Generation of an Electrochemical Proton Gradient in Streptococcus Cremoris by Lactate Efflux (Fermentation/Transport) ROEL OTTO, ANTON S

Proc. Natl. Acad. Sci. USA Vol. 77, No. 9, pp. 5502-5506, September 1980 Microbiology

Generation of an electrochemical proton gradient in Streptococcus cremoris by lactate efflux (fermentation/transport) ROEL OTTO, ANTON S. M. SONNENBERG, HANS VELDKAMP, AND WIL N. KONINGS* Department of Microbiology, Biological Centre, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands Communicated by Peter Mitchell, June 9, 1980

ABSTRACT Recently an energy-recycling model was pro- solute will occur when [(n - 1)AT - nZApH] < -Z posed that postulates the generation of an electrochemical log(A-/A-,) and the energy of the solute gradient then will be gradient in fermentative bacteria by carrier-mediated excretion into of the of metabolic end products in symport with protons. In this paper converted energy electrochemical proton gra- experimental support for this model is given. In batch cultures dient. of Streptococcus cremoris with glucose as the sole energy source During fermentation, excretion of metabolic end products the maximal specific growth rate decreased by 30% when the via a carrier in symport with proton(s) can occur only when the external lactate concentration was decreased from 50 to 90 mM. outwardly directed driving force supplied by the chemical end In the same range of external lactate concentrations the molar product gradient exceeds the inwardly directed driving force growth yield Y for glucose as measured in energy-limited che- supplied by the electrochemical gradient. The excretion of end mostat cultures also showed a 30% drop. From ynaxtose values of S. cremoris grown in the presence and absence of added lactate products will then lead to the generation of an electrochemical it was calculated that the net energy ain from the lactate efflux gradient. Michels et al. (1) calculated the generation of the system was at least 12%. Lactate effux from de-energized cells electrochemical gradient for a model cell that excreted lactate loaded with lactate could drive the uptake of leucine. This up- in symport with a variable number (1 or 2) of protons. It was take was sensitive to carbonylcyanide p-trifluoromethoxy- concluded that in these cells lactate efflux could account for an phenylhydrazone and was only partly inhibited by dicyclo- additional 30% of metabolic energy. hexylcarbodiimide (DCCD). The limited inhibition by DCCD of lactate-induced leucine uptake indicates that ATP hydrolysis In this paper experimental support is given for this energy- was not the driving force for transport of leucine. recycling model. The effects of external lactate on maximum Uptake studies with the lipophilic cation tetraphenylphos- specific growth rate, cell yield, and maintenance requirements phonium demonstrated that lactate efflux increased the elec- of Streptococcus cremoris were studied. The chemostat culture trical potential across the membrane by 51 mV. The generation technique was used in this study because it allowed a quanti- of an electrical potential by lactate efflux and the demonstration tation of the effects of lactate on energy metabolism under of a potassium efflux-induced uptake of lactate indicates that energy-limited conditions. In addition, we investigated whether lactate is translocated across the membrane by a symport system lactate efflux could generate an electrochemical gradient in with more than one proton. energy-depleted cells. The energy-recycling model recently proposed by Michels et al. (1) is an extension of the chemosmotic model given by MATERIALS AND METHODS Mitchell (2-4). The energy-recycling model postulates that Culture Conditions. S. cremoris Wg2 was obtained from the carrier-mediated excretion of metabolic end products can lead Dutch Institute of Dairy Research (Nederlands Instituut voor to the generation of an electrochemical gradient across the Zuivelouderzoek, Ecle, The Netherlands). The organism was cytoplasmic membrane, thus providing metabolic energy to routinely maintained in 