Requirement of ATP in bacterial chemotaxis.

Evidence is presented that chemotaxis requires ATP or a closely related metabolite, in addition to its known requirements of ATP for synthesis of S-adenosylmethionine (AdoMet) and maintenance of the proton motive force. Previous studies demonstrated a loss of tumbling and chemotaxis, and depletion of ATP when hisF auxotrophs of Salmonella typhimurium are starved for histidine (Galloway, R. J., and Taylor, B. L. (1980) J. Bacteriol. 144, 1068-1075). In the present study, intracellular [AdoMet], membrane potential, and [ATP] were measured in a hisF mutant of S. typhimurium. Membrane potential, determined from partitioning of [3H]tetraphenylphosphonium ion between the inside and the outside of the cell, was about -150 mV at pH 7.6, and did not decrease in histidine starvation but was slightly increased. The concentration of AdoMet decreased from 0.4 mM to 0.3 mM during starvation but when cycloleucine, an inhibitor of AdoMet synthetase, was used to decrease [AdoMet] by a similar amount in histidine-fed cells there was little change in tumbling frequency. Intracellular [ATP] was reduced from 4.5 mM to less than 0.2 mM by histidine starvation. About 0.2 mM ATP was necessary for spontaneous tumbling. A similar [ATP] was required for tumbling in arsenate-treated cells. Adenine at concentrations as low as 20 nM caused a transient increase in both tumbling frequency and [ATP] in histidine-starved cells. Thus, out of three parameters tested, only the intracellular [ATP] correlated with changes in tumbling frequency in the histidine-starved cells.

A requirement for ATP, in addition to methionine, is suggested by two lines of evidence. Arsenate, which depletes the cell of ATP, abolishes chemotaxis by preventing tumbling (Larsen et al., 1974;Aswad and Koshland, 1975a). Starvation for histidine of S. typhimurium hisF strains greatly decreases the intracellular concentration of ATP and also abolishes chemotaxis by preventing tumbling (Galloway and Taylor, 1980). Depletion of ATP could impair chemotaxis by perturbing one of the known reactions or by an unknown mechanism. Because ATP and methionine are substrates for AdoMet synthetase, earlier discussions assumed that the low ATP concentration in arsenate-treated cells caused depletion of AdoMet (Larsen et al., 1974;Aswad and Koshland, 1975a;Springer et al., 1975). Subsequently, it was reported that tumbling in certain mutants of E. coli and S. typhimurium is eliminated by arsenate treatment but not by methionine starvation (Aswad and Koshland, 1975a;Springer et al., 1975;Kondoh, 1980). This suggests that ATP might be required for chemotaxis, in addition to its role in activating methionine. The possibility that suppression of tumbling is secondary to the effect of arsenate on the proton motive force has not been ruled out. Under appropriate conditions, arsenate can decrease the proton motive force (A&+) across the inner membrane (Khan and Macnab, 1980a). A minimum proton motive force &H+ is necessary to maintain the normal probability of tumbling and when Aj.i~+ falls below this value the probability of tumbling in E. coli and S. typhimurium is diminished and may approach zero (Khan and Macnab, 1980a).
More recently, Arai (1981) reported that a small amount of phosphate can restore tumbling to arsenate-treated E. coli without greatly increasing the concentration of ATP. Furthermore, an uncA mutant has normal tumbling frequency at ATP concentrations that are lower than those in arsenatetreated wild type cells. Arai concluded that a phosphorylated compound other than ATP is required for chemotaxis. There is, therefore, considerable uncertainty about the mechanism of the arsenate effect on chemotaxis. Histidine starvation of hisF strains of S. typhimurium is particularly useful in probing the role of ATP in chemotaxis because it induces adenine starvation in contradistinction to arsenate treatment which induces phosphate starvation. Adenine and ATP are depleted in histidine starvation as a consequence of the loss of feedback inhibition of phosphoribosyltransferase, the fwst enzyme in the histidine biosynthetic pathway (Shedlovsky andMagasanik, 1962a, 1962b). In minimal medium containing glucose, hisF strains cease tumbling about 2 h after they are transferred to histidine-free medium, but vigorous motility continues for more than 24 h (Galloway and Taylor, 1980). Tumbling is rapidly restored in histidinestarved cells by addition of adenine. Swimming speed and adaptation to serine and aspartate are normal in the histidinestarved bacteria, suggesting that the tumbling defect is not 7969 the result of lower Ajk+ or AdoMet depletion. In this study, we measured the AdoMet level and APHf in histidine-starved cells and demonstrated that they are adequate to support tumbling. Close correlations between the ATP level and tunbling frequency under various conditions indicated a requirement for ATP or a closely related metabolite in chemotaxis.
