based on the stimulated or adapted state of the cell. In this situation the signaling properties of different MCP classes contribute to the common regulatory pathway. The methylesterase activity is then generally modulated by the summed contributions of all MCPs and thus to stimuli mediated by any MCP class. Opposing signals are integrated before regulation of methylesterase activity. Opposing stimuli that produced no behavioral response also produced no net change in the rate of demethylation (Fig. 5C). In the simplest interpretation, the rate of demethylation of each MCP class remained unchanged. Analysis of the data in Fig. 5A-C (see below) suggests that some integrated signal from all MCPs is used to control the methylesterase activity in the cell. If the changes in demethylation rate after Tsr stimuli are applied are regulated directly by Tsr molecules and are independent of the effects of Tar stimuli on the methylesterase (see the models above), we predict that the net change in demethylation rate after the application of opposing Tsr and Tar stimuli would be the result obtained by the simple addition of the stimuli applied individually. In a chase turnover experiment in which two stimuli were exactly equal and opposite to one another (for example, removal versus addition of 35 FLM serine [see Fig. 5B]), the peak of demethylation resulting from the negative stimulus occurred more rapidly and recovered faster to the base-line level than did the trough of inhibition in [3H]methanol production. The numerical addition of such equal and opposing stimuli predicts a peak followed by a slight trough. The predicted results for the addition of the Tsr and Tar stimuli applied in Fig. 5 were obtained as follows. The curves in panels A and B of Fig. 5 were added together, divided by two to normalize background, and graphed as the natural logarithm of volatile radioactivity (Fig. 5D). This added curve showed that a significant peak of [3H]methanol release was predicted to occur after each opposing stimulation. This result is virtually identical to the results obtained by applying the added stimuli to a mixture of cells composed of equal numbers of tsr and tar deletion mutants (see above). In contrast, the in vivo integration of the stimuli in wild-type cells showed no such peaks (Fig. SC) and suggests that each class of transducer protein does not regulate its demethylation independently of other transducer classes. The results of applying opposing stimuli (Fig. 5C) also showed that a positive Tsr stimulus and a negative Tar stimulus balanced one another to exactly the same extent as the corresponding negative Tsr stimulus and positive Tar stimulus. This indicates that the negative signal produced by the removal of 35 ,uM serine is of the same magnitude as the positive signal generated by the addition of 35 ,uM serine. The result was observed in both steady-state and chase turnover experiments. This implies that the corresponding positive and negative stimuli must produce opposite excita-
CONTROL OF ADAPTATION IN CHEMOTAXIS
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tory signals of equal size; therefore, both less and more methylated transducer molecules were effective signalers.
This enables a population of multiply methylated forms of MCPs with various levels of methylation to maintain the adapted state in a cell. The control of methylesterase activity seems to reside at a level different from sensory reception (Fig. 5). This level, which serves as an integrated signal, could represent a common regulator that is in the pathway of sensory information flow. Thus, these results are consistent with a global regulation of demethylation. It is possible, however, that the level of integration may be an interactive complex of different MCP classes that communicates sensory information before generating an excitatory signal. Such an explanation involving complexes must accommodate both the inhibition of demethylation seen with a positive stimulus to a single transducer class (Fig. 3) and integration of positive and negative stimuli to different transducer classes. This is difficult to describe in molecular terms. It seems clear that the majority of methyl groups placed on transducers after a single positive stimulus are specific to the particular transducer class which is stimulated. A global control of methylation and demethylation imparts no such specificity. Thus, there must be some component to the control of the methylation level that provides specificity and acts at the level of the transducer. This may occur in either the methylation or demethylation reaction or both. ACKNOWLEDGMENTS We thank J. S. Parkinson and M. I. Simon for providing E. coli strains; D. Chelsky for describing the vapor transfer assay for methanol before publication; workers in the laboratory of P. Von Hippel for use of computer facilities; and G. Burget for preparation of the manuscript. This work was supported by Public Health Service grant GM24792 from the National Institutes of Health to F.W.D. and Public Health Service grant A106375 from the National Institutes of Health to M.R.K. F.W.D. is the recipient of an American Cancer Society Faculty Research Award. LITERATURE CITED 1. Adler, J. 1975. Chemotaxis in bacteria. Science 166:1588-1597. 2. Berg, H. C., and R. A. Anderson. 1973. Bacteria swim by rotating their flagellar filaments. Nature (London) 245:380-382. 3. Berg, H. C., and D. A. Brown. 1972. Chemotaxis in Escherichia coli analyzed by three-dimensional tracking. Nature (London) 239:500-504. 4. Berg, H. C., and P. M. Tedesco. 1975. Transient response to chemotactic stimuli in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 72:3235-3239. 5. Boyd, A., and M. I. Simon. 1980. Multiple electrophoretic forms of methyl-accepting chemotaxis proteins generated by stimuluselicited methylation in Escherichia coli. J. Bacteriol. 143:809-815. 6. Chelsky, D., and F. W. Dahlquist. 1980. Structural studies of methyl-accepting chemotaxis proteins of Escherichia coli: evidence for multiple methylation sites. Proc. Natl. Acad. Sci. U.S.A. 77:2434-2438. 7. Chelsky, D., and F. W. Dahlquist. 1981. The methyl-accepting chemotaxis proteins of Escherichia coli are methylated at three sites in a single tryptic fragment. Biochemistry 20:977-982. 8. Chelsky, D., N. I. Gutterson, and D. E. Koshland, Jr. 1984. A diffusion assay for detection and quantitation of methylesterified proteins on polyacrylamide gels. Anal. Biochem. 141:143-148. 9. Clarke, D., and D. E. Koshland, Jr. 1979. Membrane receptors for aspartate and serine in bacterial chemotaxis. J. Biol. Chem. 254:9695-9702.
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