10% (wt/vol) skimmed milk and stored the cell. The proposed model is based on the following consid- at -20'C. From the milk cultures S. cremoris was transferred erations. to a complex MRS medium (6) and subsequently to a chemically The driving force for translocation of solute A across the defined medium (7). Batch cultures were grown anaerobically cytoplasmic membrane by a solute-proton symport system (2) at 30'C in screw-capped tubes (diameter 10 mm, length 10 cm) is the sum of the electrochemical gradient and the solute gra- or in pH-controlled 3-liter erlenmeyer flasks. Chemostat cul- dient (5): tures were grown anaerobically under N2 atmosphere in glass chemostats with a working volume of 200 ml at 30'C and Z log(A-/A- t) + (n - 1)A' - nZApH, controlled pH of 6.3 as described by Laanbroek et al. (8). in which AiT is the electrical potential and ApH is the pH Maximal Specific Growth Rate in Batch Cultures. Growth gradient across the cytoplasmic membrane, n is the number of rate was determined from the increase of OD660 during expo- protons transported in symport with A-; Z is 2.3RT/F (R, gas nential growth. OD6co was followed by placing the screw- constant; T, absolute temperature; F, Faraday constant); and capped tubes in special adaptors in a Vitatron UC 200 spec- A- and A- t are the concentrations of A- in the cell and the trophotometer (Vitatron Scientific Instruments, Dieren, The external medium, respectively. A steady-state level of accu- Netherlands). mulation is reached when this driving force is zero, thus when Cell Suspensions for Transport Studies. Suspensions were (n - 1)AT - nZApH =-Z log(Aj-/A- t). According to this obtained from 3-liter batch cultures (OD6W1 = 0.8). The cells equation accumulation of solute A- will occur when [(n - were washed twice with 2 liters of 40mM potassium phosphate, 1)AT - nZApH] > -Z log(Aj-/A- t). However, excretion of pH 7.0, at room temperature, resuspended in this buffer to a The publication costs of this article were defrayed inpart by page Abbreviations: MeSGal, methyl 1-thio-3-D-galactopyranoside; FCCP, charge payment. This article must therefore be hereby marked "ad- carbonylcyanide p-trifluoromethoxyphenylhydrazone; DCCD, di- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate cyclohexylcarbodiimide; Ph4P+, tetraphenylphosphonium. this fact. * To whom reprint requests should be addressed. 5502 Downloaded by guest on October 2, 2021 Microbiology: Otto et al. Proc. Natl. Acad. Sci. USA 77 (1980) 5503 density of 100 mg/ml (dry wt) and stored as 0.25-ml samples described by Bakker et al. (17). For batch-grown cells the in- in liquid nitrogen. tracellular volume was 3.79 ,l/mg of cell protein. De-energization of S. cremoris. A 0.25-ml cell suspension Materials. Radioactive labeled leucine and L-lactate were was thawed quickly and washed twice at room temperature obtained from the Radiochemical Centre (Amersham). [3H]- with 10 ml of choline/Hepes/KCI buffer (10 mM choline/ Ph4P+ was generously supplied by H. R. Kaback (Roche Insti- Hepes, pH 7.0, and 2 mM KCI). Methyl I-thio-f3-D-galacto- tute of Molecular Biology, Nutley, NJ). Carbonylcyanide p- pyranoside (MeSGal) was added to the washed cell suspension trifluoromethoxyphenylhydrazone (FCCP) and dicyclohex- [2.5 mg/ml (dry wt)] to a final concentration of 1 mM (9). The ylcarbodiimide (DCCD) were dissolved in absolute ethanol. cell suspension was incubated for 1 hr at room temperature. The Additions to incubation mixtures were made to maximal eth- cells were washed twice with 10 ml of choline/Hepes/KCI anol concentrations of 1% (vol/vol). buffer and finally resuspended in this buffer to a density of 100 mg/ml (dry wt). RESULTS Loading of De-energized Cells with Lactate or Chloride. Effects of External L-Lactate on Growing Cells of S. cre- De-energized cells (0.25 ml) [100 mg/ml (dry wt)] were washed moris. According to the energy-recycling model, efflux of twice with 10 ml of choline/Hepes/KCI buffer supplemented lactate from S. cremoris, growing anaerobically on glucose, with 50 mM choline L-lactate. The suspension was acidified to should result in the generation