FEMS MicrobiologyLetters 19 (1983) 111-114
Published by Elsevier
111
Lactate efflux stimulates [32p i ]ATP exchange in Streptococcus
faecalis membrane vesicles
S t e p h e n J. Simpson, R o b e r t Vink, A u b r e y F. Egan * a n d Peter J. Rogers
School of Science, Griffith University, Nathan, Q. 4111, and * C.S.LR. O. Division of Food Research, Meat Research Laboratory, Cannon
Hill, Q. 41170, Australia
Received 18 February 1983
Revision received21 March 1983
Accepted 22 March 1983
1. I N T R O D U C T I O N
Michels et al. [1] postulated that symport of
protons with metabolic end products may be coupled to energy transduction. Efflux of lactate by
the enteric bacteria [1] and some streptococci [3] is
carrier-mediated, and Ten Brink and Konings [2]
and Otto et al. [3] have shown that lactate efflux
from loaded membrane vesicles of Escherichia coli
and Streptococcus cremoris can form a transmembrane electrical potential. We have shown that the
proton:lactate stoichiometry of symport varies
from approx. 0.9 at pH 6.5 to almost 2 at pH 7.8
in intact cells of Streptococcus faecalis (Simpson,
Rogers, unpublished data). The apparent p K of
the functional group responsible for electrogenic
symport in this organism is 7.0 (Simpson, S.J. and
Rogers, P.J., unpublished data). Homolactic fermentation by the streptococci is usually characterized by high efficiency (i.e. YATO, g dry
weight/mol ATP consumed) [4]. Otto et al. [5]
showed that a high external lactate concentration
caused the molar growth yield to drop by as much
as 30%; hence end product efflux may be coupled
directly to energy transduction. Here, we report
that lactate transport can be directly coupled to
energy transduction in membrane vesicles. We have
isolated membrane vesicles from S. faecalis and
shown that lactate efflux stimulates [32 P1]ATPexchange.
2. MATERIALS A N D M E T H O D S
L-Lactate, ATP, DCCD, CCCP were obtained
from the Sigma Chemical Co., Nigericin from
Calbiochem. S. faecalis (UQ1768) was obtained
from University of Queensland Culture Collection,
and grown at 37°C, in 1% glucose, 1% Bactopeptone (Oxoid), 0.1% yeast extract (Difco) and 1%
K H z PO4, p H 7.5. Cells were harvested during late
logarithmic growth phase.
2.1. Preparation o f membrane vesicles
10 g (wet weight) S. faecalis cells were washed
and resuspended in 20 ml of 80 mM Tris-SO 4, pH
7.0. 40 ml of 80 mM Tris-SO4, 10 mM MgSO4,
pH 7.0 and 300 mg of egg lysozyme (EC 3.2.1.17)
were added and incubation at 30°C for 30 min
commenced. 15 ml of saturated K2SO 4 [3], and
140 ml of 80 mM Tris-SO4, pH 7.0 containing
RNase (11 mg) and DNase (11 mg), were added
sequentially, and the incubation continued for a
further 20 min. 25 ml of potassium-EDTA (150
mM), pH 7.0 was added followed by a 10-min
incubation. MgSO 4 was added to a final con-
0378-1097/83/0000-0000/$03.00 © 1983 Federation of European MicrobiologicalSocieties
112
centration of 20 mM, and the suspension centrifuged at 48000 × g for 30 min. The pellet was
resuspended in 50 ml of 40 m M T r i s - S O 4, 10 m M
MgSO 4, p H 7.0. Unlysed whole cells and cell
debris were removed by centrifuging at 750 g for
70 min at 4°C. The resulting supernatant containing the membrane vesicles was centrifuged at
48 000 x g for 30 min at 4°C. The bright yellow
pellet was resuspended in 40 m M Tris-SO4, 10
m M MgSO 4, p H 7.0 to a final concentration of
about 10 mg p r o t e i n / m l . 100 ttl aliquots were
snap frozen in liquid N 2 and stored at - 7 0 ° C .
Lactate-loaded membrane vesicles were prepared
as above with the exception that after the first high
speed spin, the membrane pellet was resuspended
in 40 m M Tris-SO4, p H 7.0 containing 50 m M
sodium L-lactate and 10 m M MgSO 4. Subsequent
steps were carried out as before.
The ATPase activity of membrane vesicles was
measured as released inorganic phosphate (Pi).
Membrane vesicles were diluted in 0.1 M Tris-SO 4,
2.5 m M MgSO 4, 5 m M ATP, p H 7.5, to a final
protein concentration within the range 0.02-0.8
m g / m l . Aliquots were removed and the reaction
was terminated by addition of the phosphate assay
reagent [6] in the ratio 3:1 (assay reagent:reaction suspension). To assay total ATPase activity
(i.e. activity associated with internal and external
0.8
I
o.4
g
i
10
2'0
i
Time (rain)
Fig. 1. ATPase activity of membrane vesicles of Streptococcus
faecalis at pH 7.0, 25°C., *, membranes were preincubated in
100 mM Tris-SO 4, 2.5 mM MgSO4, 5 mM ATP, pH 7.0
containing 2.0% ( w / v ) Triton X-100; II, membranes incubated
as above but without Triton.
membrane surfaces) the membranes were incubated in an equal volume of 0.2% Triton X-100
in 40 m M T r i s - S O 4, 10 mM MgSO 4, p H 7.0.
