Vol. 124, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Nov. 1975, p. 613-622
Copyright C) 1975 American Society for Microbiology
Dicarboxylic Acid Transport in Membrane Vesicles from Bacillus subtilis ARNOLD BISSCHOP,* HENK DODDEMA,' AND WIL N. KONINGS Laboratory for Microbiology, Biological Centre, University of Groningen, Kerlaan 30, Haren (Gr.), The Netherlands Received for publication 30 June 1975
Membrane vesicles isolated from Bacillus subtilis W23 catalyze active transport of the 04 dicarboxylic acids L-malate, fumarate, and succinate under aerobic conditions in the presence of the electron donor reduced ,8-nicotinamide adenine dinucleotide or the non-physiological electron donor system ascorbate-phenazine methosulfate. The dicarboxylic acids are accumulated in unmodified form. Inhibitors of the respiratory chain, sulfhydryl reagents, and uncoupling agents inhibit the accumulation of the dicarboxylic acids. The affinity constants for transport of L-malate, fumarate, and succinate are 13.5, 7.5, and 4.3 AM, respectively; these values are severalfold lower than those reported previously for whole cells. Active transport of these dicarboxylic acids occurs via one highly specific transport system as is indicated by the following observations. (i) Each dicarboxylic acid inhibits the transport of the other two dicarboxylic acids competitively. (ii) The affinity constants determined for the inhibitory action are very similar to those determined for the transport process. (iii) Each dicarboxylic acid exchanges rapidly with a previously accumulated dicarboxylic acid. (iv) Other metabolically and structurally related compounds do not inhibit transport of these dicarboxylic acids significantly, except for L-aspartate and L-glutamate. However, transport of these dicarboxylic amino acids is mediated by an independent system because membrane vesicles from B. subtilis 60346, lacking functional dicarboxylic amino acid transport activity, accumulate the C4 dicarboxylic acids at even higher rates than vesicles from B. subtilis W 23. (v) A constant ratio exists between the initial rates of transport of L-malate, fumarate, and succinate in all membrane vesicle preparations isolated from cells grown on various media. This high-affinity dicarboxylic acid transport system seems to be present constitutively in B. subtilis W23.
Active transport of metabolites across the cytoplasmic membrane has received considerable attention in the last few years. Most studies have been concerned with transport systems for amino acids and carbohydrates, and only recently has interest been directed towards transport systems for tricarboxylic acid cycle intermediates. Several of these tricarboxylic acid cycle intermediates appear to be accumulated via specific and inducible transport systems. Studies in whole cells, for instance, demonstrated the presence of specific and inducible uptake systems for citrate in Klebsiella aerogenes (4, 28), Pseudomonas spp. (20), and Bacillus subtilis (32). In Salmonella typhimurium there appear to be three specific and inducible
transport systems for the tricarboxylic acids citrate, isocitrate, cis-aconitate, and tricarballylate (9). Studies on the transport of dicarboxylic acids demonstrated the presence of specific and inducible transport systems for 04 dicarboxylic acids in Escherichia coli (13, 21) and P. putida (3). Similar studies have been done with B. subtilis (5-7). In the wild type and in mutants that lack a functional tricarboxylic acid cycle, inducible transport systems were demonstrated for L-malate, fumarate, succinate, and a-ketoglutarate. DL-Tartrate also seems to be accumulated by a C4 dicarboxylic acid transport system
(5). One of the major problems in the study of active transport of metabolites by whole cells is the rapid conversion of transported substrate
I Present address: Laboratory for Plant Physiology, Biological Centre, University of Groningen, Kerklaan 30, Haren (Gr.), The Netherlands.
upon entrance into the cells. For some metabolites this problem can be solved by studying 613
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BISSCHOP, DODDEMA, AND KONINGS
transport in cells in which a genetic block is introduced to prevent metabolism of the transported substrate. Such an approach has been taken in studies of the transport of C4 dicarboxylic acids in B. subtilis and E. coli (5, 7, 21). An alternative approach involves the use of nonmetabolizable substrate analogues, such as 3fluoro-L-erythro-malate and 2-fluoro-L-erythrocitrate, for L-malate and citrate (25, 31). A more generally applicable solution to this problem can be found in the use of membrane vesicles. These membrane vesicles consist of cytoplasmic membranes and are devoid of cytoplasmic constituents and cell wall components (10, 16). They have been shown to be physiologically active with regard to certain integrated membrane functions (1, 11, 12, 14, 18, 19, 29) such as amino acid and sugar transport, phospholipid biosynthesis, and electron transport. We have been concerned with active transport processes in membrane vesicles of B. subtilis. Membrane vesicles from this gram-positive organism can be isolated in a rather simple and gentle way (17), and it has been shown previously that these membrane vesicles actively transport many metabolites in the presence of reduced f3-nicotinamide adenine dinucleotide (NADH) or ascorbate-phenazine methosulfate (Asc-PMS) at rates comparable with those observed for whole cells (1, 14, 17-19, 23). Moreover, freeze-etch electron microscopy (17) and biochemical studies on the localization of membrane-bound enzymes (14) revealed that the vesicles have the same orientation as the cytoplasmic membrane of whole cells. During our studies on the specificity of the transport systems in vesicles from B. subtilis, it was demonstrated that at least nine distinct systems exist for the transport of amino acids (19). One of these systems is specific for the dicarboxylic amino acids L-glutamate and L-aspartate (16). The C4 dicarboxylic acids L-malate, fumarate, and succinate are transported by separate systems and inhibit transport of the dicarboxylic amino acids in a noncompetitive way (16). The existence of distinct transport systems for dicarboxylic acids has also been demonstrated in membrane vesicles from E. coli (23, 24, 26) and Pseudomonas spp. (23). It is the purpose of this paper to characterize the specificity and the kinetic properties of the transport system(s) involved in the accumulation of L-malate, succinate, and fumarate. A brief preliminary report of these studies has been presented previously (A. Bisschop and W. N. Konings, Abstr. FEBS Meet. 1974, p. 250). MATERIALS AND METHODS Bacterial strains.B. subtilis W23 was used as the