of an electrochemical proton pH 4.3 with 0.2 M L-lactic acid and incubated at room tem- gradient that contributes to the energy metabolism of the cell. perature-for 10 min. Subsequently the suspension was neu- A decrease of the lactate gradient across the cytoplasmic tralized with 0.2 M choline hydroxide and incubated for an membrane will reduce the energy yield and consequently will additional 30 min at room temperature. Finally the cells were lower the molar growth yield as well as the maximal specific concentrated to a cell density of 100 mg/ml (dry wt). Loading growth rate, if it is assumed that this is determined by the rate of de-energized cells with choline chloride was performed in of ATP supply. the same way except that choline lactate and L-lactic acid were The effect of increasing extracellular L-lactate concentrations replaced by choline chloride and HCI, respectively. on the maximal specific growth rate of S. cremoris, grown Uptake of L-Leucine and Ph4P+. A sample (1 Ml) of un- anaerobically in a synthetic medium in batch culture, is shown loaded or loaded cells [100 mg/ml (dry wt)] was diluted into in Fig. 1. This effect is clearly different from that of other 100 Ml of 10 mM choline/Hepes/KCI buffer containing 2.85 compounds, such as sodium propionate and NaCI (Fig. 1). The AM ['4C]leucine (351 mCi/mmol; 1 Ci = 3.7 X 1010 becquerels) latter compounds exhibited a significant inhibitory effect at or 40 MM [3H]tetraphenylphosphonium bromide (Ph4P+) (54 concentrations as low as 10 mM. Inhibition patterns with KC1, mCi/mmol) and 0-50mM choline L-lactate or 0-50mM cho- choline chloride, and potassium propionate did not differ sig- line chloride. Uptake was stopped by rapid dilution with 2 ml nificantly from the ones found with NaCl and sodium propio- of 0.1 M LiCl. Uptake measurements were further performed nate (data not shown). as described (10, 11). The effect of increasing L-lactate concentrations on the Uptake of L-Lactate and Ph4P+ by Valinomycin-Induced K+ Efflux. Uptake was measured essentially as described by growth yield were studied in glucose-limited chemostat cultures Schuldiner et al. (10). Cell suspension (0.25 ml) [100 mg/ml (dry wt)] was washed twice with 0.1 M potassium phosphate buffer, pH 7.0, and finally concentrated to the original cell density. Valinomycin was added to a final concentration of 0.6 nmol/mg (dry wt). The cell suspension was incubated for 1 hr on ice. Small samples (1 Ml) were diluted in 200 Ml of 0.1 M 0.5 choline phosphate, pH 7.0, or 0.1 M potassium phosphate, pH 7.0, containing 4 MM valinomycin and 10 mM L-['4C]lactate (0.5 mCi/mmol) or 40MuM [3H]Ph4P+ (54 mCi/mmol). Uptake experiments were further performed as described above. Residual Carbohydrate and Metabolic Products. These 0 compounds were determined in the medium fluid of 10-ml SW samples from the chemostat. The samples were filtered over 0- a combination of a 1.2-,um Selectron filter (type AE 95, Schleicher & Schiil, Dassel, Federal Republic of Germany) and a Whatman glass-fiber filter (GF/F). The filtrates were frozen at -20°C until further use. Glucose and lactose were deter- A~~~~~~~~~ mined with the anthrone reagent according to Fairbairn (12), using glucose as a standard. Lactate and acetate were determined in these filtrates as described (13). Dry Weight. Dry weights of cell suspensions were determined from organic carbon measured with a carbon analyzer (model 915 A, Beckman Instruments, Fullerton, CA), using a conversion factor of 52% (wt/wt) carbon per g (dry weight). ATP. Intracellular ATP was extracted as described by Mal- oney and Wilson and (14) determined according to Cole et al 50 1002A0150 (15). mM Protein. Protein was Salt, determined by the method of Herbert FIG. 1. Effects of sodium L-lactate (O.), NaCl (,&), and sodium et al. (16). propionate (3) on the maximal specific growth rate of S. cremoris Intracellular Volume. This volume was determined from grown in batch cultures in chemically defined complex medium at the distribution of [3H]water and ['4C]dextran by the procedure 300C and a constant pH of 6.3. Downloaded by guest on October 2, 2021 5504 Microbiology: Otto et al. Proc. Natl. Acad. Sci. USA 77 (1980)

