[37] Generation of a protonmotive force in anaerobic bacteria by end-product efflux

492

BACTERIALTRANSPORT

[37]

[37] G e n e r a t i o n o f a P r o t o n m o t i v e F o r c e in A n a e r o b i c Bacteria by End-Product Efflux

By BART TEN BRINK and W|L N. KONINGS

Introduction Chemiosmotic phenomena, as postulated by Mitchell,~,2 play a central role in bacterial metabolism. The chemiosmotic hypothesis postulates that proton translocation by primary proton pumps generates an electrochemical proton gradient, or protonmotive force (Ap) across the cytoplasmic membrane. This ap consists of an electrical parameter, the membrane potential (Aqj, inside negative) and a chemical parameter, the pH difference (ApH): Ap = Aqj - ZApH

(in mY)

(l)

where Z equals 2.3 RT/F and R, T, and F have their usual meaning. The protonmotive force acts as a driving force or regulatory parameter in a large number of different metabolic processes. 3 Until recently only three primary proton pumps had been described in bacteria4: membranebound electron transfer chains, 5-v the proton-translocating ATPase complex, 8,9 and, in halobacteria, the light-driven proton pump bacteriorhodopsin. ~° Strictly fermentative bacteria which do not possess electron transfer systems therefore seemed to be totally dependent on ATP hydrolysis for the generation and maintenance of a Ap. This generation of a Ap would consume a considerable fraction of the ATP'formed by substrate level phosphorylation and as a consequence less ATP would be available for biosynthetic purposes. P. Mitchell, Biol. Reo. 41, 445 (1966). 2 p. Mitchell, J. Bioenerg. 4, 63 (1973). 3 K. J. Hellingwerf and W. N. Konings, Adv. Microb. Physiol., in press (1986). 4 W. N. Konings and P. A. M. Michels, in "Diversity of Bacterial Respiratory Systems" (C. J. Knowles, ed.), p. 33. CRC Press, Boca Raton, Florida, 1980. 5 E. Padan, O. Zilberstein, and H. Rottenberg, Fur. J. Biochem. 63, 533 (1976). 6 S. Ramos and H. R. Kaback, Biochemistry 16, 848 (1977). 7 p. B. Garland, J. A. Downie, and B. A. Haddock, Biochem. J. 152, 547 (1976). 8 V. Liebeling, R. K. Thauer, and K. Jungermann, Eur. J. Biochem. 55, 445 (1975). 9 F. M. Harold, Curr. Top. Bioenerg. 6, 83 (1977). 10 W. Stoeckenius, R. H. Lozier, and R. A. Bogomolni, Biochim. Biophys. Acta 505, 215 (1979).

METHODS IN ENZYMOLOGY, VOL. 125

Copyright g2 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

[37]

GENERATION OF A PROTONMOTIVE FORCE

493

In 1979, Michels e t al. ~ proposed another mechanism for the generation of a protonmotive force in anaerobic bacteria. In their "energyrecycling model" it was postulated that metabolic end products are excreted via specific transport proteins in the cytoplasmic membrane and that together with the end products, protons are translocated from the cytoplasm to the external medium. As a result of this proton (and charge) translocation a protonmotive force is generated. Especially in fermentative bacteria, which continuously produce relatively large quantities of metabolic end products, this process could contribute significantly to the generation of a protonmotive force and consequently to the overall production of metabolic energy. Solute Transport in Bacteria Before describing the energy-recycling model in detail the most important solute transport systems of bacteria will be briefly reviewed. Solute transport across the cytoplasmic membrane of bacteria can occur by two major mechanisms. (I) Secondary transport systems: transport by these systems is driven by electrochemical gradients and will lead to the translocation of solute in unmodified form. (2) Group translocation: solute is substrate for a specific enzyme system in the membrane; the enzymatic reaction results in a chemical modification of the solute and release of the products at the cytoplasmic side. The only well-established group transiocation system is the phosphoenolpyruvate phosphotransferase system (PTS).4 Most solutes are translocated across the cytoplasmic membrane by secondary transport either passively, without the involvement of specific membrane proteins or facilitated by specific carrier proteins. This latter mechanism is often termed "active transport." Mitchell visualized three different systems for facilitated secondary transport. "Uniport": only one solute is translocated by the carrier protein. "Symport": two or more different solutes are transiocated in the same direction by the carrier protein. "Antiport": two or more different solutes are translocated by one carrier in opposite directions. The driving forces for solute transport will depend on the overall charge and the number of protons which are transiocated, as well as on the solute gradient. 4 The driving force is composed of components of the protonmotive force and of the solute gradient, and translocation of solute will proceed until the total driving force is zero. At that stage a steady~ P. A. M. Michels, J. P. J. Michels, J. Boonstra, and W. N. Konings, F E M S Microbiol. Lett. 5, 357 (1979).

494

BACTERIALTRANSPORT external medium

1.

A

2.

A"

membrane

•

3.