J. BACTERIOL.
wild-type strain. Strain JH402 (trpC2 citF2) was derived from B. subtilis 168 and is genetically blocked in succinic dehydrogenase (EC 1.3.99.1) activity. This mutant was donated by J. A. Hoch, Department of Microbiology, Scripps Clinic and Research Foundation, La Jolla, Calif. (27). Strain HS1A21 is a malic dehydrogenase (EC 1.1.1.37)-defective mutant derived from B. subtilis 168, and was donated by R. S. Hanson, Department of Bacteriology, University of Wisconsin, Madison (2). Cultures of the mutants defective in the tricarboxylic acid cycle were tested for revertants as described by Carls and Hanson (2). B. subtilis 168 (trpC2), auxotrophic for tryptophan, was obtained from G. Venema, Genetic Institute, Biological Centre, University of Groningen. B. subtilis 60346, deficient in the active transport of dicarboxylic amino acids, was a gift of M. Diesterhaft, National Institutes of Health, Bethesda, Md. Culture media. All liquid media had the basic composition (per liter): K2HPO4, 14 g; KH2PO4, 6 g; MgSO4 7H20, 0.25 g; MnSO4 H20, 0.017 g; (NH4)2SO4, 2 g; and L-glutamic acid, 0.15 g. The carbon and energy sources used were added to this basic medium as follows: tryptone (5 g/liter) and yeast extract (3 g/liter); DL-malate, fumarate, or succinate, if added to the enriched tryptone or yeast extract media, were added at a 5 mM concentration as described by R. E. Fournier et al. (5). In defined media, 0.15 g of L-glutamate per liter was replaced by 1% fumarate, DL-malate, L-glutamate, or L-aspartate. Cell growth. Cells were grown on the media described above, in a 3-liter Biotec fermenter (LKB Producter, Bromma, Sweden) under vigorous aeration at 37 C. The culture was inoculated to an absorbancy at 663 nm of 0.1 with cells grown overnight in the same medium. Growth was followed turbidimetrically, and late-log-phase cells were harvested by centrifugation. Preparation of membrane vesicles. Membrane vesicles were prepared as described previously (17) but with the following modifications. Cells were suspended in 0.05 M potassium phosphate buffer (pH 8.0) at 37 C at a concentration of 4 g (wet weight) per liter. Lysozyme, deoxyribonuclease I, and ribonuclease were added at final concentrations of 300, 10, and 10 ug/ml, respectively. After 15 min of incubation, MgSO4 was added at a final concentration of 10 mM. The incubation was continued for 30 min, after which the incubation was stopped by centrifugation of the solution at 20,000 x g for 30 min. The membranes were suspended thoroughly by means of a hypodermic syringe fitted with an 18-gauge needle, washed twice with 0.1 M potassium phosphate buffer (pH 6.6), and finally resuspended in this buffer at 4 to 6 mg of membrane protein per ml. Portions of 0.5 to 1 ml in thin-walled plastic tubes were frozen rapidly and stored in liquid nitrogen. Protein was determined by the method of Lowry et al. (22). Transport assays. Transport experiments were performed, as described previously (1, 15, 19, 23), in a final incubation volume of 100 .l at 25 C. Unless otherwise stated, transport studies were performed
with membrane vesicles ofB. subtilis W23 grown on mineral salt medium supplemented with 0.3% yeast
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VOL. 124, 1975
extract and 5 mM DL-malate. In all studies, Asc (10 mM) and PMS (100 ,uM) or NADH (10 mM) were used as the electron donor. Initial transport rates were measured after a 1-min incubation of the membrane vesicles with the electron donor. The substrates were added in the following concentrations unless stated otherwise: L-[U-'4C]glutamic acid, 18.9 ;LM (specific acitivity 265 mCi/mmol); o[U'4C]aspartic acid, 21.6 ;LM (specific activity 232 mCi/mmol); L-[U-14C]malic acid, 38.1 ,uM (specific activity 82 mCi/mmol); [2,3-'4C]fumaric acid, 217 IAM (specific activity 13 mCi/mmol); and [1,4"4C]succinic acid, 119 ,uM (specific activity 21 mCi/mmol). Materials. All 14C-labeled compounds were purchased from the Radiochemical Centre (Amersham, England). Tryptone and yeast extract were obtained from Difco (Detroit, Mich.). NADH was from Boehringer (Mannheim, Germany); PMS was from BDH Chemicals Ltd. (Poole, England); 2-heptyl-4-hy-
5-ethyl-5-isoamylbarbidroxyquinoline-N-oxide, turic acid (Amytal), carbonyl cyanide-m-chlorophenyl hydrazone, and L-malic acid were from Sigma Chemical Co. (St. Louis, Mo.); and L-lactic acid and D-lactic acid, both lithium salts, were from Calbiochem AG (Lucerne, Switzerland). Ribonuclease and deoxyribonuclease were products of Miles Laboratories, Inc. (Kankakee, Ill.). Rotenone was obtained from Aldrich-Europe (Beerse, Belgium). Selectron BA85 membrane filters, pore size 0.45 ,m, were purchased from Schleicher and Schull GMBH (Dassel, Germany). All other chemicals were purchased from E. Merck (Darmstadt, Germany).
RESULTS Active transport of dicarboxylic acids. Membrane vesicles from B. subtilis W23 grown on a mineral salts medium supplemented with yeast extract plus DL-malate as the carbon and energy source accumulate all three C4 tricarbox-
615
ylic acid cycle intermediates (L-malate, fumarate, and succinate) in the presence of an electron donor (Fig. 1). High initial rates of uptake are obtained in the presence of the non-physiological electron donor Asc-PMS and also with the physiological electron donor NADH, and steady-state levels are reached between 1.5 and 9.5 nmol/mg of membrane protein. In the absence of an electron donor, no concentration of dicarboxylic acids occurs (Fig. 1). It should be emphasized that the initial rates of transport in the presence of NADH or Asc-PMS varies among different vesicle preparations. It has been shown previously (1) that vesicle preparations isolated at different stages of the growth curve demonstrate an increase in the rate of NADH or Asc-PMS oxidation per milligram of membrane protein. The variation in the transport activity correlates with this variation in the oxidation rates (1). The dicarboxylic acids are not metabolized during accumulation as was demonstrated by extraction of the membrane vesicles after steady-state level accumulation followed by thin-layer chromatography. Scanning of the radioactivity yielded single spots with the Rf values: L-malate, 0.46; fumarate, 0.83; and succinate, 0.74. The same Rf values were obtained for the original substrates, and these values are in good agreement with the data presented by Ting and Dugger (30). Transport of these dicarboxylic acids is coupled to electron flow in the respiratory chain in a way similar to that demonstrated for amino acid transport (15, 16, 18, 19). Inhibitors of electron transfer such as amytal, 2-heptyl-4-hydroxyquinoline-N-oxide, rotenone, and cyanide
C 4.0 -
E
3.0
S
, 2.0 E5
1
2
3
4
5
1
2 3 time (min)
4
5
1
2
3
4
5
FIG. 1. Transport of dicarboxylic acid by membrane vesicles from Bacillus subtilis W23. (A). L-Malate; (B) fumarate; (C) succinate. Symbols: 0, In the presence of20 mM NADH; 0, in the presence of 10 mM Asc plus 100 M PMS; and V, in the absence of an energy source. The incubation mixture (100 ,u) contained 0.047 mg of membrane protein.
