Dec 10, 1974 - gately anaerobic organism Veillonella alcalescens contains cytochromes b, a and a2 and that cytochrome b functions in anaerobic electron ...
JOURNAL OF BACTRIOLOGY, Apr. 1975, p. 245-249 Copyright 0 1975 American Society for Microbiology
Vol. 122, No. 1 Printed in U.S.A.
Amino Acid Transport in Membrane Vesicles of Obligately Anaerobic Veillonella alcalescens W. N. KONINGS,* J. BOONSTRA, AND W. DE VRIES Laboratorium voor Microbiologie, University of Groningen, Haren,* and Microbiology Department, Biological Laboratory, Free University, Amsterdam, The Netherlands
Received for publication 10 December 1974
Membrane vesicles of Veillonella alcalescens, grown in the presence of L-lactate and KNOs, actively transport amino acids under anaerobic conditions in the presence of several electron donors and the electron acceptor nitrate. The highest initial rates of uptake are obtained with L-lactate, followed by reduced nicotinamide adenine dinucleotide, glycerol-i-phosphate, formate, and Lmalate. The membrane vesicles contain the dehydrogenases for these electron donors, and these enzymes are coupled with nitrate reductase. In membrane vesicles from cells, grown in the presence of nitrate, the dehydrogenases are not coupled with fumarate reductase, and anaerobic transport of amino acids does not occur with fumarate as electron acceptor. Under aerobic conditions none of the physiological electron donors can energize transport. However, a high rate of uptake is observed with the electron donor system ascorbate-phenazine methosulfate. This electron donor system also effectively energizes transport under anaerobic conditions in the presence of the electron acceptor nitrate.
Cytoplasmic membrane vesicles of a number of microorganisms catalyze the active transport of amino acids and a wide variety of metabolites (5). In aerobically grown microorganisms, the energy for these transport processes is derived from the oxidation of substrates like D-lactate, reduced nicotinamide adenine dinucleotide (NADH), succinate (5), and reduced phenazine methosulfate (PMS) (7, 10) via a membranebound cytochrome chain with oxygen as terminal electron acceptor. Recently, it was demonstrated that in Escherichia coli under anaerobic conditions active transport of lactose (11) and amino acids (J. Boonstra et al., manuscript in preparation) is energized by electron flow in at least two specific inducible electron transfer systems (6, 12, 15, 16), e.g., the oxidation of glycerol-i-phosphate with fumarate as electron acceptor and the oxidation of formate with nitrate as electron acceptor. In several obligately anaerobic organisms electron transfer systems have been demonstrated (2), and the question of whether a similar relationship exists between active transport and anaerobic electron transfer systems in these organisms arises. In a recent report, de Vries et al. (1) demonstrated that the obligately anaerobic organism Veillonella alcalescens contains cytochromes b, a and a2 and that cytochrome b functions in anaerobic electron transfer to nitrate or fumarate, with
L-lactate or pyruvate as electron donors. The experiments presented here demonstrate that anaerobic amino acid transport in this obligately anaerobic organism can be coupled to electron transfer, utilizing nitrate as electron acceptor. MATERIALS AND METHODS Culture media and growth conditions. V. alcalescens was obtained from the Dental Institute of the Free University, Amsterdam, The Netherlands. The strain was maintained as a stab culture as described previously (1) and was grown at 37 C in 0.5% tryptone (Difco Laboratories, Detroit, Mich.), pH 6.5 to 6.8, supplemented (per liter) with 10 g of sodium lactate, 5 g of KNO2, 10 g of yeast extract (Difco Laboratories), 0.25 g of K2HPO4, 5 mg of MnSO4, and 0.5 g of cysteine, pH 7 (sterilized separately), in 3-liter Erlenmeyer flasks filled to the top, tightly stoppered, and stirred slowly by means of a magnetic stirrer (1, 2). Preparation of membrane vesicles. Cells were harvested at an absorbancy at 660 nm of 0.8 to 0.9, and membrane vesicles were prepared as described previously (11) for anaerobically grown E. coli. Transport studies. Transport studies were performed under aerobic and anaerobic conditions as described previously (11, 14). Protein assay. The protein was determined according to the method of Lowry et al. (13). Enzyme assays. Nitrate reductase and fumarate reductase activities were assayed spectrophotometrically at 550 nm at 25 C by following the oxidation of 245
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reduced benzylviologen (BDH Chemicals Ltd., Poole, England) by nitrate or fumarate under nitrogen (12, 18). L-Lactate dehydrogenase and formate dehydrogenase were assayed spectrophotometrically at 660 nm at 25 C by following the PMS-mediated reduction of dichlorophenolindophenol (Fluka AG, Buchs, Switzerland) by L-lactate or formate under nitrogen (12). Glycerol-i-phosphate dehydrogenase was assayed spectrophotometrically at 570 nm at 25 C by following the PMS-mediated reduction of 3(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (Aldrich, Beerse, Belgium) (6). NADH dehydrogenase was measured spectrophotometrically at 420 nm at 25 C by following the anaerobic reduction of ferricyanide under nitrogen
(3).
