Generation of an electrochemical proton gradient in Streptococcus

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 77, No. 9, pp , September 198 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 3, 9751 NN Haren, The Netherlands Communicated by Peter Mitchell, June 9, 198 ABSTRACT Recently an energy-recycling model was proposed that postulates the generation of an electrochemical gradient in fermentative bacteria by carrier-mediated excretion of metabolic end products in symport with protons. In this paper experimental support for this model is given. In batch cultures of Streptococcus cremoris with glucose as the sole energy source the maximal specific growth rate decreased by 3% when the external lactate concentration was decreased from 5 to 9 mm. In the same range of external lactate concentrations the molar growth yield Y for glucose as measured in energy-limited chemostat cultures also showed a 3% drop. From ynaxtose values of S. cremoris grown in the presence and absence of added lactate it was calculated that the net energy ain from the lactate efflux system was at least 12%. Lactate effux from de-energized cells loaded with lactate could drive the uptake of leucine. This uptake was sensitive to carbonylcyanide p-trifluoromethoxyphenylhydrazone and was only partly inhibited by dicyclohexylcarbodiimide (DCCD). The limited inhibition by DCCD of lactate-induced leucine uptake indicates that ATP hydrolysis was not the driving force for transport of leucine. Uptake studies with the lipophilic cation tetraphenylphosphonium demonstrated that lactate efflux increased the electrical potential across the membrane by 51 mv. The generation of an electrical potential by lactate efflux and the demonstration of a potassium efflux-induced uptake of lactate indicates that lactate is translocated across the membrane by a symport system with more than one proton. The energy-recycling model recently proposed by Michels et al. (1) is an extension of the chemosmotic model given by Mitchell (2-4). The energy-recycling model postulates that carrier-mediated excretion of metabolic end products can lead to the generation of an electrochemical gradient across the cytoplasmic membrane, thus providing metabolic energy to the cell. The proposed model is based on the following considerations. The driving force for translocation of solute A across the cytoplasmic membrane by a solute-proton symport system (2) is the sum of the electrochemical gradient and the solute gradient (5): Z log(a-/a- t) + (n - 1)A' - nzaph, in which AiT is the electrical potential and ApH is the ph gradient across the cytoplasmic membrane, n is the number of protons transported in symport with A-; Z is 2.3RT/F (R, gas constant; T, absolute temperature; F, Faraday constant); and A- and A- t are the concentrations of A- in the cell and the external medium, respectively. A steady-state level of accumulation is reached when this driving force is zero, thus when (n - 1)AT - nzaph =-Z log(aj-/a- t). According to this equation accumulation of solute A- will occur when [(n - 1)AT - nzaph] > -Z log(aj-/a- t). However, excretion of The publication costs of this article were defrayed inpart by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact. 552 solute will occur when [(n - 1)AT - nzaph] < -Z log(a-/a-,) and the energy of the solute gradient then will be converted into energy of the electrochemical proton gradient. During fermentation, excretion of metabolic end products via a carrier in symport with proton(s) can occur only when the outwardly directed driving force supplied by the chemical end product gradient exceeds the inwardly directed driving force supplied by the electrochemical gradient. The excretion of end products will then lead to the generation of an electrochemical gradient. Michels et al. (1) calculated the generation of the electrochemical gradient for a model cell that excreted lactate in symport with a variable number (1 or 2) of protons. It was concluded that in these cells lactate efflux could account for an additional 3% of metabolic energy. In this paper experimental support is given for this energyrecycling model. The effects of external lactate on maximum specific growth rate, cell yield, and maintenance requirements of Streptococcus cremoris were studied. The chemostat culture technique was used in this study because it allowed a quantitation of the effects of lactate on energy metabolism under energy-limited conditions. In addition, we investigated whether lactate efflux could generate an electrochemical gradient in energy-depleted cells. MATERIALS AND METHODS Culture Conditions. S. cremoris Wg2 was obtained from the Dutch Institute of Dairy Research (Nederlands Instituut voor Zuivelouderzoek, Ecle, The Netherlands). The organism was routinely maintained in 1% (wt/vol) skimmed milk and stored at -2'C. From the milk cultures S. cremoris was transferred to a complex MRS medium (6) and subsequently to a chemically defined medium (7). Batch cultures were grown anaerobically at 3'C in screw-capped tubes (diameter 1 mm, length 1 cm) or in ph-controlled 3-liter erlenmeyer flasks. Chemostat cultures were grown anaerobically under N2 atmosphere in glass chemostats with a working volume of 2 ml at 3'C and controlled ph of 6.3 as described by Laanbroek et al. (8). Maximal Specific Growth Rate in Batch Cultures. Growth rate was determined from the increase of OD66 during exponential growth. OD6co was followed by placing the screwcapped tubes in special adaptors in a Vitatron UC 2 spectrophotometer (Vitatron Scientific Instruments, Dieren, The Netherlands). Cell Suspensions for Transport Studies. Suspensions were obtained from 3-liter batch cultures (OD6W1 =.8). The cells were washed twice with 2 liters of 4 mm potassium phosphate, ph 7., at room temperature, resuspended in this buffer to a Abbreviations: MeSGal, methyl 1-thio-3-D-galactopyranoside; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; DCCD, dicyclohexylcarbodiimide; Ph4P+, tetraphenylphosphonium. * To whom reprint requests should be addressed.