A preliminary account of this work has been presented (Shioi and Galloway, 1981 Research Center, Negev, Israel. Cycloleucine (l-aminocyclopentane-1-carboxylic acid), AdoMet, and SP-Sephadex C-25 were obtained from Sigma. Sodium tetraphenylboron and triphenylmethylphosphonium bromide were obtained from K and K Laboratories, Plainview, NY. Tetraphenylphosphonium bromide was obtained from Aldrich. ATP Monitoring Reagent was purchased from LKB-Wallac.

Growth and Starvation of Cells
Cells were grown at 30 "C in Vogel and Bonner medium E (Vogel and Bonner, 1956), fortified with the auxotrophic requirements of the strain and glucose (7 mg/ml) as the carbon source. Overnight cultures were diluted 1:lO into growth medium and the cells were harvested during the exponential phase of growth (ODm = 0.6 to 0.8) by centrifugation at 6000 X g for 10 min. For histidine starvation, the pellets were washed twice in starvation medium (histidine-free growth medium), resuspended in a volume of starvation medium equivalent to the initial culture volume, and incubated at 30 "C in a shaker incubator. The timing of starvation arbitrarily was commenced at the final resuspension in starvation medium.

Observation of Motility
A small drop (IO p l ) of bacterial suspension was placed on a microscope slide and the bacteria were observed at a magnification of X 500 through a Leitz Dialux trinocular microscope with dark-field optics and an objective lens (L32) with a long working distance. The tumbling frequency of the bacteria was measured by the photographic procedure of Spudich and Koshland (1975). Measurement of swimming velocity has been described previously (Galloway and Taylor, 1980).

AdoMet Assay
Isotope-labeling Method-Aliquots (7 ml) taken from a starved or unstarved culture were labeled for 20 min with 250 p~ ~-[~H]methionine in the presence of 200 pg/ml of chloramphenicol. Six milliliters of labeled culture was removed and quickly added to 4 ml of ice-cold 20% (w/v) trichloroacetic acid containing 5 mM EDTA. To quantitate recovery, unlabeled AdoMet (50 p g ) was added to the trichloroacetic acid extract and the isolated AdoMet was measured spectrophotometrically. After incubation at 4 "C for 20 min, the sample was centrifuged at 20,000 X g for 20 min at 4 "C. AdoMet was determined by the method of Glazer and Peale (1978) with the following mod& cation. The sample loaded on the SP-Sephadex C-25 column (1 X 6 cm) was washed with 50 ml of 0.2 M HCl and AdoMet was eluted with 20 ml of 0.5 M HCl. Two-milliliter fractions were collected at a flow rate of 1 ml/min in test tubes that were pretreated overnight with 0.5 M HC1 to minimize background UV absorbance. The concentration of standard AdoMet was determined from the absorbance at 257 nm. Radioactivity was measured by counting 0.5-ml aliquots in 5 ml of BetaPhase scintillation mixture (WestChem Products, San Diego, CA) with a Beckman LS7500 liquid scintillation counter. AdoMet appeared in a single peak eluted by the step pH gradient but was not present in these fractions if the trichloroacetic acid extract was heated in boiling water. In unstarved cells, about 0.1% of the radioactivity in ~-[~H]methionine edded to the external medium appeared in AdoMet isolated from the bacteria. The recovery of AdoMet was 77 f 8%. The intracellular concentration of AdoMet was calculated on the assumption that 1 A unit at 600 nm corresponds to 0.68 pl of cell volume/ml. Spectrophotometric Method-The entire 200 ml of starved or unstarved culture was centrifuged at 6,000 X g for 10 min. The pellet was resuspended in 7 ml of starvation or growth medium, and the optical density at 600 nm was determined. Six milliliters of the cell suspension was mixed with 4 ml of the ice-cold 20% (w/v) trichloroacetic acid and [3H]AdoMet was added to quantitate the recovery. AdoMet was extracted and chromatographed by the procedure used for labeled AdoMet and the concentration was determined from absorbance at 257 nm.