The coupled enzyme activities of nitrate reductase with L-lactate dehydrogenase, NADH dehydrogenase, glycerol-i-phosphate dehydrogenase, and formate dehydrogenase were assayed by measuring the increase of nitrite formation upon addition of the corresponding electron donors (17). The coupled activities of fumarate reductase with these dehydrogenases were assayed by measuring the conversion of "C-labeled fumarate to succinate (Boonstra et al., manuscript in preparation). Materials. "4C-labeled substrates were obtained from the Radiochemical Centre, Amersham, Buckinghamshire, England, e.g., L- [U-14C ]glutamate (265 mCi/mmol); L- [U- 14C Jalanine (171 mCi/mmol); L- [U14C ]serine (171 mCi/mmol); L- [U- 14C proline (540 mCi/mmol); L-[J-14C ]lysine (318 mCi/mmol); [114Cllactose (20 mCi/mmol); L- [U-14C ]malate (35 mCi/mmol); and sodium-[2-3- 4C ]fumarate (59 mCi/ mmol). Stock solutions (0.5 M) of the electron donors and the electron acceptors, neutralized to pH 7.0, were used as the following salts: lithium L-lactate, sodium glycerol-i-phosphate, sodium formate, potassium Lmalate, sodium pyruvate, potassium nitrate, and
sodium fumarate. All chemicals were reagent grade obtained from commercial sources.
RESULTS Membrane vesicles were isolated from V. alcalescens grown in a medium containing Llactate and nitrate. These membrane vesicles catalyze the anaerobic active transport of Lglutamate in the presence of the electron donor L-lactate and the electron acceptor nitrate (Fig. 1A). L-Lactate alone or nitrate alone causes a slight stimulation of L-glutamate uptake over endogenous levels (Fig. lA). In the presence of the electron acceptor, nitrate, significant stimulation of L-glutamate uptake is also observed with the electron donors NADH (Fig. 1B), glycerol-i-phosphate (Fig. 1C), formate (Fig. 1D), and also with L-malate (data not shown). No uptake of L-glutamate above endogenous levels could be obtained with pyruvate in the presence or absence of nitrate. De Vries et al. (1) observed that fumarate can function as an electron acceptor in V. alcalescens. In membrane vesicles of cells grown in the presence of nitrate, no stimulation of L-glutamate uptake could be observed upon the addition of fumarate together with the electron donor L-lactate or NADH (data not shown). In membrane vesicles of Bacillus subtilis, L-glutamate inhibits fumarate transport noncompetitively, whereas L-serine has hardly any effect on fumarate transport (8). Such an interaction between fumarate and L-glutamate might also exist in V. alcalescens. The effect of L-lactate with or without fumarate on L-serine transport was therefore investigated (Fig. 2). For this amino acid, also, a stimulation of
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2
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FIG. 1. Anaerobic transport of L-glutamate by membrane vesicles of V. alcalescens. Symbols: (A) 9, L-lactate (10 mM) + KNO, (10 mM); *, L-lactate (10 mM); A, KNO3 (10 mM); V, without electron donor or acceptor added. (B) 0, NADH (10 mM) + KNO, (10 mM); *, NADH (10 mM). (C) 0, glycerol-l-phosphate (10 mM) + KNO, (10 mM); *, glycerol-l-phosphate (10 mM). (D) 0, formate (10 mM) + KNO, (10 mM); *, formate (10 mM). Uptake experiments were performed at 25 C under nitrogen in incubation mixtures of 50 gI containing 0.05 mg of membrane protein. L-Glutamate concentration. 7.6 uM.