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BISSCHOP, DODDEMA, AND KONINGS
strongly inhibit the initial rates of transport of all three dicarboxylic acids and exert inhibitions between 40 and 90% (Table 1). Almost complete inhibition of the initial rates of transport is observed in the presence of the uncoupling agents dinitrophenol, carbonyl cyanidem-chlorophenyl hydrazone and azide. Thiol reagents such as p-chloromercuribenzoate and Nethyl maleimide are also effective inhibitors of transport. Specificity of dicarboxylic acid transport. Initial rates of L-malate, fumarate, and succinate transport as a function of the substrate concentration display simple Michaelis-Menten kinetics, and Lineweaver-Burk plots yield straight lines for all three dicarboxylic acids over a 20-fold concentration range (Fig. 2). Transport occurs via high-affinity system(s) with K,12 values of the order of 10-5 M and Vmax, values of 0.6 to 3.7 nmol!mg of membrane protein per min (Table 2). These K 12 values are the average values of five independent determinations. The transport system(s) has the highest affinity for succinate, a twofold-lower affinity for fumarate, and a threefold-lower affinity for L-malate. In Fig. 2 it is also shown that the transport of each dicarboxylic acid is inhibited competitively by the other two individual dicarboxylic acids (only one set of figures is presented). The TABLE 1. Effect of metabolic inhibitors on initial rates of C4 dicarboxylic acid transport in membrane vesicles from B. subtilis W23a Inhibio Inhibitor
Concn
(mM)
% Inhibition of initial transport rate of:
L-Malate Fumarate Succinate
Amytal HOQNO Rotenone KCN DNP PNP Sodium
0.1 10-2 0.1 10 0.1 0.5 1.0
57 82 43 49 98 99 90
60 88
10-3 1.0 0.5
100 79 40
100
37 47 97 92 91
58 77 35 45 96 99 96
azide CCCP
pCMB NEM
97 39
100 71
37
a The inhibitors were added prior to preincubation of the incubation mixture. 2-Heptyl-4-hydroxyquinoline-N-oxide (HOQNO), amytal, car-
bonyl cyanide-m-chlorophenyl hydrazone (CCCP), and rotenone were dissolved in dimethylsulfoxide. All values are stated with respect to controls having the same dimethyl sulfoxide concentration. DNP, Dinitrophenol; pCMB, p-chloromercuribenzoate; NEM, N-ethyl maleimide; PNP, p-nitrophenol.
J. BACTERIOL.
inhibition constants (Ki) calculated from these data are given in Table 2. Each value is the average of at least three experiments. The K, values are in the same order of magnitude as the K1/2 values, but in all cases the lowest values were obtained with succinate, slightly higher values with fumarate, and the highest values with L-malate as transported substrate. No explanation can be offered at this time for this observation. These observations suggest the existence of a common transport system for L-malate, fumarate, and succinate. Other lines of evidence support this contention. The effects of various structurally and metabolically related metabolites on the initial rates of transport are the same for all three dicarboxylic acids (Table 3). The strongest inhibitory effects were exerted by the dicarboxylic acids themselves (more than 80%); the dicarboxylic amino acids L-aspartate and L-glutamate were moderately inhibitory (50 to 60%), whereas other mono-, di-, and tricarboxylic acids had no significant effect on dicarboxylic acid transport. Similar observations were made with membrane vesicles from mutants of B. subtilis lacking succinic dehydrogenase (strain JH 402), lacking fumarase (strain JH 404), and lacking malic dehydrogenase (strain HS1A21), all grown on the same medium as strain W23 in Table 3, and with membrane vesicles of B. subtilis W23 grown on different carbon sources (data not shown). More evidence for the existence of a common transport system for the dicarboxylic acids was obtained from exchange experiments. In Fig. 3 it is shown that accumulated L-malate exchanges rapidly with external L-malate and also with fumarate and succinate, and that 85% of the radioactivity is lost in less than 1 min. Similar observations were made with the other C4 dicarboxylic acids (data not shown). Differences between dicarboxylic acid and dicarboxylic amino acid transport system. In Table 3 it is shown that the dicarboxylic amino acids L-glutamate and L-aspartate inhibit the transport of the dicarboxylic acids significantly. However, the two groups of metabolites do not share a common transport system because the dicarboxylic acids inhibit transport of the dicarboxylic amino acids non-competitively, as was shown previously (16). This is supported by the observation that membrane vesicles from a B. subtilis mutant defective in dicarboxylic amino acid transport do not transport the dicarboxylic amino acids while transport of the dicarboxylic acids is unimpaired (Fig. 4). Effect of the growth medium on dicarbox-
VOL. 124, 1975
DICARBOXYLIC ACID TRANSPORT IN B. SUBTILIS
617
l/[fumarateJ x 105 M-1 FIG. 2. Kinetics of competitive inhibition of C4 dicarboxylic acid transport by membrane vesicles from B. subtilis W23. The initial rates of transport were determined after a 1-min incubation with 0.047 mg of membrane protein. (A) Kinetics of L-malate transport in the presence of succinate. Concentrations of succinate used: *, 0 M; 0, 12.5 pM; A, 25 pM; x, 50 pM. (B) Kinetics of fumarate transport in the presence of Lmalate. Concentrations of L-malate used: 0, 0 p; 0, 25 p; A, 50 pM; x, 100 M. (C) Kinetics of succinate transport in the presence of fumarate. Concentrations offumarate used: 0, 0 p; 0, 10 W; A, 20 PM; x, 40
pM.