Alteration of Intracellular AdoMet Level
Cycloleucine, an inhibitor of AdoMet synthetase, was ground finely in a mortar and added directly to the cell suspension which had been preincubated with ~-[~H]methionine for 10 min in the presence of 200 pg/ml of chloramphenicol. After incubation for 30 min, cell motility was analyzed and the cell suspension was treated with cold trichloroacetic acid solution and assayed for AdoMet.

EDTA Treatment
The permeability of the intact cell to TPP+ varied from 1 day to another. The cells were consistently permeable after treatment with EDTA by the procedure of Leive (1965) or Szmelcman and Adler (1976), but these treatments partly reversed the behavioral change observed in histidine-starved cells. By simplifying the procedure as described below, the smooth motility in histidine-starved cells was successfully preserved. Cells were harvested by centrifugation at 6,000 X g for 8 min and the pellet was resuspended in 0.1 M Tris-HC1, 10 mM EDTA (pH 8.0) to one-fortieth of the initial volume. After incubation for 1 min at 30 "C, the suspension was diluted 1:lO into motility medium (pH 7.1) which consisted of 10 mM potassium phosphate, 0.1 mM EDTA, and 20 mM potassium lactate. The pH of the final suspension was 7.6 and the cell concentration was about 1.4 X lo9 cells/ml. The permeability to TPP' of S. typhimurium treated by this simplified procedure and resuspended in motility medium was similar to the permeability of cells treated by the complex procedure (Szmelcman and Adler, 1976) and of the permeable S. typhimurium mutant SL3730. The cells remained permeable for more than 30 min after treatment. S. typhimurium treated by the simplified procedure was not permeable to TPP+ if resuspended in medium of high ionic strength. Therefore, treated cells could not be transferred into growth medium or starvation medium for measurement of membrane potential.
Measurement of Membrane Potential TPP+ uptake was determined by the method of Shioi et al. (1980) with the following modification. Permeable S. typhimurium suspended in motility medium (0.25 to 0.75 ml) was incubated with 10 PM [3H]TPPf at 30 "C with vigorous shaking. The optimal concentration of cells had an optical density at 600 nm of 2 (1.4 X lo9 cells/ml).

At higher densities, the cells became anaerobic. Aliquots ( s d p l )
removed after 3, 4, and 5 min were diluted with 2 ml of the motility medium, filtered on Millipore filters (EHWP, pore size 0.45 pm), and washed with 2 ml of the motility medium. Between 20% and 40% of the total TPP' was retained on the filters. Nonspecific binding of the radioactivity to the cells was small and was corrected for after treating the cells with 2% toluene. The uptake of TPP+ reached a maximum after 3 min and gradually decreased after 5 min. Membrane potential (A+) was calculated from the partitioning of E3H]TPP' between the inside and outside of the cells using the Nernst equation. The uptake of TPMP' in the presence of sodium tetraphenylboron gave similar A+ values but equilibrated more slowly.

ATP Assay
Aliquots of the trichloroacetic acid extract were diluted more than 100-fold with 0.1 M Tris-HC1, 2 mM EDTA buffer (pH 7.75) or were extracted with ether three times. A purified luciferase ATP Monitoring Reagent (LKB-Wallac) was used to determine ATP because it produced a relatively constant luminescence. Luminescence was counted for 0.1 min with a Beckman LS7500 liquid scintillation system with the coincidence circuit off (Lundin and Thore, 1975). The minimal detectable ATP concentration was lo-" M. The assay was performed in duplicate and corrected for an extracellular ATP concentration of about 15 nM.