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TABLE 1. Enzyme activities of dehydrogenases, reductases, and coupled dehydrogenase-reductase activities in membrane vesicles of V. alcalescens grown in the presence of nitratea
_%
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O
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Coupled Coupled enzyme enzyme activity activitywt fuwith
Enzymeeaiy EnyeEnzyme act
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213 L-Lactate dehydrogenase 59 NADH dehydrogenase 17 Glycerol-P dehydrogenase 11 Formate dehydrogenase 10 L-Malate dehydrogenase 2 Pyruvate dehydrogenase 10,395 Nitrate reductase 2,159 Fumarate reductase
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0
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37 11 11 10 NDb ND
2 1 1 0 ND ND
-
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a The enzyme assays were performed as described in Materials and Methods at 25 C with 0.015 to 0.5 mg of membrane protein per ml. Results are given in nanomoles per milligram of membrane protein per minute. bND, Not determined.
01
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2
3
4
5
minutes FIG. 2. Anaerobic transport of L-serine by membrane vesicles of V. alcalescens. Symbols: 0, Llactate (10 mM) + KNOS (10 mM); A, L-lactate (10 mM); V, L-lactate (10 mM) + Na-fumarate (10 mM); 0, without electron donor or acceptor added. Uptake experiments were performed as described in legend to Fig. 1. L-Serine concentration, 11.7 AM.
uptake above endogenous levels is obtained with L-lactate in the presence of nitrate, but not in the presence of fumarate, as acceptor. In addition to L-glutamate and L-serine the uptake of other amino acids was also studied with the electron donor-acceptor combination L-lactate-nitrate, and a stimulation above endogenous levels was observed with L-alanine and L-lysine but not with L-proline (data not shown). To correlate the transport activities observed in the presence of the different electron donoracceptor combinations with the electron transfer systems present in the membranes, the enzyme activities and the coupled enzyme activities were determined (Table 1). The membrane vesicles contain a highly active L-lactate dehydrogenase and also, with lower activities,
NADH dehydrogenase, glycerol-i-phosphate dehydrogenase, formate dehydrogenase, and Lmalate dehydrogenase and a very low activity of pyruvate dehydrogenase. In addition, an extremely high activity of nitrate reductase is present. The coupled activities are consistent with these observations, although the levels of the coupled activities of all dehydrogenasereductase combinations are lower than the activities of the individual enzymes. The membrane vesicles also contain a high fumarate reductase activity, but a coupled activity of the dehydrogenases with fumarate reductase could not be detected. These observations are in good agreement with the effects of the electron donor-acceptor combinations on L-glutamate or L-serine transport (Fig. 1,2) and indicate that at least one electron transfer system is coupled to amino acid transport. Electrons can be donated to this system by at least five dehydrogenases, and electrons are transferred via nitrate reductase to nitrate. Electron transfer in V. alcalescens is strongly inhibited by 2-N-heptyl-4-hydroxy-quinolineN-oxide (HOQNO) (1). In agreement with this observation, 10 ,M HOQNO inhibits L-glutamate transport energized by L-lactate-nitrate in membrane vesicles more than 60%. In addition, the uncouplers 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone exert in concentrations of 5 and 10 ,uM, respectively, inhibitions of more than 70% (data not shown). In membrane vesicles of anaerobically grown
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E. coli, oxygen can also function as an effective electron acceptor and transport can be energized aerobically by several electron donors (11). Such an aerobic electron transfer system should not be present in membrane vesicles of this obligately anaerobic microorganism. All the physiological electron donors which are effective under anaerobic conditions with nitrate as acceptor failed to stimulate transport under aerobic conditions (Fig. 3), and no oxidation of formate and glycerol-i-phosphate occurs by
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FIG. 3. Aerobic and anaerobic transport of L-glutamate by membrane vesicles of V. alcalescens in the
