Please see attached files
Proc. Nat. Acad. Sci. USA Vol. 71, No. 10, pp. 3896-3900, October 1974 A Protonmotive Force Drives ATP Synthesis in Bacteria (themiosmotic hypothesis/membrane-bound ATPase/membrane potential/valinomycin/ATPase-negative mutants) PETER C. MALONEY, E. R. KASHKET, AND T. -HASTINGS WILSON Department of Physiology, Harvard Medical School, Boston, Massachusetts 02115 Communiwed by DeWit Stetten, Jr., July U2, 1974 ABSTRACT When cells of Streptococcus lactis or Escherichia coli were suspended in- a potassium-free medium, a membrane potential (negative inside) could be artificially generated by the addition of-the potassium ionophore, valinomycin. In 'response to this inward directed protonmotive force, ATP synthesis catalyzed by the mnembrane-bound'ATPase (EC 3.6.1.3) was observed. The formation of ATP was not found in S. lactis that had been treated 'with the ATPase inhibitor, NN'dicyclo- hexylcarbodiimide, nor was it observed in a mutant -of E. coli lacking the ATPase. Inhibition of ATP synthesis in S. lactis was also observed when the membrane potential was reduced by the' presence of external potassium, or when cells were first incubated with the proton conductor, carbonylcyanidefluoro-methoxyphenyihydrazone. These results are in agreement with predictions made by the chemiosmotic, hypothesis of Mitchell. In microorganisms the membrane-bound ATPase (EC 3.6.1.3; ATP phosphohydrolase) plays a central role in both aerobic and anaerobic energy transductions. Studies of ATPase-deficient mutants of Escherichia coli have led to the conclusion that one function of the ATPase is to catalyze the synthesis of ATP during oxidative phosphorylation (1-5). A second function of this enzvme, distinguished under anaerobic conditions, is thought to be the coupling of ATP hydrolysis to essential membrane events that require the expenditure of metabolic energy. In the absence of respiration, ATPase- negative mutants cannot utilize ATP from substrate level phosphorylations to drive the ATP-linked transhydrogenase (6, 7)'or the accumulation of various metabolites (3, 5, 8). Such anaerobic function of the ATPase is also suggested by the effects of NN'-dicyclohexylcarbodiimide (DCCD), an in- hibitor of this enzyme (9, 10). In E. coli, DCCD blocks both the ATP-linked transhydrogenase (7) and the accumulation of proline found under anaerobic conditions (11). DCCD also inhibits active transport of metabolites in Streptococci, which lack oxidative metabolism (9, 12, 13). These observations are in agreement with predictions made by the chemiosmotic. hypothesis of Mitchell (14, 15; for a re- view see ref. 16). According to this view, oxidation of sub- strates by the electron transport chain leads to the net transfer of protons (H+) from the inside to the outside of the cell. This extrusion of protons establishes a gradient of p1I (interior alkaline) as well as a membrane potential (interior negative). Mitchell has proposed that ATP synthesis during- oxidative phosphorylation occurs when protons, moving down their electrochemical' gradient, reenter the cell via the ATPase (Fig. 1A). Thus, the electrochemical potential of protons (the protonmotive force) provides the driving force for ATP syn- thesis. The alternative (anaerobic) function of the ATPase is required when protons cannot be extruded by the respiratory chain. Under these conditions, the ATPase couples the hy- drolysis of ATP to the electrogenic movement of protons out of the cell (Fig. 11B). The protonmotive force generated by ATP hydrolysis is then utilized by energy-dependent reactions such as the "ATP-linked" transhydrogenase, or the active transport of metabolites. Evidence in support of this anaerobic function of the ATPase has been presented by Harold and his collaborators, who have studied the anaerobe S. fecalis (faecium). They showed that glycolyzing cells establish both a pH gradient (interior alkaline) and a membrane potential (interior nega- tive), and that DCCD inhibits the formation of each of these components of the protonmotive force (17-19). More recently, West and Mitchell, studying membrane vesicles from E. coli, have shown that ATP hydrolysis is associated with the move- ment of protons across the membrane (20). In microorganisms, the evidence in support of the chemios- motic hypothesis remains incomplete without the direct demonstration of ATP' synthesis driven 'by a protonmotive force. The experiments reported here show that the mem- brane-bound ATPase catalyzes the synthesis of ATP when an inward directed protonmotive force is imposed across the cell membrane. MATERIALS AND METHODS Cultures of Streptococcus lactis (ATCC 7962) were grown to early stationary phase, by described methods (21). Cells were harvested by centrifugation, washed twice with 0..1 M sodium phosphate (pH 6) unless otherwise indicated, and resuspended in a small volume of this same buffer. Wild-type E. coli strain 1100 and its ATPase-negative derivative,- strain 72, were obtained from T. H.- Yamamoto. Strains 1100 and 72 were grown at 370 in medium 63 supplemented with 1% (w/v) A ATP ADP + Pi B PROTON ENTRY PROTON EXTRUSION (AEROBIC) (ANAEROBIC) FIG. 1. The ATPase of bacteria. (A) Proton entry coupled to ATP synthesis occurs in aerobic organisms or in facultative anaerobes (e.g., E. coli). (1B) Proton extrusion coupled to ATP hydrolysis occurs in anaerobes (e.g., S. lactis) or in Jacultative anaerobes. 3896 Abbreviations: DCCD, NN'-dicyclohexylcarbodiimide; CCFP, carbonylcyanidefluoromethoxyphenylhydrazone. ATP Synthesis Driven by a Protonmotive Force 3897 2E 3.0 |\ 0. B GLUCOSEIE-0 2.0 -J VALINOMMINUE FIG. 2.