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BISSCHOP, DODDEMA, AND KONINGS
J. BACTERIOL.
10
20
30
1/[succinate] x 105 M1 FIG. 2C TABLE 2. Kinetic constants for transport of C4 dicarboxylic acids in membrane vesicles from B. subtilis W23 substrate
K~ "' Ki(A)
L-Malate Fumarate Succinate
13.5 (+1.5) 7.5 (±0.8) 4.3 (±0.2)
Transported
Vmaz (nmol/nin
x
KK
mg of protein)
L-Malate
1.6 3.7 0.6
7.6 (±1.8) 5.3 (±0.3)
ylic acid transport. To investigate the inducibility of the C4 dicarboxylic transport system, we measured initial transport rates of L-malate, fumarate, and succinate in membrane vesicles from B. subtilis W23 grown under different nutrient conditions. In all cases, membrane vesicles were prepared by a standardized procedure, as described above, to avoid variations due to experimental conditions. However, variations in the stage of growth at which the cells were harvested for the membrane isolation were unavoidable. These variations, together with variations in the respiratory chain activity due to the different growth conditions, might account for slight differences in the transport activities as was shown previously (1). Membrane vesicles from cells grown on the media listed in Table 4 perform active transport of L-malate, fumarate, and succinate with AscPMS as the electron donor. In all cases the ratio between the initial rates of transport of L-mal-
of the competitive inhibitor
(jLM)
Fumarate
Succinate
8.5 (±1.8)
14.0 (±5.5) 9.0 (±2.6)
3.8 (±0.1)
ate, fumarate, and succinate is about 2:4:1. Membrane vesicles from cells grown in the presence of DL-malate or fumarate demonstrate the same transport activity as vesicles grown in the presence of L-glutamate or L-aspartate. The effect of succinate as the sole carbon source on the initial rates of C4 dicarboxylic acid transport could not be tested due to the low growth rate on this substrate. Membrane vesicles prepared from B. subtilis W23 grown on a rich medium containing tryptone transport C4 dicarboxylic acids at relatively high rates. Addition of dicarboxylic acids seems to cause repression of dicarboxylic acid transport; a strong decrease of transport is observed especially in the presence of succinate. Supplementation of yeast extract medium with L-malate or fumarate has just the opposite effect (Table 5). A threefold stimulation is observed in B. subtilis W23 and a 30-fold increase is observed in B. subtilis JH 402 (lacking suc-
DICARBOXYLIC ACID TRANSPORT IN B. SUBTILIS
VOL. 124, 1975
TABLE 3. Effect of structurally and metabolically related compounds on the initial rates oftransport of L-malate, fumarate, and succinate in membrane vesicles from B. subtilis W23
619
c
.UCL 10,0-
% Inhibition of the initial
Inhibitor (1.0 mM)
L-Malate Succinate Fumarate L-Glutamate L-Aspartate meso-Tartrate Maleate Malonate Oxaloacetate a-Ketoglutarate 1)-Lactate L-Lactate n-Butyrate Oxalate Citrate
rate of transport of: L-Malate Fumarate Succinate 94 93
78 89
86 87
93 52
89 48
87
54
50 15 0 2
3 13
2 19 0 9
56 60
'- 8,0 7.0,6.
3 10
9
1 4
0
10
12
19
11
10
17
5
5 10 0
9
7 10
E 9.0
60-
5,0
5 9
4.0
0.5 1
2
3
4 *-
minues
FIG. 3. Transport of L-malate by membrane vesicles from B. subtilis W23 grown on a medium containing 0.3% yeast extract. At the time indicated by the arrow, 1 p1 ofunlabeled L-malate, fumarate, or succinate was added to a final concentration of 1.0 mM. The 100-pi incubation mixture contained 0.046 mg of membrane protein. Symbols: *, Uptake with 10 mM Asc and 100 p PMS; 0, uptake without electron donor; V, exchange upon the addition of -malate; O, exchange upon the addition offumarate; A, exchange upon the addition of succinate.
cinic dehydrogenase) upon the addition of DLmalate. Stimulation of the transport activity is also observed in the presence of fumarate, but this effect is only about one-quarter of that observed with malate.
5
minutes
FIG. 4. Transport of dicarboxylic acids and dicarboxylic amino acids by membrane vesicles from B. subtilis 60346. Open symbols, in the presence of 10 mM ascorbate plus 100 juM PMS; closed symbols, in the absence of energy source. (0) L-Malate; (A) fumarate; (O) succinate; (V) L-glutamate; (O) Laspartate. The 100-p incubation mixture contained 0.083 mg of membrane protein.
DISCUSSION The C4-dicarboxylic acids L-malate, fumarate, and succinate are actively transported by membrane vesicles of B. subtilis in the presence of the electron donors NADH or Asc-PMS. Calculated on the basis of an internal membrane vesicle volume of 3 p.1/mg of membrane protein (19), the internal concentrations of these dicarboxylic acids at steady-state levels are 6 to 45 times the initial external concentrations. These observations indicate that transpart of these dicarboxylic acids is coupled to the respiratory chain in a way similar to that dem-
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BISSCHOP, DODDEMA, AND KONINGS
TABLE 4. Initial rates of L-malate, fumarate, and succinate transport by membrane vesicles from B. subtilis W23 grown on different media Additions to mineral salt medium
Initial rates of transport (nmol/min x mg of protein) L-Malate Fumarate Succinate
1% Glutamate 1% DL-Malate 1% Fumarate 0.5% Tryptone 0.5% Tryptone + 5 mM DL-malate 0.5% Tryptone + 5 mM fumarate 0.5% Tryptone + 5 mM succinate a
1.30 1.50 1.15 0.98 0.88
2.10 ND 1.40 2.76 2.46
NDa ND 0.70 0.90 0.45
0.54
1.40
0.30
0.44
1.35
0.27
ND, Not determined.
onstrated for the transport of amino acids (15, 16, 18, 19). The inhibitory effect of respiratory chain inhibitors on the transport of dicarboxylic acids supports this conclusion. No detectable metabolism of the dicarboxylic acids occurs during or after transport. This observation also holds for succinate despite the fact that the vesicles contain a relatively high activity of succinic dehydrogenase (14). This enzyme is, however, very loosely coupled to the respiratory chain. Moreover, electron flow from succinic dehydrogenase to this respiratory chain might be inhibited further by the electron flow supplied by the oxidation of NADH or AscPMS under the conditions of the transport assay.