H÷ A H÷

cytoplasm

[37]

driving force (mV)

steady state

ADA/F

A~A/F = 0

L~A/F + &~

A~A/F = - g~

AVA/F - ZApH

A~A/F = Z&pH

&~A/F + A~ - ZApH

APA/F = -At' + ZApH

f~ J

FIG. 1. Schematic presentation of four s e c o n d a r y transport processes. 1. Passive transport o f a neutral solute; 2. passive transport of a cation; 3. facilitated transport of an anion in s y m p o r t with one proton; 4. facilitated transport o f a neutral solute in s y m p o r t with one proton. For each p r o c e s s the driving force and the steady-state level of accumulation are given.

state level of accumulation is reached. Figure 1 shows schematically four different translocation processes. In example 1 transport of a neutral solute via passive secondary transport is shown. The driving force of this transport will be supplied only by the solute gradient and will be equal to A I . t A / F = Z Iog[Ain]/[Aout]. A steady state will be reached when the solute gradient is dissipated (A/.~A = 0) and the internal solute concentration equals the external concentration. Example 2 shows transport of a monovalent positively charged solute by passive secondary transport. This transport will not only depend on the chemical concentration gradient of solute but also on the electrical potential. The driving force therefore will be A t Z A / F = g log[Ain]/[Aout] + At0 and a steady state will be reached when Z log[Ai.]/[Aout] = -A@. Unless the electrical potential is dissipated by movement of other ions across the membrane in the steady state, the internal concentration of solute will not be equal to the external concentration. In the absence of active primary transport systems net transport will already stop when the internal solute concentration is lower than the external concentration. However, when a A¢ (interior negative) is generated by other systems accumulation of solute can occur. In a way similar to that shown above, the driving forces and steadystate levels of transport can be derived for other transport processes, a This is done in example 3 for a symport system by which a negatively charged solute is translocated together with one proton. The overall charge during this translocation process is zero and due to the translocation of one proton only the ApH will contribute to the driving force of this translocation process. In example 4 the driving force is determined for a

[37]

GENERATION OF A PROTONMOTIVE FORCE

495

neutral solute symported with one proton. In this process charge and protons are translocated and consequently the total protonmotive force (Ark and ApH) contributes to the driving force. A general equation for the driving force for transport of solute A with charge m in symport with n protons can be derived. This driving force is nAp + A/~A/F

(in mV)

(2)

Combination of Eqs. (1) and (2), and substitution of electrochemical gradient of A m (A/2A/F) by the sum of the chemical gradient of A (A/2A/F = Z log[Ai./Aout]) plus the electrical component (mAq0 yields driving force = Af~A/F + (n + m)Atk - n Z A p H

(in mV)

(3)

The Energy-Recycling Model In essence, the energy-recycling model describes the reversed process of solute uptake via a secondary transport system. The chemiosmotic hypothesis and the carrier model of Rottenberg ~2postulate that secondary transport processes can proceed in both directions across the membrane. The direction of transport is determined entirely by the directionality of its driving force. During solute uptake the energy present in Ap is used to drive the accumulation of solute into the bacterial cell, whereas during end-product excretion the energy in the product concentration gradient is used to generate a protonmotive force. Since metabolism of the energy source in fermentative bacteria leads to a continuous efflux of metabolic end products, a continuous generation of a protonmotive force takes place. The components of the protonmotive force which will be formed during the efflux process will vary with the translocated solute. If the efflux process leads to a net translocation of positive charges, a A~ will be generated. A ApH will be formed if a net translocation of protons takes place. For the generation of both a A~ and a ApH a net translocation of both protons and charges has to occur (see also Fig. 1). In the detailed mathematical description of the energy-recycling modeP ~ Michels et al. took a homofermentative lactate-producing organism as a model system. The energy source glucose is accumulated by a PEP phosphotransferase system and during the translocation process converted to glucose 6-phosphate. The production of metabolic energy during glucose metabolism by such organisms can be divided in two distinctly different parts: substrate level phosphorylation and lactate excretion (see also Fig. 2). ~-~H. Ronenberg, FEBS Lett. 66, 159 (1976).

496

BACTERIALTRANSPORT

[37]

acid ®

oui FIG. 2. Schematic presentation of the energy-recycling model in an organism with a homolactic glucose fermentation. Glucose is taken up by a phosphoenolpyruvate-dependent group translocation system (PTS).

Substrate level phosphorylation: glucoseo.t + 2 ADP~n + 2 Pm--~ 2 lactate~n + 2 H~ + 2 ATPm + 2 H20

(4)

Lactate excretion: lactatei. + n H~ --~ lactate,,,,, + n H~ut

(5)

where n equals the number of protons translocated together with lactate (the H+/lactate stoichiometry) during carrier-mediated lactate excretion. The overall process is glucoseo.t + 2 ADPi. + 2 Pin + 2(n - I) H + 2 lactateo.t + 2 ATPi. + 2n H~t + 2 H20

(6)

From the overall reaction it will be clear that ifn equals 2, 4 protons and 2 positive charges are translocated across the cytoplasmic membrane per glucose molecule fermented. As a consequence both a A~b and a ApH can be generated by the lactate excretion process, and less ATP has to be hydrolyzed for the generation of a protonmotive force. However, if n equals !, only 2 protons and no charges are translocated. In that case lactate excretion will not result in the generation of a membrane potential and only a small pH difference will be created as a result of the acidification of the external medium by the produced lactic acid. This example clearly demonstrates that the H+/solute stoichiometry during end-product excretion is a very important parameter in the energy-recycling process.