- Comparison of ATP levels in S. latit treated with valinom'ycin 'or glucose. Cells were washed and resuspended in 0.1 M sodium phosphate (pH 6) and diluted with this same buffer (final volume of 5 ml) to a cell density of 176 -Mett units. After samples were removed for measurement of zero-time ATP levels, either 0.1 ml of 1.25 M glucose (25 mM final concentration) or 5 ;1of 10 mM valinomycin (101AuM final concentration) was added. At the indicated titnes, aliquots were removed for the determi- nation of intracellular ATP concentrations. Difco-Bacto Tryptone, 0.5% (w/v) glucose, and 1 ;4g/ml of thiamine. All experimental procedures were done at 23°_ unless other- wise indicated. ATP was measured by use of firefly extract by the procedure of Cole et al. (22). Cell density was determined turbidimetrically with a Kle-tt-Summerson calorimeter (no. 42 filter). The intracellular concentration of ATP was calculated from the known relationship between intracellular water volume and cell density. For S. lactis, 1 ml of a cell suspension of 100 Klett units is equivalent to 0.24 IAI of intracellular water or 165 ug dry weight (23). The 'corresponding relationship for E. coli is 0.6 IAI of cell water or 220 ;&g dry weight (24). Firefly extract (FLE-50) wa's obtained from Sigma Chem- ical Co. Valinomycin was purchased from Calbiochem. Corp., and DCCD was obtained from Baker Chemical Co. Carbonyl- cyanidefluoromethoxyphenylhydrazone (CCFP) was a gift of Dr. E. P. Kennedy. Valinomycin, DCCD, and CCFP were added to cell suspensions as small volumes of stock solutions in 95%O ethanol; final ethanol concentrations were never more than 0.2%. RESULTS Valinomycin-Induced ATP Synthesis in Streptococcus lacti8. An inward directed protonmotive force was artificially gen- erated by treatment of cells with valinomycin. This ionophore makes the cell membrane highly permeable to the potassium ion, and the efflux of K+ establishes a membrane potential, interior negative (25). The basic observation is illustrated by the results presented in Fig. 2. Cells from- the stationary phase of growth were washed and resuspended in a potassiumn-free medium. After they were sampled to determine the basal level of ATP, valinomycin was added. The' addition of valinomycin resulted in a rapid increase in the intracellular level of ATP, followed by a somewhat slower decline. The peak level (2.8 mM ATP) i -0.8 0. CONTROL j 0.6 sdu0.4Zp l u 5~~~~~~~~~~1feter9%ehaoCr0.CDCDn9%etao a z 0.2 + DCCD 00 ~~~MINUTES3 4 5 FIG. 3. Effect of DCCD on ATP sy-nthesis in valinomycin- treated S. lactig. Cells were washed and resuspended in 0.1 M sodium phosphate (pH 8). To 0.6 ml of cells (13000 Klett units), 5 ,d of either 95% ethanol or 0.1 M. DCCD in 95% ethanol was added (final concentration of DCCD was 0.83 mM). After 30 min the cells were centrifuged and resuspended in 0.6 ml of 0.1 M sodium phosphate (pH 5). Fifteen minutes later they were diluted with this same buffer to a final cell density of 260 Klett units. After samples were removed for measurement of zero-time ATP, each suspension (either DCCD- or ethanol-treated) was divided into two portions. To one portion, valinomycin (10 pM final concentration) was added and samples were removed at the indicated times. (Inset) To the second portion, glucose was added (25 mM final concentration) and samples were removed after 25 min for determination of ATP levels. was attained after 1 min and represented a 19-fold increase over the initial level (0.15mM ATP). Within 5 min, ATP had returned to about its original concentration. In this experi- ment the valinomycin-induced synthesis of ATP was com- pared to that found when cells were presented with glucose. The ATP generated by glycolytic reactions attained a stable level of about 2.4mM ATP after an initial "overshoot." Thus, the maximum level of ATP observed in valinomycin-treated cells was comparable to the steady-state level of ATP found in glycolyzing cells. Other experiments showed that the valino- mycin-induced ATP synthesis was dependent on the pH of the incubation medium. Between pH 4 and pH 6, the kinetics of formation of ATP were as described in Fig. 2. At pH 7, maximal levels of ATP were only 3-fold increased over the basal value; at pH 8, no ATP synthesis was detected. Inhibition of Valinomycin-Induced ATP Synthesis in Streptococcus lacti. To determine whether the valinomycin- induced synthesis of ATP required the activity of the ATPase, the effect of the inhibitor, DCCR, was studied (Fig. 3). In cells previously exposed to DCCD, no ATP formation was found after addition of valinomycin. However, DCCD-treated cells retained the capacity to generate ATP from substrate level phosphorylations. When incubated with glucose, both control and DCCD-treated cells attained similar levels ofATP (inset to Fig. 3). The ratio of intracellular to extracellular potassium deter- mines the size of the membrane potential present in valino- mycin-treated cells. If ATP synthesis in such cells depends on Proc. Nat. Acad. Sci. USA 71 (1974) 3898 Microbiology: Maloney et al. f 2.0 z 1.0 0 mM K 1 mMK 0 1 2 3 4 5 MINUTES FIG. 4. Effect of external potassium pd ATP synthesis in valinomycin-treated S. lactis. Cells were washed and resuspended in 0.1 M sodium phosphate (pH 6) and diluted to a final cell density of 240 Klett units using this same buffer containing 1KC0 at the indicated concentrations. After samples were removed for measurements of zero-time ATP levels, vallnomycin was added (10 sM final concentration) and later samples were withdrawn at the indicated times. No ATP synthesis was observed after valinomycin was added to cells