The results described in this paper indicate that transport of the C4 dicarboxylic acids occurs by one specific transport system with a high affinity for all three dicarboxylic acids. Lineweaver-Burk plots demonstrate simple saturation kinetics over a 20-fold concentration range, indicating that, most probably, only one saturable site is involved. The affinity constants (K112) for L-malate, fumarate, and succinate are 13.5, 7.5, and 4.3 txM, respectively. These affinity constants are of the same order of magnitude as those observed for the amino acid transport systems in membrane vesicles of B. subtilis (16, 19). They are in sharp contrast, however, to the affinity constants determined by other investigators in whole cells of B. subtilis. Fournier et al. (5) obtained affinity constants of 400 ,uM for L-malate and of 700 ,uM for fumarate transport, and Ghei and Kay (7) obtained affinity constants of 100 ,uM for succinate in a succinic dehydrogenase-deficient mutant and for fumarate of 140 ,uM in a fumarasedeficient mutant. Willecke and Lange (31),
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with the non-metabolizable L-malate analogue 3-fluoro-L-erythro-malate, obtained affinity constants for uninduced cells of 230 ,uM and in cells induced for L-malate transport affinity constants of 20 ,tM. Only this last value is of the same order of magnitude as those obtained in our experiments. At this time, no explanation can be offered for the discrepancy in affinity constants measured in whole cells and membrane vesicles. It is of interest, however, that affinity constants determined in whole cells of other organisms are of the same order of magnitude as our determinations. In P. putida the affinity constants for succinate transport are 11.6 ,M in uninduced cells and 12.5 ,uM in induced cells (3), and those for the transport of dicarboxylic acids in E. coli are all in the range of 15 to 30 ,uM (21). Similar values have been found in membrane vesicles from this same strain of E. coli (24), and in membrane vesicles of E. coli K-12 an affinity constant of 5 ,tM for succinate transport was found (23). The involvement of a common transport system for the three dicarboxylic acids is suggested by the observation that each of these C4 dicarboxylic acids competitively inhibits transport of the other two and that the affinity constants determined during the transport process (K+) are very similar to those determined during the inhibitory action (K). Moreover, after accumulation each dicarboxylic acid exchanges rapidly with external dicarboxylic acid. Furthermore, only the dicarboxylic amino acids inhibit transport of the dicarboxylic acids significantly; other mono-, di-, and tricarboxylic acids are only slightly inhibitory. However, transport of the dicarboxylic amino acids is mediated by a highly specific system, and the inhibition exerted by these amino acids on dicarboxylic acid transport is noncompetitive (16). The best evidence for the existence of separate transport systems for the dicarboxylic acids and the dicarboxylic amino acids comes from studies of memTABLE 5. Initial rates of L-malate transport by membrane vesicles from several B. subtilis strains grown on different media L-Malate transport rate Additions to salt medium
0.3% Yeast extract 0.3% Yeast extract + 5
(nmol/min x mg of protein) W23
168 168
JH402
0.45 1.20
1.90 2.66
0.17 5.20
NDa
ND
1.25
mM DL-malate 0.3% Yeast extract + 5 mM fumarate a
ND, Not determined.
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VOL. 124, 1975
brane vesicles from B. subtilis 60346, a mutant lacking dicarboxylic amino acid transport. Membrane vesicles of this mutant transport the dicarboxylic acids at rates that are even higher than those observed in wild-type B. subtilis W23 (Fig. 4). Evidence for the presence of one common transport system for C4 dicarboxylic acids was also presented for P. putida (3) and E. coli (21, 24). In contrast to these observations, Ghei and Kay reported competitive inhibition of dicarboxylic acid transport by the dicarboxylic amino acid L-aspartate in whole cells of B. subtilis 168 (6, 7) and inferred the existence of one system predominant in L-malate-grown cells, whose affinity is L-malate > fumarate > succinate > aspartate. Moreover, they also presented evidence for a second transport system in citrate-grown cells which transports fumarate more efficiently than L-malate (6, 7). C4 dicarboxylic acid transport activity seems to be present constitutively in B. subtilis W23, but the presence of the dicarboxylic acids in the growth medium influences this transport activity. In agreement with the observations made with intact cells (5), an increase in transport activity was observed upon the supplementation of yeast extract with DL-malate or fumarate. Supplementation of tryptone with these dicarboxylic acids results, however, in a decreased transport activity. Ghei and Kay (7) reported an induction of dicarboxylic acid transport in the presence of the dicarboxylic acids or precursors of these dicarboxylic acids. Malate was found to be the best dicarboxylic acid transport inducer in mutants lacking a functional tricarboxylic acid cycle. Our experiments cannot exclude the fact that the dicarboxylic acids are indeed the inducers for their own transport. The apparently conflicting observations for vesicles from cells grown on yeast extract or tryptone might possibly be explained by the low vitamin content of tryptone. Preliminary results indicate that supplementation of minimal media with vitamins results in vesicles with higher transport activities. Such a role of vitamins in the activity of amino acid transport has already been reported for Lactobacillus plantarum (8). ACKNOWLEDGMENTS This study was supported by the Netherlands Organization for Advancement of Pure Scientific Research (ZWO). We are grateful to M. Diesterhaft, G. Venema, J. A. Hoch, and R. S. Hanson for sending us the mutant strains of Bacillus subtilis. LITERATURE CITED 1. Bisschop, A., L. de Jong, M. E. Lima Costa, and W. N. Konings. 1975. Relation between reduced nicotinamide adenine dinucleotide oxidation and amino acid
621