[37]

GENERATION OF A PROTONMOTIVE FORCE

497

Experimental Approach In the past 5 years a steadily increasing body of experimental evidence in favor of the energy-recycling model has been presented. These results were mainly obtained from studies on lactate excretion in streptococci. However, the excretion of other end products in other fermentative bacteria will most likely also lead to the generation of a protonmotive force. In this chapter the general approach and experimental techniques which can be used to study the generation of a protonmotive force by endproduct efflux have been described. Some results obtained in Escherichia coli and Streptococcus cremoris will be given as experimental examples.

Role of Specific Transport Proteins in End-Product Translocation It has been demonstrated above for lactate efflux that an effective generation of a protonmotive force by the excretion of neutral or negatively charged end products requires the net translocation of positive charges from the cytoplasm to the external medium. This can be achieved only if the end product is translocated in symport with protons via a specific transport protein. Evidence for the involvement of such a carrier in the end-product translocation can be obtained from studies on the translocation in the opposite direction, i.e., uptake studies of the end product. This can be achieved only if the driving force for end-product translocation is directed from the medium to the cytoplasm. In whole cells this can be realized by using starved cells with no internal end product present and imposing artificially a protonmotive force, inside negative and/or alkaline. For some end products it might be better to use isolated membrane vesicles instead of intact cells, in order to avoid effects of the metabolism. Isolation procedures of membrane vesicles have been described for a large number of organisms.~3-16 In cells and membrane vesicles a driving force for electrogenic secondary transport can be generated artificially by imposing a potassium diffusion potential across the membrane. 17This is usually achieved by incubating concentrated cell or membrane vesicle preparations (30-50 mg protein/ml) with the K+-ionophore valinomycin (1-2 nmol/mg protein) in the presence of 100 to 200 mM potassium ions and diluting small aliquots 100- to 200-fold in K+-free buffer. The efflux of the K + ions will result in the generation of a AqJ. 13 H. R. Kaback, this series, Vol. 22, p. 99. 14 W. N. Konings, Adv. Microbiol. Physiol. 15, 175 (1977). ~5 R. Otto, R. G. Lageveen, H. Veldkamp, and W. N. Konings, J. Bacteriol. 149, 733 0982). ~6 W. N. Konings, this series, Vol. 56, p. 370. ~7 S. Schuldiner and H. R. Kaback, Biochemistry 14, 5451 0976).

498

BACTERIALTRANSPORT

[37]

A driving force for H+-solute symport can also be created by the artificial formation of a ApH. This can be achieved by diluting cells or membrane vesicles, incubated in a buffer of high pH (7 to 8) into a buffer with a lower pH (4.5-6). Uptake of end products can be studied by adding radioactively labeled end products to the dilution media. Preferentially the end product with the highest radioactive label available should be used at different concentrations in the p,M to mM range. The uptake is measured after filtration of the cells or vesicles over 0.45-tzm pore size filters, followed by washing as described.18 In Streptococcus cremoris cells the A0 generated by K + efflux can drive the uptake and accumulation of the end product lactate at pH 7.0,19 indicating that under these conditions lactate uptake is a carrier mediated and electrogenic process (the H+/lactate stoichiometry is higher than !). If the organism under study contains a proton-translocating electron transfer chain, a Ap can be generated by addition of the appropriate electron donor and acceptor. In membrane vesicles from anaerobically grown Escherichia coli lactate uptake could be driven by the addition of ascorbate/phenazine methosulfate under aerobic conditions (Fig. 3). The uptake of lactate has been studied in the pH range 5.5 to 8 in the presence of the ionophore nigericin. Under these conditions no npH exists since nigericin exchanges H + for K ÷ ions across the membrane but the A0 is constant at about - 100 mV in this pH range. Addition of nigericin (0.5 p,M) to these vesicles at pH 5.5 almost completely inhibited lactate uptake, but had no effect at pH 8.0fl ° This result strongly indicates that at pH 5.5 lactate uptake is mainly ApH driven, while at pH 8.0 the A0 is the main driving force. This implies that in E. coli membrane vesicles at pH 5.5 lactate uptake is an electroneutral process (1 H ÷ per lactate symported), while at pH 8.0 the translocation is electrogenic (more than one H ÷ per lactate symported). These observations supply strong evidence for a pH-dependent variation in the H+/lactate stoichiometry during lactate transport in E. coli vesicles. The most likely explanation for this variation in stoichiometry is that the external pH affects the dissociation state of the carrier. 12.21 Completely different techniques to study lactate uptake were used by Simpson et al. 22 with Streptococcusfaecalis. They investigated the initial ~8 H. R. K a b a c k , this series, Vol. 31, p. 698. J9 R. Otto, A. S. M. Sonnenberg, H. Veldkamp, and W. N. Konings, Proc. Natl. Acad. Sci. U.S.A. 77, 5502 (1980). 20 B. ten Brink and W. N. Konings, Eur. J. Biochem. 111, 59 (1980). 2~ W. N. Konings, Trends Biochem. Sci. 6, 257 (1981). 2,_ S. J. Simpson, M. R. Bendall, A. F. Egan, and P. J. Rogers, Eur. J. Biochem. 136, 63 (.'983).