2 Microbiology: Otto et al. density of 1 mg/ml (dry wt) and stored as.25-ml samples in liquid nitrogen. De-energization of S. cremoris. A.25-ml cell suspension was thawed quickly and washed twice at room temperature with 1 ml of choline/hepes/kci buffer (1 mm choline/ Hepes, ph 7., and 2 mm KCI). Methyl I-thio-f3-D-galactopyranoside (MeSGal) was added to the washed cell suspension [2.5 mg/ml (dry wt)] to a final concentration of 1 mm (9). The cell suspension was incubated for 1 hr at room temperature. The cells were washed twice with 1 ml of choline/hepes/kci buffer and finally resuspended in this buffer to a density of 1 mg/ml (dry wt). Loading of De-energized Cells with Lactate or Chloride. De-energized cells (.25 ml) [1 mg/ml (dry wt)] were washed twice with 1 ml of choline/hepes/kci buffer supplemented with 5 mm choline L-lactate. The suspension was acidified to ph 4.3 with.2 M L-lactic acid and incubated at room temperature-for 1 min. Subsequently the suspension was neutralized with.2 M choline hydroxide and incubated for an additional 3 min at room temperature. Finally the cells were concentrated to a cell density of 1 mg/ml (dry wt). Loading of de-energized cells with choline chloride was performed in the same way except that choline lactate and L-lactic acid were replaced by choline chloride and HCI, respectively. Uptake of L-Leucine and Ph4P+. A sample (1 Ml) of unloaded or loaded cells [1 mg/ml (dry wt)] was diluted into 1 Ml of 1 mm choline/hepes/kci buffer containing 2.85 AM ['4C]leucine (351 mci/mmol; 1 Ci = 3.7 X 11 becquerels) or 4 MM [3H]tetraphenylphosphonium bromide (Ph4P+) (54 mci/mmol) and -5mM choline L-lactate or -5mM choline chloride. Uptake was stopped by rapid dilution with 2 ml of.1 M LiCl. Uptake measurements were further performed as described (1, 11). Uptake of L-Lactate and Ph4P+ by Valinomycin-Induced K+ Efflux. Uptake was measured essentially as described by Schuldiner et al. (1). Cell suspension (.25 ml) [1 mg/ml (dry wt)] was washed twice with.1 M potassium phosphate buffer, ph 7., and finally concentrated to the original cell density. Valinomycin was added to a final concentration of.6 nmol/mg (dry wt). The cell suspension was incubated for 1 hr on ice. Small samples (1 Ml) were diluted in 2 Ml of.1 M choline phosphate, ph 7., or.1 M potassium phosphate, ph 7., containing 4 MM valinomycin and 1 mm L-['4C]lactate (.5 mci/mmol) or 4MuM [3H]Ph4P+ (54 mci/mmol). Uptake experiments were further performed as described above. Residual Carbohydrate and Metabolic Products. These compounds were determined in the medium fluid of 1-ml samples from the chemostat. The samples were filtered over 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 -2 C until further use. Glucose and lactose were determined 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 Maloney and Wilson (14) and determined according to Cole et al (15). Protein. Protein was determined by the method of Herbert et al. (16). Intracellular Volume. This volume was determined from the distribution of [3H]water and ['4C]dextran by the procedure Proc. Natl. Acad. Sci. USA 77 (198) 553 described by Bakker et al. (17). For batch-grown cells the intracellular volume was 3.79,l/mg of cell protein. Materials. Radioactive labeled leucine and L-lactate were obtained from the Radiochemical Centre (Amersham). [3H]- Ph4P+ was generously supplied by H. R. Kaback (Roche Institute of Molecular Biology, Nutley, NJ). Carbonylcyanide p- trifluoromethoxyphenylhydrazone (FCCP) and dicyclohexylcarbodiimide (DCCD) were dissolved in absolute ethanol. Additions to incubation mixtures were made to maximal ethanol concentrations of 1% (vol/vol). RESULTS Effects of External L-Lactate on Growing Cells of S. cremoris. According to the energy-recycling model, efflux of lactate from S. cremoris, growing anaerobically on glucose, should result in the generation of an electrochemical proton gradient that contributes to the energy metabolism of the cell. A decrease of the lactate gradient across the cytoplasmic membrane will reduce the energy yield and consequently