Proc. Nat. Acad. Sci. USA
Vol. 71, No. 10, pp XXXXXXXXXX, October 1974
A Protonmotive Force Drives ATP Synthesis in Bacteria
(themiosmotic hypothesis/mem
ane-bound ATPase/mem
ane 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 o
Escherichia coli were suspended in- a potassium-free
medium, a mem
ane potential (negative inside) could
e artificially generated by the addition of-the potassium
ionophore, valinomycin. In 'response to this inward
directed protonmotive force, ATP synthesis catalyzed by
the mnem
ane-bound'ATPase (EC XXXXXXXXXXwas observed.
The formation of ATP was not found in S. lactis that had
een treated 'with the ATPase inhibitor, NN'dicyclo-
hexylca
odiimide, 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 mem
ane potential
was reduced by the' presence of external potassium, o
when cells were first incubated with the proton conductor,
ca
onylcyanidefluoro-methoxyphenyihydrazone. These
esults are in agreement with predictions made by the
chemiosmotic, hypothesis of Mitchell.
In microorganisms the mem
ane-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 XXXXXXXXXXA
second function of this enzvme, distinguished under anaerobic
conditions, is thought to be the coupling of ATP hydrolysis to
essential mem
ane 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'-dicyclohexylca
odiimide (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
y 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 transfe
of protons (H+) from the inside to the outside of the cell. This
extrusion of protons establishes a gradient of p1I (interio
alkaline) as well as a mem
ane potential (interior negative).
Mitchell has proposed that ATP synthesis during- oxidative
phosphorylation occurs when protons, moving down thei
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
equired 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 mem
ane potential (interior nega-
tive), and that DCCD inhibits the formation of each of these
components of the protonmotive force XXXXXXXXXXMore recently,
West and Mitchell, studying mem
ane vesicles from E. coli,
have shown that ATP hydrolysis is associated with the move-
ment of protons across the mem
ane (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-
ane-bound ATPase catalyzes the synthesis of ATP when an
inward directed protonmotive force is imposed across the cell
mem
ane.
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
A
eviations: DCCD, NN'-dicyclohexylca
odiimide; CCFP,
ca
onylcyanidefluoromethoxyphenylhydrazone.
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 buffe
(final volume of 5 ml) to a cell density of 176 -Mett units. Afte
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 XXXXXXXXXXCell density was determined
tu
idimetrically with a Kle-tt-Summerson calorimeter (no. 42
filter). The intracellular concentration of ATP was calculated
from the known relationship between intracellular wate
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 wate
or 165 ug dry weight (23). The 'co
esponding relationship fo
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. Ca
onyl-
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 mem
ane highly permeable to the potassium
ion, and the efflux of K+ establishes a mem
ane 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. Afte
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
y 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 XXXXXXXXXXKlett 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 afte
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
eturned 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
asal 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 mem
ane potential present in valino-
mycin-treated cells. If ATP synthesis in such cells depends on
Proc. Nat. Acad. Sci. USA XXXXXXXXXX)
3898 Microbiology: Maloney et al.
f 2.0
z
1.0
0 mM K
1 mMK
XXXXXXXXXX
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 fo
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 afte
valinomycin was added to cells