[37]

GENERATION OF A PROTONMOTIVE FORCE pH 55

pH 8.0

pH 66 I

E .o_

499

i

80

~6 I

,3O

60

,3

40

d

20

L

12

3456

i

otI

i

1 2 3 4 5 6

J

B

i

l

l

i

12345

Time (minutes)

FIG. 3. Time course of L-lactate accumulation by membrane vesicles of Escherichia coli ML 308-225, under aerobic conditions at different pH values. In all experiments the membrane vesicles were energized by the oxidation of the electron donor potassium ascorbate (10 mM) plus phenazine methosulfate (100 txM). (O) No further additions; (©) plus nigericin (0.5/xM). Taken from ten Brink and Konings, Eur. J. Biochem. 111, 59 (1980), with permission.

rates of uptake of protons and lactate anions resulting from the addition of high concentrations of lactate to resting intact cells. [~4C]Lactate was used to measure the rate of lactate influx. The proton influx was calculated from the changes in the external and cytoplasmic pH as determined with the 3Jp NMR technique. 23,24 The initial rate of lactate uptake was independent of the external pH, while the rate of lactate influx induced proton influx strongly increased with the external pH. The ratio between proton influx and lactate influx rate, which equals the H+/lactate stoichiometry (n), increased from about 1 at pHo 6.5 to about 2 at pHo 7.5.22 This indicates that also in S. faecalis cells lactate uptake is a carrier mediated and electrogenic process at pH values near or above 7.0. This conclusion was confirmed by the observations that especially at high pH the addition of lactate strongly accelerated the rate of membrane depolarization in resting S. faecalis cells with an inside negative AqJ.22

23 G. Navon, S. Ogawa, R. G. Shulman, and T. Yamane, Proc. Natl. Acad. Sci. U.S.A. 74, 888 (1977). 24 K. Ugurbil, H. Rottenberg, P. Glynn, and R. G. Shulman, Proc. Natl. Acad. Sci. U.S.A. 75, 2244 (1978).

500

BACTERIAL TRANSPORT

[37]

End-Product Efflux under Nonphysiological Conditions The main postulate of the energy-recycling model is that excretion of end products is carrier-mediated and that with the end product positive charges and/or protons are translocated from the cytoplasm to the external medium. To study this efflux process cells or membrane vesicles have to be loaded with high concentrations of end product. Usually this can be achieved by incubating deenergized cells or membrane vesicles for several hours at room temperature with the metabolic end product. J9,20If the translocation of an acidic end product is investigated, incubation at low pH may shorten the incubation time needed, since then a large part of the product is present in the undissociated form, which is more membrane permeable. Membrane vesicles can also easily be loaded during the membrane vesicle isolation by performing the lysis of the protoplasts or spheroplasts in the presence of the end product. 2°.25The end product then will be entrapped in the membrane enclosed volume. Dilution of "loaded" cells or membrane vesicles into end product-free medium will create an outwardly directed end-product concentration gradient. Efflux of the end product then can lead to the generation of a protonmotive force. The generation of a membrane potential (inside negative) can be recorded by performing the dilution in a medium containing the radioactive labeled lipophilic cation tetraphenylphosphonium (TPP +) at micromolar concentrations. For measurements of a ApH (inside alkaline) radioactively labeled weak acids like acetate or benzoate at micromolar concentrations have to be present in the dilution medium. The uptake by the cells or the membrane vesicles can be followed after filtration and counting of the radioactivity. In a similar way the uptake of a solute driven by the protonmotive force generated by the efflux process can be followed. When lactate loaded starved cells of S. cremoris were diluted 100-fold into lactate-free buffer containing [J4C]leucine, a significant accumulation of leucine could be observed. ~9However, if a lactate gradient was not created, virtually no uptake of leucine occurred (Fig. 4A), indicating that lactate efflux and not ATP hydrolysis supplied the energy for leucine uptake. This could also be concluded from the observation that the ATPase inhibitor dicyclohexylcarbodiimide (DCCD) only slightly inhibited the leucine uptake in the presence of a lactate gradient (Fig. 4B). Figure 4C shows that lactate efflux induced leucine uptake is completely inhibited by the uncoupler carbonyi cyanide p-trifluoromethoxyphenylhydrazone (FCCP). This suggests that lactate efflux results in the generation of a protonmotive force, 25 S. J. Simpson, R. Vink, A. F. Egan, and P. J. Rogers, FEMS Microbiol. Lett. 5, 85 (1983).