will lower the molar growth yield as well as the maximal specific growth rate, if it is assumed that this is determined by the rate of ATP supply. The effect of increasing extracellular L-lactate concentrations on the maximal specific growth rate of S. cremoris, grown anaerobically in a synthetic medium in batch culture, is shown in Fig. 1. This effect is clearly different from that of other compounds, such as sodium propionate and NaCI (Fig. 1). The latter compounds exhibited a significant inhibitory effect at concentrations as low as 1 mm. Inhibition patterns with KC1, choline chloride, and potassium propionate did not differ significantly from the ones found with NaCl and sodium propionate (data not shown). The effect of increasing L-lactate concentrations on the growth yield were studied in glucose-limited chemostat cultures SW.5 - A~~~~~~~~~ 5 1 2A 15 Salt, mm FIG. 1. Effects of sodium L-lactate (O.), NaCl (,&), and sodium propionate (3) on the maximal specific growth rate of S. cremoris grown in batch cultures in chemically defined complex medium at 3C and a constant ph of 6.3.

3 554 Microbiology: Otto et al. Proc. Natl. Acad. Sci. USA 77 (198) 2. o 25 Q ED._a 2 t, la '' U) U_ 1.5 ' vz 1. E _ Cd *1 co E I5-4 E bo U 1' Ia 5 Lactate, mm 1 FIG. 2. Effect of sodium L-lactate on molar growth yield () and the production of lactate (A) by S. cremoris during growth on glucose. S. cremoris was cultivated at 3'C in an energy-limited chemostat (dilution rate.22 hr-1) in a chemically defined complex medium at ph 6.3 containing glucose at 2.5 g/liter and various concentrations of sodium L-lactate. The molar growth yield and the production of lactate were determined after a steady state was established. (Fig. 2). These were run- at a dilution rate of.22 hr-'; glucose was converted 86% into L-lactate and 2% into acetate. This fermentation pattern remained constant at all external L-lactate concentrations studied (Fig. 2), indicating that the same metabolic pathways were involved in the metabolism of glucose at different L-lactate concentrations. The molar growth yield on glucose (Yglucose) showed a dependency on the external L- lactate concentration similar to that of the specific growth rate. Energy Yield from L-Lactate Efflux. In energy-limited cells Yglucoae is a function of the amount of ATP formed in glucose dissimilation. During homolactic fermentation, S. cremoris forms 2 mol of ATP at the substrate level for each mol of glucose fermented. And, as shown above, there were no indications that substrate-level ATP generation was affected by external lactate concentrations (Fig. 2). The decrease of Yglucose with increasing external lactate concentration therefore might be due to an increased maintenance requirement, a decrease in the energy yield through lactate efflux, or both, as predicted by the energy-recycling model. To test these possibilities, the maintenance requirement of S. cremoris was determined in a lactose-limited chemostat at different dilution rates. The steady-state lactate concentration at all dilution rates (= specific growth rates) was approximately 3 mm, or 9mM in experiments in which 6 mm lactate was added to the inflow medium of the chemostat. The maintenance requirements in both cases were determined graphically, applying the following equation (18) /y Qlactose = ylactow = H/lactose ma + me, in which Qlactose is the specific lactose consumption rate [mol of lactose/g (dry wt) per hr], ju is specific growth rate (h-'), Ylactose is molar growth yield observed, Ylactose is molar growth Specific growth rate, hr-1 FIG. 3. Relationship between the specific lactose consumption rate (Qlactose) and the specific growth rate of S. cremoris determined for the condition that no sodium L-lactate is added to the inflow medium () and for the condition that 6 mm sodium L-lactate is supplemented in the inflow medium (A&). S. cremoris was cultivated in a chemically defined medium supplemented with lactose at 2.5 g/liter as described in the legend to Fig. 2. yield corrected for maintenance requirement, and me is the