RESULTS
Behavior in Histidine Starvation-S. typhimurium ST171 has auxotrophic requirements for histidine and thymine, and has a constantly tumbling phenotype in which the bacterium performs an erratic tumbling motion in the unstimulated state. Various treatments induce translational movement termed "smooth swimming." The motility was quantitated photographically using a 1-s exposure of bacteria illuminated with a 4.7 Hz stroboscopic light source (Macnab and Koshland, 1972;Spudich and Koshland, 1975). Each swimming bacterium was represented on the photographic negative by a sequence of five images. The bacterium was scored as "tumbling" if the images were superimposed or indicated an acute change in direction, or "smooth" if the images formed a straight, or gently curving line. Smooth tracks were essen-tiaUy absent from photographs of a normal motility of ST171. When ST171 was starved for histidine in minimal medium containing glucose and thymine, the total population of cells changed to smooth swimming over a short time interval, about 2 h after washing (Fig. 1). On the other hand, thymine starvation in the growth medium did not change the behavior at a l l (data not shown).
Some requirements for suppression of tumbling by starvation were investigated (Fig. 1). There was little change in tumbling frequency when ST171 was starved for histidine in chemotaxis buffer (10 m~ potassium phosphate, pH 7.1, 0.1 m~ EDTA, 7 mg/ml of glucose). The addition of Mg' to the chemotaxis buffer slightly accelerated the decrease in tumbling frequency during starvation and the addition of NH4' caused a 50% decrease in tumbling. The simultaneous addition of M P and NH,' resulted in an even greater effect on The cells were harvested, washed, resuspended in the histidine-free media indicated, and incubated at 30 "C. At intervals, motility was photographed using a 1-s exposure as described in the text. For each datum, more than 50 traces of swimming bacteria were examined and traces that had one or more abrupt changes in direction or superimposed images were scored as tumbling. 0, glucose motility medium (10 mM potassium phosphate, pH 7.0,O.l m~ EDTA, 39 mM glucose, 160 PM thymine); 0, motility medium with added MgS04 (1 mM); A, motility medium with added (NH4)2S04 (10 mM); 0, motility medium with added MgS04 and (NH4)zSOa; ., Vogel and Bonner medium E with glucose and thymine. Histidine starvation and analysis of behavior was in medium E as described in Fig. 1. AdoMet (0) was determined by the isotopelabeling method described under "Experimental Procedures."  Comparison of tumbling frequency and intracellular concentration of AdoMet in S. typhimurium ST171 in the preaence of cycloleucine. ALiquots (7 ml) of a L-histidine-fed culture of S. typhzmuriurn ST171 were incubated at 30 "C and chloramphenicol (final concentration, 200 pg/ml) and cycloleucine (at the indicated final concentration) were added at 0,5, and 15 min, respectively. After a further 30-min incubation, tumbling was analyzed (see Fig. 1) and the cell suspension was extracted with cold trichloroacetic acid (8%) for AdoMet assay (see Fig. 2). 0, intracellular concentration of AdoMet; 0, percentage of tumbling cells.
tumbling in histidine starvation. It is likely that a nitrogen source stimulated protein synthesis and thereby facilitated depletion of the intracellular histidine pool. In the following experiments, bacteria were starved for histidine in medium E which includes both NH4+ and M e .
AdoMet Level in Histidine Starvation-To determine whether smooth swimming was caused by AdoMet depletion, the intracellular concentration of AdoMet was monitored during the course of histidine starvation. In the isotope-labeling procedure, ~-[~H]methionine added to the medium was rapidly transported into the cell and incorporated into [3H] AdoMet. Due to a rapid turnover of AdoMet, it was necessary to add at least 0.25 mM methionine to the medium in order to achieve a steady state level of intracellular r3H]AdoMet (data not shown). The steady state AdoMet concentration of unstarved S. typhimurium ST171 was a function of the extracellular methionine concentration, and was 0.4 mM in the presence of 0.25 mM methionine and 1.4 mM in the presence of 5 mM methionine. Using the spectrophotometric procedure, the intracellular AdoMet concentration was found to be 0.32 mM in methionine-free medium.
When ST171 was starved for histidine, there was a 30% decrease in the intracellular concentration of AdoMet that was concomitant with the change of behavior (Fig. 2). The observed decline in the AdoMet level was not an artifact of the isotope-labeling method and was confirmed using the spectrophotometric assay for AdoMet (data not shown).