[37]

GENERATION OF A PROTONMOTIVE FORCE F

Bl

A

501

(~I

5

4

g3"3 E

\

< 1

•

30

60

3'0 Time, sec

60

30

6*0

FIG. 4. Time course of L-lactate efflux-induced leucine uptake by S. cremoris. Deenergized cells [100 mg/ml (dry wt)] were diluted i : 100 into choline/HEPES/KCI buffer at 25°. (A) Cells loaded with 50 mM choline L-lactate were diluted into choline/HEPES/KCI buffer (O) or into choline/HEPES/KCI buffer containing 50 mM choline L-lactate (O). Cells loaded with 50 mM choline chloride were diluted into choline/HEPES/KCI buffer (+). (B) Cells were preincubated with 25/*M DCCD for 30 rain at room temperature. Cells loaded with 50 mM choline L-lactate were diluted into choline/HEPES/KCI buffer containing 25 /xM DCCD (~') or into the same medium supplemented with 50 mM choline L-lactate (IS]). (C) Cells loaded with 50 mM choline L-lactate were diluted into choline/HEPES/KCI buffer containing 10 p M FCCP (A) or into the same medium containing 50 mM choline L-lactate (×). Taken from Otto et al., Proc. Natl. Acad. Sci. U.S,A. 77, 5502 (1980), with permission.

which subsequently drives the uptake of leucine. Similar results were obtained with membrane vesicles of S. cremoris 15 and E. coli2°: lactate efflux resulted in uncoupler sensitive amino acid uptake. Direct evidence for the generation of a Ap by lactate efflux was obtained from experiments in which the uptake of the lipophilic cation tetraphenylphosphonium (TPP +) was studied. When E. coil membrane vesicles loaded with 50 mM lactate at pH 6.6 were diluted 100-fold into a buffer of pH 6.6 containing the same concentration of lactate, only a low level of TPP* accumulation was observed. 2° On the other hand, when these loaded vesicles were diluted into lactate-free buffer, a high level of TPP + accumulation (24fold) was reached within 20 sec, followed by rapid efflux of the accumulated TPP + (Fig. 5). Here, in analogy with the lactate efflux-induced amino acid uptake, the uncoupler FCCP also completely inhibited TPP + uptake. These results clearly showed that in membrane vesicles ofE. coli

502

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[37]

80 25

'6 20 E

o=

+a_ 15 O_

.o

1--

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60

4O

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20 0 i

30 60

120

180

i

240

Time (sec.)

FIG. 5. Time course of L-lactate efflux induced tetraphenylphosphonium accumulation by Escherichia coli ML 308-225 membrane vesicles at pH 6.6 and 25°. Membrane vesicles loaded with 50 mM K L-lactate were diluted 100-fold into (O) buffer; (O) buffer + 50 mM K L-lactate; (&) buffer + 10/xM FCCP. Taken from ten Brink and Konings, Eur. J. Biochem. 111, 59 (1980), with permission.

a A~b (inside negative) can be formed by efflux of lactate from the vesicles. The lactate efflux-induced A~b was calculated with the Nernst equation from the increased level of TPP ÷ accumulation 2° to be about - 5 5 mV. This means that at a pH near neutrality not only the uptake but also the efflux of the monovalent negatively charged lactate ion is carrier mediated and occurs in symport with more than one proton (n > l). Similar results have been obtained with intact cells ~9and membrane vesicles 15of S. cremoris. This phenomenon of solute efflux induced amino acid uptake and A~ generation was not only observed for the end product lactate: efflux of thiomethylgalactoside or gluconate from E. coli ceils 26.27 and lactose from E. coli vesicles 28 also resulted in Ap formation. The generation of a protonmotive force by carrier-mediated solute efflux therefore seems well established and justifies an examination of the transport mechanism of other metabolic end products. 26 j. L. Flagg and T. H. Wilson, Membr. Biochem. 1, 61 (1978). z7 M. Bentaboulet, A. Robin, and A. Kepes, Biochem. J. 178, 103 (1979). 2s G. J. Kaczozowski and H. R. Kaback, Biochemistry 18, 3691 (1979).

[37]