transport in membrane vesicles from Bacillus subtilis. J. Bacteriol. 121:807-813. 2. Carls, R. A., and R. S. Hanson. 1971. Isolation and characterization of tricarboxylic acid cycle mutants of Bacillus subtilis. J. Bacteriol. 106:848-855. 3. Dubler, R. E., W. A. Toscano, Jr., and R. A. Hartline. 1974. Transport of succinate by Pseudomonas putida. Arch. Biochem. Biophys. 160:422-429. 4. Eagon, R. G., and L. S. Wilkerson. 1972. A potassiumdependent citric acid transport system in Aerobacter aerogenes. Biochem. Biophys. Res. Commun. 46:1944-1950. 5. Fournier, R. E., M. N. McKillen, A. B. Pardee, and K. Willecke. 1972. Transport of dicarboxylic acids in Bacillus subtilis. Inducible uptake of L-malate. J. Biol. Chem. 247:5587-5595. 6. Ghei, Om. K., and W. W. Kay. 1972. A dicarboxylic acid transport system in B. subtilis. FEBS Lett. 20:137140. 7. Ghei, Om. K., and W. W. Kay. 1973. Properties of an inducible C4-dicarboxylic acid transport system in Bacillus subtilis. J. Bacteriol. 114:65-79. 8. Holden, J. T., 0. Hild, Y. L. W. Leung, and G. Rouser. 1970. Reduced lipid content as the basis for defective amino accumulation capacity in pantothenate- and biotin-deficient Lactobacillus plantarum. Biochem. Biophys. Res. Commun. 40:123-128. 9. Imai, K., T. Iijima, and T. Hasegawa. 1973. Transport of dicarboxylic acids in Salmonella typhimurium. J. Bacteriol. 114:961-965. 10. Kaback, H. R. 1970. Preparation and characterization of bacterial membranes, p. 99-120. In W. B. Jakoby (ed.), Methods in enzymology, vol. 22. Academic Press Inc., New York. 11. Kaback, H. R. 1974. Transport in bacterial membrane vesicles. Science 186:882-892. 12. Kaback, H. R., and J. S. Hong. 1973. Membranes and transport. C.R.C. Crit. Rev. Microbiol. 2:333-376. 13. Kay, W. W., and H. L. Kornberg. 1971. The uptake of C4-dicarboxylic acids by Escherichia coli. Eur. J. Biochem. 18:274-281. 14. Konings, W. N. 1975. Localization of membrane proteins in membrane vesicles ofBacillus subtilis. Arch. Biochem. Biophys. 167:570-575. 15. Konings, W. N., E. M. Barnes, Jr., 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. 16. 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. 17. 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. 18. Konings, W. N., and E. Freese. 1971. L-serine transport in membrane vesicles of B. subtilis energized by NADH or reduced phenazine methosulfate. FEBS Lett. 14:65-68. 19. Konings, W. N., and E. Freese. 1972. Amino acid transport in membrane vesicles ofBacillus subtilis. J. Biol. Chem. 247:2408-2418. 20. Lawford, H. G., and G. R. Williams. 1971. The transport of citrate and other tricarboxylic acids in two species of Pseudomonas. Biochem. J. 123:571-577. 21. Lo, T. C. Y., M. K. Rayman, and B. D. Sanwal. 1972. Transport of succinate in Escherichia coli. Biochemical and genetic studies of transport in whole cells. J. Biol. Chem. 247:6323-6331. 22. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.
622
23.
24.
25.
26.
27.
BISSCHOP, DODDEMA, AND KONINGS
Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Matin, A., and W. N. Konings. 1972. Transport of lactate and succinate by membrane vesicles of E. coli, B. subtilis and a Pseudomonas species. Eur. J. Biochem. 34:58-67. Murakawa, S., K. Izaki, and H. Takahashi. 1973. A transport system for C4-dicarboxylic acids in isolated membrane preparations from E. coli. Agric. Biol. Chem. 37:1905-1914. Oehr, P., and K. Willecke. 1974. Citrate-Mg2+ transport in Bacillus subtilis. Studies with 2-fluoro-erythro-citrate as a substrate. J. Biol. Chem. 249:2037-2042. Rayman, M. K., T. C. Y. Lo, and B. D. Sanwal. 1972. Transport of succinate in E. coli. II. Characteristics of uptake and energy coupling with transport in membrane preparations. J. Biol. Chem. 247:6332-6339. Rutberg, B., and J. A. Hoch. 1970. Citric acid cycle:gene-enzyme relationships in Bacillus subtilis. J.
J. BACTERIOL. Bacteriol. 104:826-833. 28. Sachan, D. S., and J. R. Stern. 1971. Studies of citrate transport in Aerobacter aerogenes: binding of citrate by a membrane bound oxalo acetate dicarboxylase. Biochem. Biophys. Res. Commun. 45:402-408. 29. Thomas, C. L., H. Weissbach, and H. R. Kaback. 1973. Further studies on metabolism of phosphatidic acid of isolated E. coli membrane vesicles. Arch. Biochem. Biophys. 150:797-806. 30. Ting, J. P., and W. M. Dugger, Jr., 1965. Separation and detection of organic acids on silicagel. Anal. Biochem. 12:571-578. 31. Willecke, K., and R. Lange. 1973. C4-dicarboxylate transport in Bacillus subtilis studied with 3-fluoro-Lerythro-malate as a substrate. J. Bacteriol. 117:373378. 32. Willecke, K., and A. B. Pardee. 1971. Inducible transport of citrate in a Gram-positive organism Bacillus subtilis.J. Biol. Chem. 246:1032-1040.
JOURNAL OF BACTERIOLOGY, Nov. 1975, p. 613-622
Copyright C) 1975 American Society for Microbiology
Dicarboxylic Acid Transport in Membrane Vesicles from Bacillus subtilis ARNOLD BISSCHOP,* HENK DODDEMA,' AND WIL N. KONINGS Laboratory for Microbiology, Biological Centre, University of Groningen, Kerlaan 30, Haren (Gr.), The Netherlands Received for publication 30 June 1975
Membrane vesicles isolated from Bacillus subtilis W23 catalyze active transport of the 04 dicarboxylic acids L-malate, fumarate, and succinate under aerobic conditions in the presence of the electron donor reduced ,8-nicotinamide adenine dinucleotide or the non-physiological electron donor system ascorbate-phenazine methosulfate. The dicarboxylic acids are accumulated in unmodified form. Inhibitors of the respiratory chain, sulfhydryl reagents, and uncoupling agents inhibit the accumulation of the dicarboxylic acids. The affinity constants for transport of L-malate, fumarate, and succinate are 13.5, 7.5, and 4.3 AM, respectively; these values are severalfold lower than those reported previously for whole cells. Active transport of these dicarboxylic acids occurs via one highly specific transport system as is indicated by the following observations. (i) Each dicarboxylic acid inhibits the transport of the other two dicarboxylic acids competitively. (ii) The affinity constants determined for the inhibitory action are very similar to those determined for the transport process. (iii) Each dicarboxylic acid exchanges rapidly with a previously accumulated dicarboxylic acid. (iv) Other metabolically and structurally related compounds do not inhibit transport of these dicarboxylic acids significantly, except for L-aspartate and L-glutamate. However, transport of these dicarboxylic amino acids is mediated by an independent system because membrane vesicles from B. subtilis 60346, lacking functional dicarboxylic amino acid transport activity, accumulate the C4 dicarboxylic acids at even higher rates than vesicles from B. subtilis W 23. (v) A constant ratio exists between the initial rates of transport of L-malate, fumarate, and succinate in all membrane vesicle preparations isolated from cells grown on various media. This high-affinity dicarboxylic acid transport system seems to be present constitutively in B. subtilis W23.