In order to evaluate the significance of a 30% decrease in AdoMet, the intracellular concentration of AdoMet in unstarved S. typhimurium ST171 was decreased by addition of cycloleucine, an inhibitor of AdoMet synthetase (Lombardini et al., 1970). A logarithmic increase in the concentration of cycloleucine, over the range 10 mM to 400 mM, caused a linear decrease in the concentration of AdoMet (Fig. 3). The unstimulated behavior of ST171 was affected by reduction of AdoMet to low concentrations but a 30% decrease in AdoMet level had little effect on the tumbling frequency, even though the same decrease in AdoMet doubled the response times to L-serine and L-aspartate. Therefore, the 30% decrease of AdoMet level cannot explain the drastic decrease in tumbling frequency in histidine starvation.
Confirmation of this conclusion was found in two additional experiments. Tumbling was restored to histidine-starved cells within a few seconds of the addition of 10 PM adenine, but more than 15 s was required for restoration of the AdoMet level (Fig. 4A). In the presence of 100 mM cycloleucine, adenine restored tumbling quickly, but did not increase the AdoMet level (Fig. 4B).

The Proton Motive Force in Histidine Staruation-Whenever the probability of tumbling in bacteria is diminished as the result of a decrease in A j i~+ ,
the swimming speed is also diminished (Khan and Macnab, 1980b;Laszlo and Taylor, 1981). The swimming speed of S. typhimurium ST171 was found to be constant during histidine starvation although the tumbling frequency changed drastically (Fig. 5A). This suggested that A j i~+ remained above the saturating level for swimming velocity and for tumbling.
The membrane potential of intact ST171, determined from the distribution of C3H]TPP+ between the intracellular space and the extracellular medium, was about -150 mV and was slightly increased, rather than decreased, by histidine starva- Histidine starvation and analysis of tumbling frequency were carried out as described in Fig. 1. A, swimming speed (0) and tumbling frequency (0) measured during starvation. The photographic procedure for measuring swimming speed was similar to the procedure for determining tumbling frequency except that serine (final concentration 1 RIM) was added to ST171 to induce smooth swimming. The photographic negative was projected and the speed was calculated from the migration of a bacterium during light flashes (Galloway and Taylor, 1980). B, A$ (A) and tumbling frequency (0) during starvation. At intervals, an aliquot of the culture was removed, treated by the simplified EDTA procedure (see "Experimental Procedures"), and photographed for tumbling analysis. A$ was determined from TPP" uptake as described under "Experimental Procedures." tion ( Fig. 5B). Because the membrane potential (A+) is the only significant component of the proton motive force at an external pH of 7.6 (Zilberstein et al., 1979 Khan andMacnab, 1980b;Felle et al., 1980;Slonczewski et al., 1981), the results indicated that AjiH+ of the cells was not diminished by histidine starvation. Correlation of ATP Concentration and Tumbling Frequency-In contrast to AJ, and the concentration of AdoMet, the intracellular concentration of ATP was greatly reduced in histidine starvation (Fig. 6A). The ATP level began to decline during cell washing and was 10% of normal after 1 h of histidine starvation. When the ATP level decreased to less than 0.3 mM (5% of normal), there was a steep drop in tumbling frequency, and very low concentrations of ATP correlated with complete suppression of tumbling.
The addition of 1 VM adenine to histidine-starved cells restored tumbling within 3 s with a gradual increase in ATP concentration in the cells (Fig. 6B). Even 0.1 VM adenine induced a transient recovery of tumbling. Adenine uptake also increased the concentration of ATP, and, at the lowest concentration of adenine, the time course of the change in ATP corresponded to the transient change in tumbling frequency. Although the change in ATP level induced by 0.1 VM adenine was small, it was nevertheless significant as is evident from a similar experiment where the concentration of ATP was plotted on an expanded scale (Fig. 7).
In Fig. 7, the concentration of adenine added to histidinestarved ST171 varied from 10 nM to 100 nhf; and at each concentration, there was a correlation between the percentage of tumbling cells and the concentration of ATP. The increase in intracellular ATP concentration from 0.15 mM to 0.17 m~ in Fig. 7B is equivalent to depletion of 35% of the 20 nM extracellular adenine. In the previous figures, cells were scored as tumbling if they tumbled once or more during a 1-s exposure used to record motility. This measure of total tumbling cells included constantly tumbling cells and the random cells that tumbled during the exposure. In Fig. 7, the total tumbling cells and the constantly tumbling cells are shown separately, and both estimates of tumbling frequency correlate with the ATP level.