GENERATION OF A PROTONMOTIVE FORCE

503

End-Product Efflux under Physiological Conditions Experiments as described above can be used to investigate the basic assumptions of the energy-recycling model. Once these assumptions have been confirmed, it is of interest to study the end-product excretion process under physiological conditions, where an outwardly directed endproduct gradient will be created by the bacterial metabolism and not artificially. This requires techniques to determine the magnitude of Ap, the end-product gradient and the H+/end-product stoichiometry in growing or actively metabolizing bacteria, A variety of methods has been developed to measure the magnitude and composition of the protonmotive force in bacteria. In most cases the distribution of permeant ions and weak acids (or bases) is used in the determination of the AqJ and ApH, respectively. The distribution of the probe molecules can be determined chemically or by using radioactively labeled probes, either by measuring the internal or the external concentration of the probes. Usually a physical separation of both compartments by filtration or centrifugation is required which can result in leakage of the probe from the cytoplasm. To measure changes in the external concentration of the probe, either a specific electrode z9,3° or the flow dialysis technique 31,3z can be used. Measurements of the components of the Ap and an end-product gradient under identical conditions, can best be performed with a procedure which is rapid and avoids washing steps or other manipulations that can change the actual gradients. A modified version of the silicon oil centrifugation technique 33 is such a procedure: the metabolizing cells are incubated with AqJ and ApH probes, separated from the external medium by centrifugation through a layer of silicon oil and collected in perchloric acid (PCA). The ApH and AO values can be calculated from the accumulation of [14C]benzoate and [3H]TPP +, respectively. Portions of 0.5 ml cell suspension (50-500 mg protein/ml) are incubated for 5 min with 0.5/~Ci [14C]benzoate (16/zM final concentration) and/or 2.5/~Ci [3H]TPP+ (5 p,M final concentration) and transferred to microfuge tubes containing 0.5 mi of silicon oil (density 1.02 g/ml) on top of 0.1 ml 11% (w/w) PCA (density 1.07 g/ml). After 3 min centrifugation at 12,000 g virtually all cells are present in the PCA fraction. In PCA, rapid disruption of the cells, inhibi29 T. Shinbo, N. K a m o , K. Kurihara, and Y. Kobatake, Arch. Biochem. Biophys. 187, 414 (1978). 3o D. B. Kel[, P. John, M. C. Sorgato, and S. J. Ferguson, FEBS Lett. 86, 294 (1978). 3~ S. R a m o s , S. Schuldiner, and H. R. K a b a c k , Proc. Natl. Acad. Sci. U.S.A. 73, 1892 (1976). 3-" K. J. Hellingwerf and W. N. Konings, Eur. J. Biochem. 106, 431 (1980). 33 E. J. Harris and K. van Darn, Biochem. J. 106, 759 (1968).

504

BACTERIALTRANSPORT

[37]

tion of further metabolism, and release of intracellular metabolites and probe molecules occur. Samples (50/xM) of the clear supernatant and the PCA fraction are taken and assayed for radioactivity. During the centrifugation also some extracellular fluid passes with the cells to the PCA fraction. In control experiments the volume of extraceilular water can be estimated by adding membrane-impermeable 14C compounds such as dextran, inulin, or taurine to the incubation medium. The total water volume that is transferred to the PCA fraction can be labeled with 3H20. From the data on intracellular water and extracellular water in the PCA fraction and the amounts of radioactivity in the supernatants and PCA fractions, the accumulation ratios of the [J4C]benzoate and [3H]TPP+ can be calculated. However, before calculating the TPP + accumulation a correction for binding of TPP + to cell components has to be made. 34 This binding can be determined by incubating cells which are deenergized by a butanol treatment (7% n-butanol for 5 min) with [3H]TPP ÷. If also the concentrations of the metabolic end product under study are determined (i.e., enzymatically) in the supernatant and PCA fractions, the internal end-product concentration and therefore also the end-product gradient can be calculated. From the data on A~b,ApH and the end-product concentration gradient (A/2A/F), the H+/end-product stoichiometry during end-product excretion (n) can easily be calculated, if the assumption is made that during growth the driving force for the translocation process [Eq. (3)] is very close to zero. Rearrangement of Eq. (3) then yields n = (mAyO- A/2A/F)Ap

(7)

We used the procedure described above to study the energy-recycling model in more detail in cells of Streptococcus cremoris growing in batch and chemostat culture 35.36with lactose as sole energy source. Under these conditions lactate is the only end product of the energy metabolism. In these cells an outwardly directed lactate gradient is always present. Internal lactate concentrations between 50 and 200 mM were measured at external concentrations between 8 and 40 mM. Simultaneous measurements of Ark, ApH, and the lactate gradient in growing S. cremoris revealed that n was influenced by the external pH and the external lactate concentration. 35,36During pH regulated growth in batch culture at pH 6.34 the magnitude and composition of Ap are essentially constant, but the 34 j. S. Lolkema, K. J. Hellingwerf, and W. N. Konings, Biochim. Biophys. Acta 681, 85 (1982). 35 B. ten Brink and W. N. Konings, J. Bacteriol. 152, 682 (1982). 36 B. ten Brink, R. Otto, U.-P. Hansen, and W. N. Konings, J. Bacteriol. 162, 383 (1985).

[37]