Active transport of metabolites across the cytoplasmic membrane has received considerable attention in the last few years. Most studies have been concerned with transport systems for amino acids and carbohydrates, and only recently has interest been directed towards transport systems for tricarboxylic acid cycle intermediates. Several of these tricarboxylic acid cycle intermediates appear to be accumulated via specific and inducible transport systems. Studies in whole cells, for instance, demonstrated the presence of specific and inducible uptake systems for citrate in Klebsiella aerogenes (4, 28), Pseudomonas spp. (20), and Bacillus subtilis (32). In Salmonella typhimurium there appear to be three specific and inducible
transport systems for the tricarboxylic acids citrate, isocitrate, cis-aconitate, and tricarballylate (9). Studies on the transport of dicarboxylic acids demonstrated the presence of specific and inducible transport systems for 04 dicarboxylic acids in Escherichia coli (13, 21) and P. putida (3). Similar studies have been done with B. subtilis (5-7). In the wild type and in mutants that lack a functional tricarboxylic acid cycle, inducible transport systems were demonstrated for L-malate, fumarate, succinate, and a-ketoglutarate. DL-Tartrate also seems to be accumulated by a C4 dicarboxylic acid transport system
(5). One of the major problems in the study of active transport of metabolites by whole cells is the rapid conversion of transported substrate
I Present address: Laboratory for Plant Physiology, Biological Centre, University of Groningen, Kerklaan 30, Haren (Gr.), The Netherlands.
upon entrance into the cells. For some metabolites this problem can be solved by studying 613
614
BISSCHOP, DODDEMA, AND KONINGS
transport in cells in which a genetic block is introduced to prevent metabolism of the transported substrate. Such an approach has been taken in studies of the transport of C4 dicarboxylic acids in B. subtilis and E. coli (5, 7, 21). An alternative approach involves the use of nonmetabolizable substrate analogues, such as 3fluoro-L-erythro-malate and 2-fluoro-L-erythrocitrate, for L-malate and citrate (25, 31). A more generally applicable solution to this problem can be found in the use of membrane vesicles. These membrane vesicles consist of cytoplasmic membranes and are devoid of cytoplasmic constituents and cell wall components (10, 16). They have been shown to be physiologically active with regard to certain integrated membrane functions (1, 11, 12, 14, 18, 19, 29) such as amino acid and sugar transport, phospholipid biosynthesis, and electron transport. We have been concerned with active transport processes in membrane vesicles of B. subtilis. Membrane vesicles from this gram-positive organism can be isolated in a rather simple and gentle way (17), and it has been shown previously that these membrane vesicles actively transport many metabolites in the presence of reduced f3-nicotinamide adenine dinucleotide (NADH) or ascorbate-phenazine methosulfate (Asc-PMS) at rates comparable with those observed for whole cells (1, 14, 17-19, 23). Moreover, freeze-etch electron microscopy (17) and biochemical studies on the localization of membrane-bound enzymes (14) revealed that the vesicles have the same orientation as the cytoplasmic membrane of whole cells. During our studies on the specificity of the transport systems in vesicles from B. subtilis, it was demonstrated that at least nine distinct systems exist for the transport of amino acids (19). One of these systems is specific for the dicarboxylic amino acids L-glutamate and L-aspartate (16). The C4 dicarboxylic acids L-malate, fumarate, and succinate are transported by separate systems and inhibit transport of the dicarboxylic amino acids in a noncompetitive way (16). The existence of distinct transport systems for dicarboxylic acids has also been demonstrated in membrane vesicles from E. coli (23, 24, 26) and Pseudomonas spp. (23). It is the purpose of this paper to characterize the specificity and the kinetic properties of the transport system(s) involved in the accumulation of L-malate, succinate, and fumarate. A brief preliminary report of these studies has been presented previously (A. Bisschop and W. N. Konings, Abstr. FEBS Meet. 1974, p. 250). MATERIALS AND METHODS Bacterial strains.B. subtilis W23 was used as the