of different electron donors and acceptors. Symbols: 0, ascorbate (10 mM) + PMS (10 uM) under aerobic conditions; *, ascorbate (10 mM) + PMS (10 AsM) under nitrogen in the presence of KNO3 (10 mM); A, L-lactate (10 mM) under aerobic conditions; A, without electron donor under aerobic conditions. Uptake experiments were performed as described in legend to Fig. 1. L-Glutamate concentration, 7.6 ,M. presence
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membrane vesicles. However, L-lactate (10 mM) and NADH (5 mM) are oxidized by the membrane vesicles at rates of 14 and 56 nmol of oxygen per mg of membrane protein per min, respectively. The electron donor system ascorbate-PMS is an effective energy source for aerobic transport in membrane vesicles of several organisms (7, 10, 14), but not for anaerobic transport in the presence of nitrate in vesicles of anaerobically grown E. coli (Boonstra et al., manuscript in preparation). To our surprise, however, ascorbate-PMS proved to be a very effective energy source for transport in membrane vesicles of V. alcalescens, both under anaerobic conditions with nitrate and under aerobic conditions (Fig. 3).
DISCUSSION The production of energy by electron transfer in the respiratory chain of aerobic organisms has been thoroughly established, and this energy can be used for adenosine 5'-triphosphate synthesis or for active transport of metabolites through the cytoplasmic membrane (4, 5). Only indirect evidence has been presented (2) for a coupling between electron flow in anaerobic electron transfer systems and adenosine 5'-triphosphate synthesis. With isolated membrane vesicles it was possible to demonstrate in the facultatively anaerobic E. coli a direct coupling between anaerobic transport of lactose and amino acids and anaerobic electron transfer (Boonstra et al., manuscript in preparation) (11). This paper provides strong evidence that in the obligately anaerobic V. alcalescens, also, active transport of amino acids can be energized by electron flow in an aerobic electron transfer system. In the presence of nitrate, anaerobic transport of amino acids is catalyzed at a high rate by L-lactate, and also, although at a lower rate, by NADH, glycerol-i-phosphate, formate, and L-malate. The membrane vesicles contain the dehydrogenases specific for these substrates, and a coupling between these dehydrogenases and nitrate reductase was demonstrated. In addition, amino acid transport energized by L-lactate-nitrate is strongly inhibited by the electron transfer inhibitor HOQNO and by the uncouplers 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Moreover, a high initial rate of anaerobic amino acid transport is observed with a non-physiological electron donor system, ascorbate-PMS, in the presence of nitrate. Evidence was presented that cytochrome b is involved in this electron transfer system (1), and the respiratory chain inhibitor HOQNO inhibits both the reduction of cytochrome b and
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amino acid transport. De Vries et al. (1) observed that the extent of oxidation of cytochrome b with fumarate is small in membrane suspensions of V. alcalescens grown in the presence of nitrate. In agreement with this observation, no coupling of any of the dehydrogenases with fumarate reductase could be detected, and amino acid transport was not stimulated by any of the electron donors with fumarate as acceptor. Amino acid transport in membrane vesicles of V. alcalescens is also stimulated by ascorbatePMS with nitrate as electron acceptor. With oxygen as electron acceptor, an even higher stimulation of transport is obtained with this electron donor (Fig. 3). These observations indicate that reduced PMS effectively donates electrons to the membrane-bound electron transfer system and that electrons can be transferred both to nitrate and to oxygen. However, none of the physiological electron donors were able to energize transport under aerobic conditions, although L-lactate and NADH are oxidized by the membrane preparation. These oxidations might be due to autooxidations of components of the electron transfer system. At this moment no explanation can be given for the effect of ascorbate-PMS on transport under aerobic conditions. ACKNOWLEDGMENT The excellent technical assistance of Roby Kalsbeek is highly appreciated.