GENERATION OF A PROTONMOTIVE FORCE

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~-L

600

TIME (rain) F1G. 6. Growth ofS. cremoris Wg2 at 30° and pH 6.34 in batch culture on a rich medium supplemented with lactose (3 g/liter). The dotted line represents the time course of cell density measured at 663 nm. (A) Time course of the internal (A) and external (A) lactate concentration and the lactate gradient (x) during growth. (B) Time course of the electrochemical proton gradient. The line for Ap was constructed by adding AtO(O) and ApH (O). Taken from ten Brink, Otto, Hansen, and Konings)6 lactate gradient decreases sharply as the external lactate concentration increases (Fig. 6). As a c o n s e q u e n c e n [calculated from Eq. (7)] decreases with increasing external lactate concentration (Fig. 7) from 1.44 to about 0.9. If growth occurred at a higher pH, the H+/lactate stoichiometry was higher o v e r the entire lactate concentration range. The effects of the external p H and lactate concentration on n were determined in detailed studies with glycolyzing cells of S. cremoris36: increasing the external proton and lactate concentration results in lower values of n. n Values b e t w e e n a b o u t 2 (high p H , low external lactate) and 0.7 (low p H , high external lactate) could be determined. 36 It should be evident that low n values result in a lower energy production f r o m the lactate excretion process. Such a decrease of the n value is, h o w e v e r , essential since it prevents the cells from " s e l f - p o i s o n i n g . " If the H+/lactate stoichiometry would be fixed at 2.0, lactate excretion from cells with a Ap c o m p o s e d of a Aqj = - 8 0 mV and ApH = - 2 0 mV and 50 m M external lactate could o c c u r only if the internal lactate concentration e x c e e d e d the unphysiologically high value of 5 M! [as can be calculated f r o m Eq. (7)]. It should be noted that n values significantly lower than 1 indicate that less protons are excreted together with lactate than are f o r m e d internally during glycolysis. The remaining protons then have to be extruded by the action of the m e m b r a n e bound ATPase, at the expense of ATP. H+/lactate stoichiometries below 1 would therefore result in an energy loss.

506

BACTERIALTRANSPORT I 1.5

I

[37]

I

I

P

1.4

.<

1.3

-

1.2

-

1.1

-

1.0

-

0.9

-

\

d

,--,,.. t (-

0 ~

I

I

[

10

2o

3o

EXTERNAL

LACTATE

0 ~

I

4o (mNl)

FIG. 7. Effect of the external lactate concentration on the H+/lactate stoichiometry (n) in S. cremoris Wg2, grown in batch culture at pH 6.34. Taken from ten Brink, Otto, Hansen, and Konings?6

Metabolic Energy Yield by End-Product Efflux The production of metabolic energy by substrate level phosphorylation processes during the breakdown of the energy source and by the excretion of the metabolic end products can be calculated if the metabolic pathways and the H+/end-product stoichiometries are known. The energy present in excreted protons can be "translated" into ATP equivalents by dividing the number of protons excreted by p, the number of protons which have to flow back from the external medium to the cytoplasm per molecule of ATP synthesized via the membrane-bound ATPase. For instance, the overall production of metabolic energy by lactose fermentation and lactate excretion by S. cremoris can be calculated with the following equation: ATP equivalents produced ......... 4 + 4(n - 1)/p tool of lactose consumed

(8)

where n represents the H+/lactate stoichiometry during lactate excretion. Per tool of lactose 4 molecules of ATP are synthesized by substrate level phosphorylation and 4 molecules of lactate are produced. Since per molecule lactate also one proton is formed intracellularly and lactate is ex-

[37]

GENERATION OF A PROTONMOTIVE FORCE

507

creted in symport with n protons, 4(n - 1) represents the number of translocated protons which contribute to the generation of the protonmotive force. The calculated number of ATP equivalents produced per mol lactose consumed is shown in Fig. 8 for different values of n and p. If n and p both equal 2, then 6 ATP equivalents are formed, indicating that theoretically an energy gain of 50% can be the result of the lactate excretion process in S. cremoris. For a number of well-known glucose fermentations the theoretical energy gain by end-product excretion has been calculated (Table I), assuming that per neutral end product (organic acids in undissociated form) one extra proton is translocated and that two protons are translocated per ATP synthesized. Table I shows that in theory between 17 and 100% extra metabolic energy can be produced by the energy-recycling process in these fermentations. A prerequisite for this energy gain is the presence of carriers for the alcohols and weak acids in the fermenting organism. Hardly any evidence for the existence of such carriers has been presented. If the metabolic energy production of a microorganism is partly supplied by an end-product efflux process, changes in the H+/end-product stoichiometry should be reflected in changes of the cell yield. In order to investigate this implication of the energy-recycling model, detailed yield i

o

i

I

I

6

p=2

5

p:_Z,

o

o

E \

L

'-

3

/

0

>:D

2

n

1 I

I

I

I

05

1.0

1.5

2,0

(H+/tactate) FIG. 8. Effect of the H+/lactate stoichiometry (n) on the theoretical number of ATP equivalents produced per tool of lactose consumed by S. cremoris Wg2. p is the number of protons translocated per molecule of ATP synthesized. The lines were calculated from Eq. (8).

508

[37]

BACTERIAL TRANSPORT TABLE I ENERGY GAIN BY END-PRODUCT EXCRETION IN A NUMBER OF GLUCOSE FERMENTATIONS

Fermentation a b c d e

f g

Products (tool/tool glucose) 2 ethanol, 2 CO2 2 lactate 1 lactate 1 ethanol, 1 CO2 1 lactate 1.5 acetate i acetate I ethanol 2 formate 1 butyrate 2 CO2, 2 Hz 0.5 butanol, 2 H2 0.5 acetone, 2.5 CO2

ATP produced by substrate level phosphorylation (mol/mol glucose)

ATP equivalents produced by energy-recycling Eq/mol (glucose)

Energy gain

(%)