The increase in ATP concentration and tumbling frequency induced by adenine was not affected by the presence of 100 mM cycloleucine (data not shown), although tbe increase in AdoMet concentration was completely abolished. Guanine restored tumbling poorly and cyclic AMP and cyclic GMP were ineffective in restoring tumbling. Adenine was not a repellent because it did not induce tumbling in ST23, the che+ strain of S. typhimurium that is isogenic with ST171. ATP Level in Arsenate-treated Cells-The controversy over the mechanism by which arsenate suppresses tumbling in bacteria has been reviewed (see the introduction). Arsenate (10 mM) suppresses tumbling and depletes E. coli of ATP. The addition of 1 mM phosphate to the arsenate-treated cells restores tumbling with only a slight increase in the ATP level (Arai, 1981). As a result of these observations, it has been proposed that a phosphorylated compound other than ATP is required for chemotaxis (Arai, 1981). However, we converted the ATP concentrations obtained by Arai (1981) into intracellular concentrations (millimolar) by assuming that 1 mg of protein is equivalent to 1.5 p1 of cell volume. The results indicated that the ATP concentration required for tumbling in E.coli (0.3 nmol/mg of protein or 0.2 mM) is similar to the level required in S. typhimurium (0.2 m) in Fig. 6. For further confmation of the ATP requirement for tumbling, we treated S. typhimurium ST171 with 10 mM aresenate and then added 1 mM phosphate (Fig. 8). The correlation between ATP concentration and tumbling frequency was similar to FIG. 8. Intracellular ATP concentration (0) and tumbling frequency (0) in arsenate-treated S. typhimurium ST171 before and after the addition of sodium phosphate. ST171 cells, grown as described in Fig. 1, were washed and resuspended in medium (pH 7.0) containing 10 mM sodium arsenate, 0.1 mM EDTA, and 10 rrm sodium lactate (Arai, 1981). The cells were incubated at 30 "C for 130 min before 1 mM sodium phosphate (pH 7.0) was added to the cell suspension. Motility and ATP assays were performed as described in Fig. 6. After 4 h of L-histidine starvation, various concentrations of adenine were added to the culture. ATP concentrations (0) were assayed as described in Fig. 6. Behavior was assayed as described in Fig. 1. A constantly tumbling cell (V) was defined as one that appeared as a blurred spot on the photographic negative (see "Experimental Procedures" for details).  Fig. 1, were washed and resuspended in medium E supplemented with succinate (20 m~) and thymine (160 VM). Tumbling frequency (0) and ATP (0) assays were performed as described in Fig. 6 FIG. 10. Effect of chloramphenicol on the thistidine-starved S. typhimurium ST171. ST171 was starved for histidine for 4 h as described in Fig. 1. After chloramphenicol (200 pg/ml) was added at time zero, tumbling frequency (0) and ATP (0) were assayed by the procedures described in Fig. 6. that observed in histidine-starved cells (Figs. 6 and 7), and it is likely that the concentration of ATP regulates tumbling frequency in arsenate-treated cells.
Preservation of Tumbling in Histidine-starved Bacteria-Several variables counteract the suppression of tumbling in histidine starvation. These include addition of chloramphenicol, simultaneous starvation for methionine and histidine, and the carbon source (Galloway and Taylor, 1980). Chloramphenicol inhibition and methionine starvation were assumed to decrease utilization of L-histidine and ATP by stopping protein synthesis. If tumble suppression is the result of ATP depletion, conditions that prevent or reverse the loss of tumbling should increase the concentration of ATP in histidine-starved cells. This was found to be true for two conditions that we investigated. When histidine starvation of S. typhimurium ST171 was carried out in succinate medium rather than glucose medium, the loss of tumbling was delayed by approximately 3 h (Fig. 9). Compared with cells in glucose medium (Fig. 2), there was a slower decline in ATP level in ST171 in succinate medium and this accounted for the delayed loss of tumbling. Chloramphenicol (200 pg/ml) restored tumbling in histidine-starved ST171 and there was a concomitant increase in the ATP level (Fig. 10).