rate of lactose consumption for maintenance purposes [mol of lactose/g (dry wt) per hr]. Ylactose and me were determined by plotting Q against,u for cells grown at low and high external lactate concentrations, respectively (Fig. 3). From Fig. 3 the following data were derived. For cells growing in the presence of lactate (3 mm) formed by fermentation only, Yma"x = 56.3 and me = 36 X 1-5, and for cells growing in the presence of extra lactate (steady-state concentration of 9 mm) these values were Ymax 5.2 and me = 58 X 1. The decrease of Ymaxa appears to be specific for lactate because similar experiments performed with 6 mm NaCl added to the inflow medium yielded a ymawt)/mol lactose o 69g(r t/o lactose and a me of 5 X 1-5 mol lactose/g (dry wt) per hr. In all experiments 92% of lactose C was recovered as lactate and 2% as acetate, which means that in all cases 4.12 mol of ATP were formed at the substrate level per mol of lactose fermented. The above results show that the molar growth yields corrected for maintenance are different in cells grown in the presence of high (9 mm) and low (3 mm) external concentrations of lactate. And this difference can be explained only in terms of differences in energy gained by lactate efflux. The amount of energy thus obtained in cells grown at a relatively low external lactate concentration can be estimated as follows. The total number of ATP equivalents formed equals Ymax IYmx, in which YmTx is the growth yield per mol of ATP corrected for maintenance requirement. If it is assumed that Ymx is the same for cells grown at low and high lactate concentrations, and that the energy gain by lactate efflux in cells grown in the presence of high external lactate concentration is negligible, then the energy gain by lactate efflux in cells grown at lower lactate concentration can be estimated as follows: Ymax 'ATP (Ymax)LA/(4.12 = 'lactoself ~UlactoseiHi412 + X) = (ymax )/4

4 Microbiology: Otto et al. Proc. Natl. Acad. Sci. USA 77 (198) 555 #4-D co r., Time, sec in which (YmlxO)L is the molar growth yield corrected for maintenance at low lactate concentration, (Y=.')H is the value found in cells grown with high external lactate concentration, and x is ATP equivalents formed by lactate efflux in cells grown in the presence of low external lactate concentration. The value for x thus found is.5 mol of ATP per mol of lactose. This means that lactate efflux in cells grown with 3 mm external lactate results in an extra energy gain of at least 12%. This indirect evidence obtained from growth studies was supported by the transport studies described below. Lactate Efflux-Induced Leucine Accumulation. When cells of S. cremoris, starved for endogenous energy, were diluted into leucine-containing choline/hepes/kci buffer, no concentration of leucine was observed. A small level of leucine accumulation was observed in cells loaded with 5 mm choline L-lactate upon dilution in choline/hepes/kci buffer supplemented with 5 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 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 specifically by lactate efflux and not by ATP hydrolysis, because significantly lower levels of leucine accumulation were observed in the presence of 5 mm extracellular choline lactate. Moreover, no stimulation above the levels of unloaded cells was observed in cells loaded with 3 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 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 by ATP hydrolysis via the membrane-bound ATPase (19). In our experiments ATP hydrolysis can play a major role in the energization of leucine uptake observed only in the absence of a lactate gradient (Fig. 4A). DCCD, which has been shown to inhibit leucine uptake by more than 95% in freshly harvested cell suspensions (data not shown), inhibited leucine uptake in the presence of a lactate gradient only to a small extent (Fig. 4B). FIG. 4. Time course of L-lactate efflux-induced leucine uptake by S. cremoris. De-energized cells [1 mg/ml (dry wt)] were diluted 1:1 into choline/hepes/kcl buffer at 25C. (A) Cells