Assay of [32pi]ATP exchange in S. faecalis inverted membrane vesicles was based on the method
of Kagawa and Sone [7]. Each assay contained 1.0
ml of 40 m M Tris-SO 4, p H 7.0, 50 m M sodium
L-lactate, 10 mM MgSO4, 20 m M phosphate (32P~,
0.3 /~Ci/ttmol phosphate) and 10 mM ATP. The
assay was initiated by adding an aliquot of concentrated vesicles to a final protein concentration
of 0.2 m g / m l . The reaction mixture was sampled
over the next 5 min. The reaction was terminated
by addition of trichloroacetic acid to a final concentration of 5% ( w / v ) .
Incorporation of 3 2 p i w a s determined as previously described [7]. Protein was estimated by the
method of Lowry et al. [8] using bovine serum
albumin as standard.
3. RESULTS A N D D I S C U S S I O N
The yield of membrane vesicles was typically
about 10 mg vesicle p r o t e i n / g dry weight cells.
The orientation of membrane vesicles depends
upon the organism and the method of preparation
[9]. ATPase activity in the presence and absence of
Triton X-100 [10], was measured to assess the
orientation of the complex in our vesicles (Fig. 1).
ATPase activity was higher when 0.2% Triton was
present; no further increase in activity occurred
when the concentration was increased to 0.7%.
This effect is unlikely to be caused by solubilization of the enzyme since inhibition by DCCD,
which is specific for membrane-bound ATPase,
was about 85% for both control and detergenttreated vesicles. Earlier studies [10,11] found that
0.2% Triton did not significantly stimulate ATPase
activity ( < 10%) [10] in everted vesicles of S.
faecalis. Also estimates of vesicle heterogeneity
from either freeze-structure studies or ATPase distribution [11] gave comparable results, suggesting
that right side in ATPase behaves similarly towards Triton. The two fold increase in ATPase
activity observed in the presence of Triton probably indicates that we have a random population of
everted and right-side in vesicles.
113
Lactate
nH+
\ L a c t a t e
nH
n
Fig. 2. Coupling of ATP synthesis to lactate/proton transport
down an inwardly directed concentration gradient.
Otto et al. [5] showed that in S. ¢remoris, lactate
efflux from loaded vesicles can drive uptake of
leucine. Uptake was inhibited by CCCP, and did
not depend on ATPase activity. In addition, Llactate efflux generated an electrical potential Aq~,
indicating that p r o t o n : l a c t a t e symport stoicheiometry was > 1. According to Maloney et al. [12]
and Kaback [13], A~ can be artificially generated
2~
'~ 2C
O~
IE
uL
CX:~
-E
10
rn
5
Time (rain)
Fig. 3. Time course for the incorporation of 32pi into ATP as a
result of L-lactate transport. Lactate-free vesicles in buffer
containing L-lactate (50 mM), without additions (O); plus (e)
CCCP (10/~m); (11)DCCD (200 ~M); sodium L-lactateloaded
vesicles (50 mM) in buffer containing 50 mM sodium L-lactate
(A).
by K + diffusion gradients in S. lactis and coupled
to CCCP- and DCCD-sensitive [32pi]ATP exchange. Thus the A / ~ formed by lactate efflux
may also be coupled to energy transduction and
thus measurable by [ 32Pi ]ATP exchange. If lactate
is added to our vesicle preparation, transport of
lactate and protons by the symporter should generate A / ~ . For an ATPase which is located internally, A / ~ will be of opposite polarity to that which
can be used for ATP synthesis (Fig. 2) [12,14].
Membrane vesicles are generally impermeable to
A T P [13], so little should be present inside the
vesicles. Any that is will be hydrolysed by the
ATPase, and subsequent outward extrusion of
protons will only stimulate further lactate uptake,
until a steady state is reached ( A / ~ + A~L = 0,
where A/~L is the lactate electrochemical potential).
However, with the reverse orientation of ATPase,
lactate uptake can be coupled to ATP synthesis
(Fig. 2).
Fig. 3 shows the kinetics of [32pi]ATP exchange
at p H 7.8, caused by L-lactate addition to vesicle
suspensions. In the presence of 50 m M external
L-lactate, lactate transport into lactate-free vesicles
produced maximal [32Pi]ATP exchange. Accumulation was rapid and thereafter rapidly decreased.
This is consistent with electrogenic transport forming a large A / ~ which is coupled to ATP synthesis.
A T P will be hydrolysed as uptake of lactate diminishes the concentration gradient and A / ~ collapses.
With preloaded vesicles (internal lactate approx. 50 mM), the level of exchange was only
about 10% of the former value. Both the ionophore
CCCP, and the membrane-bound ATPase inhibitor, DCCD, inhibited exchange in the presence of
a lactate gradient, indicating that both intact
membranes and ATP synthase are necessary for
the exchange. Addition of cyanide or azide caused
no effect, as expected, since this organism is a
homolactic fermenter.
The conclusion from these experiments is that
the transmembrane electrochemical proton potential formed by lactate proton symport [2,3] can be
coupled to ATP synthesis. The data may rationalize the high molar growth efficiencies of many of
the streptococci and related organisms [4] during
homolactic fermentation of glucose. This supports
114
t h e o r i g i n a l e n e r g y r e c y c l i n g p r o p o s a l of M i c h e l s
et al. [1].
ACKNOWLEDGEMENT
We thank the Australian Research Grants
C o m m i t t e e for s u p p o r t ( P J R , D 2 8 2 1 5 7 8 1 ) .
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