DISCUSSION
In bacterial chemotaxis, migrational behavior is accomplished by modulating the frequency of tumbling in the swimming cells. As a result, bacteria that are deficient in tumbling are also deficient in chemotaxis. In S. typhimurium and E. coli, a minimum concentration (0.2 mM) of intracellular ATP was required to maintain spontaneous tumbling and chemotaxis. A direct relationship between the ATP concentration and tumbling frequency is indicated by the following evidence. (i) In cells that were starved for histidine in minimal medium containing glucose, cessation of tumbling occurred after 2 h and was concurrent with depletion of ATP (Fig. 6). (ii) In succinate medium, the ATP level declined more slowly in histidine starvation and suppression of tumbling was delayed until ATP decreased to about 0.3 m~ (Fig. 9). (iii) Low concentrations of adenine rapidly and specifically restored tumbling in histidine-starved bacteria. The increase in tumbling after addition of 20 nM to 100 n~ adenine was transient and paralleled a transient increase in ATP (Fig. 7). (iv) The addition of chloramphenicol to histidine-starved cells restored tumbling and the recovery of normal behavior was coincident with an increase in ATP level (Fig. 10). (v) The relationship between ATP and tumbling frequency was quantitatively similar in histidine starvation and in arsenate treatment (compare Figs. 6 and 8). That is, depletion of adenine and depletion of phosphorylated compounds have similar effects on behavior. This strongly suggests that a phosphorylated adenylate compound is required for tumbling. In adenine-depleted bacteria, factors which increased the energy charge (e.g. inhibition of protein synthesis by chloramphenicol) also aided recovery of tumbling. We conclude that the essential adenylate compound is ATP, although it is possible that a metabolite derived from ATP is the active agent.
The ATP requirement in chemotaxis is independent of the requirement for AdoMet and Aj&+, In histidine starvation, the level of AdoMet decreased by 30%, but on the basis of Fig. 3, this would account for a 3% decrease in tumbling frequency, not the 90% decrease in tumbling that was observed (Fig. 2). Furthermore, recovery of tumbling after addition of adenine was independent of change in AdoMet concentration (Fig. 4). Histidine starvation did not alter the swimming speed of S. typhimurium although it increased membrane potential slightly (Fig. 5). An increase in potential would not cause prolonged smooth swimming.
The elimination of AdoMet or membrane potential as the basis for the loss of tumbling in adenine-depleted cells, implies that ATP has a novel, unidentified role in chemotaxis. This laboratory recently demonstrated that a methylation-independent sensory adaptation mechanism is involved in aerotaxis and in chemotaxis to phosphotransferase substrates (Ni-wan0 and Taylor, 1982). A methylation-independent adaptation to repellents was reported by Stock et al. (1981). It is possible that ATP is required in one of these adaptation mechanisms and histidine starvation wiII be a useN probe to explore this possibility.
The ATPase-deficient E.coZi AN120 (uncA) continues to tumble in arsenate medium until the intracellular ATP concentration decreases to extremely low levels (0.03 nmol/mg of protein or 0.02 mM) (Arai, 1981). This suggests that the ATP threshold for tumbling is lower in AN120 than in uric+ strains. The minimum concentration of ATP required for chemotaxis might also be lowered by adaptation. In histidine starvation, we consistently observed that the ATP level required to support a given tumbling frequency was slightly lower after S. typhimurium adapted to depletion of ATP. For example, compare the ATP levels in the depletion and recovery phases in Fig. 8. Although these differences were small they were observed in all our experiments.
The relationship between AdoMet concentration and spontaneous tumbling frequency suggests that the steady state tumbling frequency is also regulated by the absolute level of methylation of the methyl-accepting chemotaxis proteins. If cycloleucine, which is not a chemoeffector, was added to S. typhimurium ST171 that had previously adapted to attractants in the medium, the spontaneous tumbling frequency in ST171 was only slightly affected by small changes in the concentration of AdoMet (Fig. 3), but if AdoMet was decreased to very low concentrations the probability of tumbling was sharply reduced (data not shown). This is consistent with the phenotype of strains which are defective in methylation (Springer et aZ., 1979;Koshland, 1981;Parkinson, 1977).