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0 0 Ia 0.1 0.2 0.3 0.4 Specific growth rate, hr-1 FIG. 3. Relationship between the specific lactose consumption rate (Qlactose) and the specific growth rate of S. cremoris determined 50 100 for the condition that no sodium L-lactate is added to the inflow Lactate, mM medium (0) and for the condition that 60 mM sodium L-lactate is in the inflow medium S. cremoris was cultivated FIG. 2. Effect of sodium L-lactate on molar growth yield (0) and supplemented (A&). in a chemically defined medium supplemented with lactose at 2.5 the production of lactate (A) by S. cremoris during growth on glucose. as in to 2. S. cremoris was cultivated at 30'C in an energy-limited chemostat g/liter described the legend Fig. (dilution rate 0.22 hr-1) in a chemically defined complex medium at pH 6.3 containing glucose at 2.5 g/liter and various concentrations yield corrected for maintenance requirement, and me is the rate of sodium L-lactate. The molar growth yield and the production of of lactose consumption for maintenance purposes [mol of lac- lactate were determined after a steady state was established. tose/g (dry wt) per hr]. Ylactose and me were determined by plotting Q against ,u for (Fig. 2). These were run- at a dilution rate of 0.22 hr-'; glucose cells grown at low and high external lactate concentrations, was converted 86% into L-lactate and 2% into acetate. This respectively (Fig. 3). From Fig. 3 the following data were de- fermentation pattern remained constant at all external L-lactate rived. For cells growing in the presence of lactate (30 mM) concentrations studied (Fig. 2), indicating that the same met- formed by fermentation only, Yma"x = 56.3 and me = 36 X abolic pathways were involved in the metabolism of glucose 10-5, and for cells growing in the presence of extra lactate at different L-lactate concentrations. The molar growth yield (steady-state concentration of 90 mM) these values were Ymax on a on L- glucose (Yglucose) showed dependency the external 50.2 and me = 58 X 10. lactate concentration similar to that of the specific growth The decrease of Ymaxa appears to be specific for lactate be- rate. cause similar experiments performed with 60 mM NaCl added Yield from L-Lactate Efflux. In cells Energy energy-limited to the inflow medium yielded a ymawt)/mollactose o 69g(r t/o Yglucoae is a function of the amount of ATP formed in glucose lactose and a me of 50 X 10-5 mol lactose/g (dry wt) per hr. In dissimilation. During homolactic fermentation, S. cremoris all experiments 92% of lactose C was recovered as lactate and forms 2 mol of ATP at the substrate level for each mol of glucose 2% as acetate, which means that in all cases 4.12 mol of ATP fermented. And, as shown above, there were no indications that were formed at the substrate level per mol of lactose fer- substrate-level ATP generation was affected by external lactate mented. concentrations (Fig. 2). The above results show that the molar growth yields cor- The decrease of Yglucose with increasing external lactate rected for maintenance are different in cells grown in the to an mainte- concentration therefore might be due increased presence of high (90 mM) and low (30 mM) external concen- nance requirement, a in energy decrease the yield through trations of lactate. And this difference can be explained only lactate efflux, or both, as predicted by the energy-recycling in terms of differences in energy gained by lactate efflux. The model. amount of energy thus obtained in cells grown at a relatively To test these the maintenance requirement of possibilities, low external lactate concentration can be estimated as fol- S. cremoris was determined in a lactose-limited chemostat at lows. different dilution rates. The steady-state lactate concentration The total number of ATP equivalents formed equals at all dilution rates (= specific growth rates) was approximately Ymax IYmx, in which YmTx is the growth yield per mol of ATP 30 mM, or 90mM in experiments in which 60 mM