loaded with 5 mm choline L-lactate were diluted into choline/hepes/kci buffer () or into choline/hepes/kcl buffer containing 5 mm choline L-lactate (). Cells loaded with 5 mm choline chloride were diluted into choline/hepes/kcl buffer (+). (B) Cells were preincubated with 25,M DCCD for 3 min at room temperature. Cells loaded with 5 mm choline L-lactate were diluted into choline/hepes/kcl buffer containing 25 gm DCCD (v) or into the same medium supplemented with 5 mm choline L-lactate (). (C) Cells loaded with 5 mm choline L-lactate were diluted into choline/hepes/kcl buffer containing 1 um FCCP (A) or into the same medium containing 5 mm choline L-lactate (X). Fig. 4C shows that lactate efflux-induced leucine uptake is completely inhibited by the uncoupler FCCP. This observation indicates that lactate efflux leads to the generation of an electrochemical proton gradient. Direct evidence for the generation of one of the components of this electrochemical proton gradient, the At, was supplied by uptake studies of the lipophilic cation Ph4P+. Upon dilution of cells loaded with 5 mm choline L-lactate into [3H]Ph4P+-containing choline/hepes/kci buffer a maximal accumulation level of Ph4P+ of 35-fold was found, while dilution of these cells into [3H]Ph4P+-containing choline/hepes/kci buffer supplemented with 5 mm choline L-lactate resulted in a maximal level of Ph4P+ accumulation of about 5-fold. From the increased level of Ph4P+ accumulation it can be calculated with the Nernst equation that lactate efflux increased the At by 51 mv. A '4 ~ ~ ~~ ~ ~ ~ ~ ~ ~~~~~~~~~~~~~~~~~~~~~~~~ Time, sec FIG. 5. Time course of K+ effux-induced uptake of Ph4P+ and L-lactate. De-energized cells [1 mg ml/(dry wt)] were incubated with.6 nmol of valinomycin per mg (dry wt) in.1 M potassium phosphate, ph 7., for 1 hr on ice. The K-loaded cells were diluted 1:2 at 25 C into.1 M choline phosphate, ph 7., containing Ph4P+(2) or L-lactate W- or into.1 M potassium phosphate, ph 7., containing Ph4P+ Cu) or L-lactate Cv). B.3 4-4

5 556 Microbiology: Otto et al. In contrast to the lactate efflux-induced Ph4P+ uptake, a decrease of Ph4P+ uptake was observed in cells preloaded with 5 mm choline chloride and diluted in choline chloride-free medium. Carrier-Mediated Lactate Transport. Efflux of lactate could occur by passive diffusion of the undissociated acid. Such a translocation process would be dependent only on the chemical proton gradient, the ApH. An essential feature of the energyrecycling model is that lactate efflux is mediated by a carrier in symport with protons and that this translocation is symmetrical and reversible. It has been argued in our previous publication (1) that a significant energy gain from lactate efflux can be expected only when lactate efflux occurs in symport with more than one proton. Under these conditions lactate translocation will depend not only on the ApH but also on the AI. Strong support for the energy-recycling model will therefore be supplied when AI-driven lactate uptake can be demonstrated in S. cremoris, especially because this organism cannot grow on lactate or utilize lactate as a carbon source. In Fig. 5 such evidence is presented. In energy-depleted cells of S. cremnris a A'I is generated by valinomycin-induced K+ efflux, as is shown by the uptake of Ph4P+ (Fig. 5A). Fig. 5B shows that the At, generated by K+ efflux, drives the uptake of L-lactate into these energy-depleted cells. DISCUSSION A few reports have appeared which show that uptake of amino acids by intact cells of Escherichia coil can be driven by carrier-mediated efflux of MeSGal and gluconate (2, 21). Recently, Kaczorowski et al. (22, 23) demonstrated in membrane vesicles from E. coil that carrier-mediated efflux of lactose results in the generation of an electrical potential across the cytoplasmic membrane. Similar results were obtained in E. colf membrane vesicles with lactate efflux (unpublished results). The generation of an electrochemical proton gradient by carrier-mediated solute efflux seems, therefore, well established. Such efflux