LITERATURE CITED 1. de Vries, W., W. M. C. van Wijck-Kapteyn, and S. K. H. Oosterhuis. 1974. The presence and function of cytochromes in Selenomonas ruminantium, Anaerovibrio lipolytica and Veillonella alcalescens. J. Gen. Microbiol. 81:69-78. 2. de Vries, W., W. M. C. van Wijck-Kapteyn, and A. H. Stouthamer. 1973. Generation of ATP during cytochrome-linked anaerobic electron transport in propionic acid bacteria. J. Gen. Microbiol. 76:31-41. 3. Enoch, H. G., and R. L. Lester. 1972. Effects of molybdate, tungstate, and selenium compounds on formate
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dehydrogenase and other enzyme systems in Escherichia coli. J. Bacteriol. 110:1032-1040. 4. Harold, F. M. 1972. Conservation and transformation of energy by bacterial membranes. Bacteriol. Rev. 36:172-230. 5. Kaback, H. R., and J-S. Hong. 1973. Membranes and transport. CRC Crit. Rev. Microbiol. 2:333-376. 6. Kistler, W. S., and E. C. C. Lin. 1971. Anaerobic L-a-glycerolphosphate dehydrogenase of Escherichia coli: its genetic locus and its physiological role. J. Bacteriol. 108:1224-1234. 7. Konings, W. N., E. M. Barnes, and H. R. Kaback. 1971. Mechanisms of active transport in isolated membrane vesicles. III: The coupling of reduced phenazine methosulfate to the concentrative uptake of ,-galactosides and amino acids. J. Biol. Chem. 246:5857-5861. 8. Konings, W. N., A. Bisschop, and M. C. C. Daatselaar. 1972. Transport of L-glutamate and L-aspartate by membrane vesicles of Bacillus subtilis W23. FEBS Lett. 24:260-264. 9. Konings, W. N., A. Bisschop, M. Veenhuis, and C. A. Vermeulen. 1973. New procedure for the isolation of membrane vesicles of Bacillus subtilis and an electron microscopy study of their ultrastructure. J. Bacteriol. 116:1456-1465. 10. Konings, W. N., and E. Freese. 1971. Amino acid transport in membrane vesicles of Bacillus subtilis. J. Biol. Chem. 247:2408-2418. 11. Konings, W. N., and H. R. Kaback. 1973. Anaerobic transport in Escherichia coli membrane vesicles. Proc. Nat. Acad. Sci. U.S.A. 70:3376-3381. 12. Lester, R. L., and J. A. DeMoss. 1971. Effects of molybdate and selenite on formate and nitrate metabolism in Escherichia coli. J. Bacteriol. 105:1006-1014. 13. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 14. Matin, A., and W. N. Konings. 1973. Transport of lactate and succinate by membrane vesicles of Escherichia coli, Bacillus subtilis and a Pseudomonas species. Eur. J. Biochem. 34:58-67. 15. Miki, K., and E. C. C. Lin. 1973. Enzyme complex which couples glycerol-3-phosphate dehydrogenation to fumarate reduction in Escherichia coli. J. Bacteriol. 114:767-771. 16. Ruiz-Herrera, J., M. K. Showe, and J. A. DeMoss. 1969. Nitrate reductase complex of Escherichia coli K-12: isolation and characterization of mutants unable to reduce nitrate. J. Bacteriol. 97:1291-1297. 17. Showe, M. K., and J. A. DeMoss. 1968. Localization and regulation of synthesis of nitrate reductase in Escherichia coli. J. Bacteriol. 95:1305-1313. 18. Spencer, M. E., and J. R. Guest. 1973. Isolation and properties of fumarate reductase mutants of Escherichia coli. J. Bacteriol. 114:563-570.