lactate was corrected for maintenance requirement. If it is assumed that added to the inflow medium of the chemostat. Ymx is the same for cells grown at low and high lactate con- The maintenance requirements in both cases were deter- and that the lactate efflux in cells mined graphically, applying the following equation (18) centrations, energy gain by grown in the presence of high external lactate concentration /y ma in Qlactose = ylactow = H/lactose + me, is negligible, then the energy gain by lactate efflux cells grown at lower lactate concentration can be estimated as fol- in which Qlactose is the specific lactose consumption rate [mol lows: of lactose/g (dry wt) per hr], ju is specific growth rate (h-'), = = 12 Ylactose is molar growth yield observed, Ylactose is molar growth Ymax'ATP (Ymax)LA/(4.12'lactoself ~UlactoseiHi412+ X) (ymax )/4 Downloaded by guest on October 2, 2021 Microbiology: Otto et al. Proc. Natl. Acad. Sci. USA 77 (1980) 5505

0 #4-Dco FIG. 4. Time course of L-lactate efflux-induced leucine r., uptake by S. cremoris. De-energized cells [100 mg/ml (dry -4 wt)] were diluted 1:100 into choline/Hepes/KCl buffer at 4.2 250C. (A) Cells loaded with 50 mM choline L-lactate were diluted into choline/Hepes/KCI buffer (0) or into choline/Hepes/KCl buffer containing 50 mM choline L-lactate (0). Cells loaded with 50 mM choline chloride were diluted into choline/Hepes/KCl buffer (+). (B) Cells were prein- cubated with 25 ,M DCCD for 30 min at room temperature. Cells loaded with 50 mM choline L-lactate were diluted into choline/Hepes/KCl buffer containing 25 gM DCCD (v) or into the same medium supplemented with 50 mM choline L-lactate (0). (C) Cells loaded with 50 mM choline L-lactate were diluted into choline/Hepes/KCl buffer containing 10 uM FCCP (A) or into the same me- Time, sec dium containing 50 mM choline L-lactate (X).

in which (YmlxO)L is the molar growth yield corrected for Fig. 4C shows that lactate efflux-induced leucine uptake is maintenance at low lactate concentration, (Y=.')H is the value completely inhibited by the uncoupler FCCP. This observation found in cells grown with high external lactate concentration, indicates that lactate efflux leads to the generation of an elec- and x is ATP equivalents formed by lactate efflux in cells grown trochemical proton gradient. Direct evidence for the generation in the presence of low external lactate concentration. The value of one of the components of this electrochemical proton gra- for x thus found is 0.5 mol of ATP per mol of lactose. This means dient, the At, was supplied by uptake studies of the lipophilic that lactate efflux in cells grown with 30 mM external lactate cation Ph4P+. Upon dilution of cells loaded with 50 mM choline results in an extra energy gain of at least 12%. This indirect L-lactate into [3H]Ph4P+-containing choline/Hepes/KCI buffer evidence obtained from growth studies was supported by the a maximal accumulation level of Ph4P+ of 35-fold was found, transport studies described below. while dilution of these cells into [3H]Ph4P+-containing cho- Lactate Efflux-Induced Leucine Accumulation. When cells line/Hepes/KCI buffer supplemented with 50 mM choline of S. cremoris, starved for endogenous energy, were diluted into L-lactate resulted in a maximal level of Ph4P+ accumulation leucine-containing choline/Hepes/KCI buffer, no concentra- of about 5-fold. From the increased level of Ph4P+ accumulation of leucine was observed. A small level of leucine accumu- tion it can be calculated with the Nernst equation that lactate lation was observed in cells loaded with 50 mM choline L-lactate efflux increased the At by 51 mV. upon dilution in choline/Hepes/KCI buffer supplemented with 50 mM choline L-lactate (Fig. 4A). However, a significantly higher level of leucine accumulation occurred upon dilution