2 2

1 I

50 50

1

1

100

2.5

1.25

50

3

2

67

3

0.5

17

2

0.5

25

Alcoholic fermentation (yeasts). b Homolactic fermentation (lactate acid bacteria). c Heterolactic bacteria (lactobacilli). d Bifidum pathway (Bifidobacterium bifidum). Mixed acid fermentation (streptococci). f Butyrate fermentation (clostridia). g Butanol-acetone fermentation (clostridia). See text for the calculation of the ATPequivalents production.

studies have to be performed, using chemostat cultures under energy limiting conditions. The molar growth yield corrected for maintenance requirement (ymax, in g cell dry weight produced per tool energy source consumed) can be determined from a graph in which the specific energy source consumption rate (q, in mol energy source consumed per g dry weight per hr) is plotted as a function of the specific growth rate (/~, hr- ~), according to the following equation37: q =/x/ymax + me

(9)

where me is the maintenance coefficient (in mol energy source consumed per g cell dry weight produced per hr). By varying the dilution rate of the 37 K. L. Schulze and R. S. Lipe, Arch. Mikrobiol. 48, I (!%3).

[37]

GENERATION OF A PROTONMOTIVE FORCE

509

TABLE II EFFECT OF GROWTH pH AND EXTERNAL LACTATE CONCENTRATION ON ]/lactose OF Slreplococcus cremoris GROWN IN CONTINUOUS CULTURE max Ylactose

pH

External lactate (mM)

(g dry weight/ tool lactose)

7.0 6.4 5.7 6.3 6.3

27 27 27 25 85"

61.2 55.2 51.2 56.3 50.2

60 mM Na-lactate was added to the inflow medium. In the control experiment 60 mM NaCI was added.

chemostat /z can be varied. Yield studies performed in this way with lactose fermenting S. cremoris 19,36 showed that decreasing the growth p H 36 as well as increasing the external lactate concentration ~9resulted in lower yjmatXse values, as could be expected on basis of the changes in n (Table II). A beneficial effect of lowering the external lactate concentration (and therefore increasing the lactate gradient and the value of n) was also demonstrated in continuous cultures of S. cremoris where the introduction of a lactate consuming Pseudomonas species increased the molar growth yield of the streptococci by over 50%. 38 This shows that detailed information on end-product excretion processes is necessary before bioenergetic parameters such as ymax can be determined. A protonmotive force generated by end-product excretion can in theory be used to drive the synthesis of ATP. 39 This ATP synthesis at the expense of a Ap, generated by end-product efflux, can be demonstrated in membrane vesicles or starved cells with low internal concentrations of ATP (low phosphorylation potential). A somewhat different approach is to study the effect of end-product influx on extravesicular ATP synthesis using inside-out membrane vesicles. Influx of lactate into inside-out vesicles of S. faecalis resulted in extravesicular ATP synthesis. 25 This ATP synthesis was determined from the incorporation of 32p~ into ATP. 4° The 38 R. Otto, J. Hugenholtz, W. N. Konings, and H. Veldkamp, F E M S Microbiol. Lett. 5, 85 (1980). 39 p. C. Maloney and T. H. Wilson, J. Mernbr. Biol. 25, 285 (1975). 40 T. Kagawa and N. Sone, this series, Vol. 55, p. 364.

510

BACTERIALTRANSPORT

[38]

lactate influx induced 32p incorporation was almost completely inhibited by both the protonophore CCCP and DCCD, indicating that indeed a Ap (inside positive and acid) was generated by lactate influx. 25 Conclusions A number of experimental approaches can supply information about the generation of metabolic energy by end-product efflux: 1. Uptake experiments, to demonstrate carrier activity and to obtain information on the H÷/end-product stoichiometry. 2. Efflux experiments from preloaded membranes, to demonstrate end-product efflux driven Ap generation (and subsequent ATP synthesis). 3. Simultaneous measurements of the end-product gradient and Ap in growing or actively metabolizing cells, to obtain information on the H÷/ end-product stoichiometry and possible contributions of energy recycling to the overall production of metabolic energy. 4. Yield experiments under various conditions. Acknowledgments The studies performed in the a u t h o r ' s laboratory were supported by the Dutch Organization o f Pure Scientific Research (ZWO). The help of Mrs. M. Pras and Mrs. M. BroensErenstein in the preparation of the manuscript is highly appreciated.

[38] L i g h t - D r i v e n C h l o r i d e T r a n s p o r t in H a l o r h o d o p s i n - C o n t a i n i n g Cell E n v e l o p e Vesicles

By BRIGITTE SCHOBERT and JANOS K. LANYI Halorhodopsin is the second retinal protein discovered in the plasma membrane of Halobacterium halobium. Like bacteriorhodopsin, it functions as a light-driven ion pump in the cytoplasmic membrane of this organism, but its substrate is chloride instead of protons, and it transports this ion into the cells, rather than outward. For unambiguous results the halobacterial strain ~ used in assays of halorhodopsin should be defective in bacteriorohodopsin, as is strain L-33, but such strains usually contain G. W a g n e r , D. Oesterhelt, G. Krippahl, and J. K. Lanyi, FEBS Lett. 131, 341 (1981).

METHODS IN ENZYMOLOGY, VOL. 125

Copyright © 1986by AcademicPress, Inc. All rights of reproduction in any form reserved.