processes can contribute significantly to the metabolic energy of a cell only when efflux of solutes is a continuous process. The energy-recycling model postulates that the excretion of metabolic end products is such a continuous carrier-mediated efflux process and that this process is an important mechanism for the generation of metabolic energy during fermentation. In this publication experimental support for this model is presented. These studies demonstrate that lactate efflux in S. cremoris is carrier mediated and results in the generation of an electrical potential across the membrane. This lactate efflux-induced electrochemical proton gradient increases the energy yield during growth on lactose by at least 12%. In our previous publication we had calculated the additional energy yield by lactate efflux from a model cell. Several assumptions and simplifications were made. The rate of lactate production was assumed to be constant at all external lactate concentrations. The results presented in Fig. 2, however, show that this assumption is not correct and that the Qiactate increases with increasing external lactate concentrations. The pk of the carrier was assumed to be 6.8. Information about this aspect is not supplied in this investigation. However, the observation that a AI is generated by lactate efflux at an external ph of 7. Proc. Natl. Acad. Sct. USA 77 (198) indicates that at this external ph the H+ per lactate stoichiometry is more than 1. A drastic decrease of the specific growth rate and growth yield was observed above external lactate concentrations of 5 mm. Up to 5 mm external L-lactate the cells are most likely capable of maintaining a high internal ph, whereas above 5 mm external L-lactate the internal ph decreases and more L-lactate will leave the cells by a passive diffusion process (or by a carrier-mediated process with a lactate per H+ stoichiometry of 1). Concomitantly the contribution to the generation of an electrochemical proton gradient will decrease. When the external lactate concentration has reached 9 mm, essentially all lactate molecules leave the cells together with one proton and the contribution of lactate efflux to the energy yield has reached its minimal value. This study was financially supported by the Dutch Institute for Dairy Research (Nederlands Instituut voor Zuivelonderzoek). 1. Michels, P. A. M., Michels, J. P. J., Boonstra, J. & Konings, W. N. (1979) FEMS Microbiol. Lett. 5, Mitchell, P. (1973) Bloenergetics 4, Mitchell, P. (1976) J. Theor. Biol. 62,, Mitchell, P. (197) Symp. Soc. Gen. Microbiol. 2, Konings, W. N. & Michels, P. A. M. (1979) in Diversity of Bacterial Respiratory Systems, ed. Knowles, D. C. J. (CRC, Cleveland, OH), in press. 6. De Man, J. C., Rogosa, M. & Sharpe, M. E. (196) J. Appl. Bacteriol. 23, Rogosa, M., Franklin, J. C. & Perry, K. D. (1961) J. Gen. Microbiol. 25, Laanbroek, H. J., Kingma, W. & Veldkamp, H. (1977) FEMS Microbiol. Lett. 1, Thompson, J. & Thomas, T. D. (1977) J. Bacteriol. 13, Schuldiner, S. & Kaback, H. R. (1976) Biochemistry 14, Matin, A. & Konings, W. N. (1973) Eur. J. Biochem. 43, Fairbairn, N. J. (1953) Chem. Ind. (London), Holdeman, L. V., Cato, E. P. & Moore, W. E. C., eds. (1977) Anaerobe Laboratory Manual (Virginia Polytechnic Institute and State University, Anaerobic Laboratory, Blacksburg, VA), p Maloney, P. C. & Wilson, T. H. (1975) J. Memb. Biol. 25, Cole, H. A., Wimpenny, J. W. T. & Hughes, D. E. (1967) Biochim. Biophys. Acta 143, Herbert, D., Phipps, P. J. & Strange, R. E. (1971) in Methods in Microbiology, eds. Norris, J. R. & Ribbons, D. W. (Academic, New York), Vol. 5B, pp Bakker, E. P., Rottenberg, H. & Kaplan, S. R. (1976) Biochim. Biophys. Acta 44, Schulze, K. L. & Lipe, R. S. (1964) Arch. Mikrobiol. 48, Asgar, S. S., Levin, E. & Harold, F. M. (1973) J. Biol. Chem. 248, Flagg, J. L. & Wilson, T. H. (1978) Membr. Biochem. 1, Bentaboulet, M., Robin, A. & Kepes, A. (1979) Biochem. J. 178, Kaczorowski, J. G. & Kaback, H. R. (1979) Biochemistry 18, Kaczorowski, J. G., Robertson, D. E. & Kaback, H. R. (1979) Biochemistry 18,