of these loaded cells into a lactate-free medium. The maximal A B level of accumulation was 5-fold after 15 sec; after this time a rapid efflux of accumulated leucine occurred. The energy for leucine uptake appeared to be supplied .3 specifically by lactate efflux and not by ATP hydrolysis, because '4~~~~~~~~~ ~ ~~ ~ ~ ~ ~ ~ ~~~~~~~~~~~~~~~~~~~~~~~~. 0 0 significantly lower levels of leucine accumulation were ob- 4-4 served in the presence of 50 mM extracellular choline lactate. Moreover, no stimulation above the levels of unloaded cells was 50- observed in cells loaded with 30 mM choline chloride and diluted into a choline chloride-free medium (Fig. 4A). Additional evidence that ATP hydrolysis is not the main energy source for leucine uptake comes from ATP measurements in S. cremoris. ATP levels as low as 0.14-0.18 Mmol of ATP per g (dry wt) were found in the starved cells and the same levels were found after preloading of the cells or directly after dilution of preloaded cells. Evidence has been presented that the energy for amino acid uptake in a related organism, S. faecalis, was supplied solely 60 120 60 120 180 by ATP hydrolysis via the membrane-bound ATPase (19). In Time, sec our experiments ATP hydrolysis can play a major role in the energization of leucine uptake observed only in the absence of FIG. 5. Time course of K+ effux-induced uptake of Ph4P+ and a lactate gradient (Fig. 4A). DCCD, which has been shown to L-lactate. De-energized cells [100 mg ml/(dry wt)] were incubated with 0.6 nmol of per wt) in 0.1 M more in valinomycin mg (dry potassium phos- inhibit leucine uptake by than 95% freshly harvested phate, pH 7.0, for 1 hr on ice. The K-loaded cells were diluted 1:200 cell suspensions (data not shown), inhibited leucine uptake in at 25°C into 0.1 M choline phosphate, pH 7.0, containing Ph4P+(2) the presence of a lactate gradient only to a small extent (Fig. or L-lactate W- or into 0.1 M potassium phosphate, pH 7.0, containing 4B). Ph4P+ Cu)or L-lactate Cv). Downloaded by guest on October 2, 2021 5506 Microbiology: Otto et al. Proc. Natl. Acad. Sct. USA 77 (1980)

In contrast to the lactate efflux-induced Ph4P+ uptake, a indicates that at this external pH the H+ per lactate stoichi- decrease of Ph4P+ uptake was observed in cells preloaded with ometry is more than 1. 50 mM choline chloride and diluted in choline chloride-free A drastic decrease of the specific growth rate and growth medium. yield was observed above external lactate concentrations of 50 Carrier-Mediated Lactate Transport. Efflux of lactate could mM. Up to 50 mM external L-lactate the cells are most likely occur by passive diffusion of the undissociated acid. Such a capable of maintaining a high internal pH, whereas above 50 translocation process would be dependent only on the chemical mM external L-lactate the internal pH decreases and more proton gradient, the ApH. An essential feature of the energy- L-lactate will leave the cells by a passive diffusion process (or recycling model is that lactate efflux is mediated by a carrier by a carrier-mediated process with a lactate per H+ stoichi- in symport with protons and that this translocation is sym- ometry of 1). Concomitantly the contribution to the generation metrical and reversible. It has been argued in our previous of an electrochemical proton gradient will decrease. When the publication (1) that a significant energy gain from lactate efflux external lactate concentration has reached 90 mM, essentially can be expected only when lactate efflux occurs in symport with all lactate molecules leave the cells together with one proton more than one proton. Under these conditions lactate translo- and the contribution of lactate efflux to the energy yield has cation will depend not only on the ApH but also on the AI. reached its minimal value. Strong support for the energy-recycling model will therefore be supplied when AI-driven lactate uptake can be demon- This study was financially supported by the Dutch Institute for Dairy strated in S. cremoris, especially because this organism cannot Research (Nederlands Instituut voor Zuivelonderzoek). grow on lactate or utilize lactate as a carbon source. In Fig. 5 such evidence is presented. In energy-depleted cells of S. cre- 1. Michels, P. A. M., Michels, J. P. J., Boonstra, J. & Konings, W. N. mnris a A'I is generated by valinomycin-induced K+ efflux, (1979) FEMS Microbiol. Lett. 5, 357-364. as is shown by the uptake of Ph4P+ (Fig. 5A). Fig. 5B shows that 2. Mitchell, P. (1973) Bloenergetics 4,63-91. the At, generated by K+ efflux, drives the uptake of L-lactate 3. Mitchell, P. (1976) J. Theor. Biol. 62,,327-8. into these energy-depleted cells. 4. Mitchell, P. (1970) Symp. Soc. Gen. Microbiol. 20,121-166. 5. Konings, W. N. & Michels, P. A. M. (1979) in Diversity of Bac- DISCUSSION terial Respiratory Systems, ed. Knowles, D. C. J. (CRC, Cleve- land, OH), in press. A few reports have appeared which show that uptake of amino 6. De Man, J. C., Rogosa, M. & Sharpe, M. E. (1960) J. Appl. Bac- acids by intact cells of Escherichia coil can be driven by car- teriol. 23,130-135. rier-mediated efflux of MeSGal and gluconate (20, 21). Re- 7. Rogosa, M., Franklin, J. C. & Perry, K. D. (1961) J. Gen. Micro- cently, Kaczorowski et al. (22, 23) demonstrated in membrane biol. 25,473-482. vesicles from E. coil that carrier-mediated efflux of lactose 8. Laanbroek, H. J., Kingma, W. & Veldkamp, H. (1977) FEMS results in the generation of an electrical potential across the Microbiol. Lett. 1, 99-102. cytoplasmic membrane. Similar results were obtained in E. colf 9. Thompson, J. & Thomas, T. D. (1977) J. Bacteriol. 130, 583- membrane vesicles with lactate efflux (unpublished results). 595. & H. R. 14,5451- generation of an proton gradient by car- 10. Schuldiner, S. Kaback, (1976) Biochemistry The electrochemical 5461. rier-mediated solute efflux seems, therefore, well estab- 11. Matin, A. & Konings, W. N. (1973) Eur. J. Biochem. 43, 58- lished. 67. Such efflux processes can contribute significantly to the 12. Fairbairn, N. J. (1953) Chem. Ind. (London), 86. metabolic energy of a cell only when efflux of solutes is a con- 13. Holdeman, L. V., Cato, E. P. & Moore, W. E. C., eds. (1977) tinuous process. The energy-recycling model postulates that Anaerobe Laboratory Manual (Virginia Polytechnic Institute the excretion of metabolic end products is such a continuous and State University, Anaerobic Laboratory, Blacksburg, VA), carrier-mediated efflux process and that this process is an im- p. 134. portant mechanism for the generation of metabolic energy 14. Maloney, P. C. & Wilson, T. H. (1975) J. Memb. Biol. 25, during fermentation. In this publication experimental support 285-310. that 15. Cole, H. A., Wimpenny, J. W. T. & Hughes, D. E. (1967) Bio- for this model is presented. These studies demonstrate chim. Biophys. Acta 143, 445-453. lactate efflux in S. cremoris is carrier mediated and results in 16. Herbert, D., Phipps, P. J. & Strange, R. E. (1971) in Methods in the generation of an electrical potential across the membrane. Microbiology, eds. Norris, J. R. & Ribbons, D. W. (Academic, This lactate efflux-induced electrochemical proton gradient New York), Vol. 5B, pp. 149-250. increases the energy yield during growth on lactose by at least 17. Bakker, E. P., Rottenberg, H. & Kaplan, S. R. (1976) Biochim. 12%. Biophys. Acta 440,557-572. In our previous publication we had calculated the additional 18. Schulze, K. L. & Lipe, R. S. (1964) Arch. Mikrobiol. 48, 1-20. energy yield by lactate efflux from a model cell. Several as- 19. Asgar, S. S., Levin, E. & Harold, F. M. (1973)J. Biol. Chem. 248, sumptions and simplifications were made. The rate of lactate 5225-5233. 20. L. & T. H. Membr. Biochem. 61- was assumed to be constant at all external lactate Flagg, J. Wilson, (1978) 1, production 72. concentrations. The results presented in Fig. 2, however, show 21. Bentaboulet, M., Robin, A. & Kepes, A. (1979) Biochem. J. 178, that this assumption is not correct and that the Qiactate increases 103-107. with increasing external lactate concentrations. The pK of the 22. Kaczorowski, J. G. & Kaback, H. R. (1979) Biochemistry 18, carrier was assumed to be 6.8. Information about this aspect is 3691-3696. not supplied in this investigation. However, the observation that 23. Kaczorowski, J. G., Robertson, D. E. & Kaback, H. R. (1979) a AI is generated by lactate efflux at an external pH of 7.0 Biochemistry 18, 3697-3704. Downloaded by guest on October 2, 2021