J. BACTERIOL.
wild-type strain. Strain JH402 (trpC2 citF2) was derived from B. subtilis 168 and is genetically blocked in succinic dehydrogenase (EC 1.3.99.1) activity. This mutant was donated by J. A. Hoch, Department of Microbiology, Scripps Clinic and Research Foundation, La Jolla, Calif. (27). Strain HS1A21 is a malic dehydrogenase (EC 1.1.1.37)-defective mutant derived from B. subtilis 168, and was donated by R. S. Hanson, Department of Bacteriology, University of Wisconsin, Madison (2). Cultures of the mutants defective in the tricarboxylic acid cycle were tested for revertants as described by Carls and Hanson (2). B. subtilis 168 (trpC2), auxotrophic for tryptophan, was obtained from G. Venema, Genetic Institute, Biological Centre, University of Groningen. B. subtilis 60346, deficient in the active transport of dicarboxylic amino acids, was a gift of M. Diesterhaft, National Institutes of Health, Bethesda, Md. Culture media. All liquid media had the basic composition (per liter): K2HPO4, 14 g; KH2PO4, 6 g; MgSO4 7H20, 0.25 g; MnSO4 H20, 0.017 g; (NH4)2SO4, 2 g; and L-glutamic acid, 0.15 g. The carbon and energy sources used were added to this basic medium as follows: tryptone (5 g/liter) and yeast extract (3 g/liter); DL-malate, fumarate, or succinate, if added to the enriched tryptone or yeast extract media, were added at a 5 mM concentration as described by R. E. Fournier et al. (5). In defined media, 0.15 g of L-glutamate per liter was replaced by 1% fumarate, DL-malate, L-glutamate, or L-aspartate. Cell growth. Cells were grown on the media described above, in a 3-liter Biotec fermenter (LKB Producter, Bromma, Sweden) under vigorous aeration at 37 C. The culture was inoculated to an absorbancy at 663 nm of 0.1 with cells grown overnight in the same medium. Growth was followed turbidimetrically, and late-log-phase cells were harvested by centrifugation. Preparation of membrane vesicles. Membrane vesicles were prepared as described previously (17) but with the following modifications. Cells were suspended in 0.05 M potassium phosphate buffer (pH 8.0) at 37 C at a concentration of 4 g (wet weight) per liter. Lysozyme, deoxyribonuclease I, and ribonuclease were added at final concentrations of 300, 10, and 10 ug/ml, respectively. After 15 min of incubation, MgSO4 was added at a final concentration of 10 mM. The incubation was continued for 30 min, after which the incubation was stopped by centrifugation of the solution at 20,000 x g for 30 min. The membranes were suspended thoroughly by means of a hypodermic syringe fitted with an 18-gauge needle, washed twice with 0.1 M potassium phosphate buffer (pH 6.6), and finally resuspended in this buffer at 4 to 6 mg of membrane protein per ml. Portions of 0.5 to 1 ml in thin-walled plastic tubes were frozen rapidly and stored in liquid nitrogen. Protein was determined by the method of Lowry et al. (22). Transport assays. Transport experiments were performed, as described previously (1, 15, 19, 23), in a final incubation volume of 100 .l at 25 C. Unless otherwise stated, transport studies were performed
with membrane vesicles ofB. subtilis W23 grown on mineral salt medium supplemented with 0.3% yeast
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VOL. 124, 1975
extract and 5 mM DL-malate. In all studies, Asc (10 mM) and PMS (100 ,uM) or NADH (10 mM) were used as the electron donor. Initial transport rates were measured after a 1-min incubation of the membrane vesicles with the electron donor. The substrates were added in the following concentrations unless stated otherwise: L-[U-'4C]glutamic acid, 18.9 ;LM (specific acitivity 265 mCi/mmol); o[U'4C]aspartic acid, 21.6 ;LM (specific activity 232 mCi/mmol); L-[U-14C]malic acid, 38.1 ,uM (specific activity 82 mCi/mmol); [2,3-'4C]fumaric acid, 217 IAM (specific activity 13 mCi/mmol); and [1,4"4C]succinic acid, 119 ,uM (specific activity 21 mCi/mmol). Materials. All 14C-labeled compounds were purchased from the Radiochemical Centre (Amersham, England). Tryptone and yeast extract were obtained from Difco (Detroit, Mich.). NADH was from Boehringer (Mannheim, Germany); PMS was from BDH Chemicals Ltd. (Poole, England); 2-heptyl-4-hy-
5-ethyl-5-isoamylbarbidroxyquinoline-N-oxide, turic acid (Amytal), carbonyl cyanide-m-chlorophenyl hydrazone, and L-malic acid were from Sigma Chemical Co. (St. Louis, Mo.); and L-lactic acid and D-lactic acid, both lithium salts, were from Calbiochem AG (Lucerne, Switzerland). Ribonuclease and deoxyribonuclease were products of Miles Laboratories, Inc. (Kankakee, Ill.). Rotenone was obtained from Aldrich-Europe (Beerse, Belgium). Selectron BA85 membrane filters, pore size 0.45 ,m, were purchased from Schleicher and Schull GMBH (Dassel, Germany). All other chemicals were purchased from E. Merck (Darmstadt, Germany).
RESULTS Active transport of dicarboxylic acids. Membrane vesicles from B. subtilis W23 grown on a mineral salts medium supplemented with yeast extract plus DL-malate as the carbon and energy source accumulate all three C4 tricarbox-
615
ylic acid cycle intermediates (L-malate, fumarate, and succinate) in the presence of an electron donor (Fig. 1). High initial rates of uptake are obtained in the presence of the non-physiological electron donor Asc-PMS and also with the physiological electron donor NADH, and steady-state levels are reached between 1.5 and 9.5 nmol/mg of membrane protein. In the absence of an electron donor, no concentration of dicarboxylic acids occurs (Fig. 1). It should be emphasized that the initial rates of transport in the presence of NADH or Asc-PMS varies among different vesicle preparations. It has been shown previously (1) that vesicle preparations isolated at different stages of the growth curve demonstrate an increase in the rate of NADH or Asc-PMS oxidation per milligram of membrane protein. The variation in the transport activity correlates with this variation in the oxidation rates (1). The dicarboxylic acids are not metabolized during accumulation as was demonstrated by extraction of the membrane vesicles after steady-state level accumulation followed by thin-layer chromatography. Scanning of the radioactivity yielded single spots with the Rf values: L-malate, 0.46; fumarate, 0.83; and succinate, 0.74. The same Rf values were obtained for the original substrates, and these values are in good agreement with the data presented by Ting and Dugger (30). Transport of these dicarboxylic acids is coupled to electron flow in the respiratory chain in a way similar to that demonstrated for amino acid transport (15, 16, 18, 19). Inhibitors of electron transfer such as amytal, 2-heptyl-4-hydroxyquinoline-N-oxide, rotenone, and cyanide
C 4.0 -
E
3.0
S
, 2.0 E5
1
2
3
4
5
1
2 3 time (min)
4
5
1
2
3
4
5
FIG. 1. Transport of dicarboxylic acid by membrane vesicles from Bacillus subtilis W23. (A). L-Malate; (B) fumarate; (C) succinate. Symbols: 0, In the presence of20 mM NADH; 0, in the presence of 10 mM Asc plus 100 M PMS; and V, in the absence of an energy source. The incubation mixture (100 ,u) contained 0.047 mg of membrane protein.
616
BISSCHOP, DODDEMA, AND KONINGS
strongly inhibit the initial rates of transport of all three dicarboxylic acids and exert inhibitions between 40 and 90% (Table 1). Almost complete inhibition of the initial rates of transport is observed in the presence of the uncoupling agents dinitrophenol, carbonyl cyanidem-chlorophenyl hydrazone and azide. Thiol reagents such as p-chloromercuribenzoate and Nethyl maleimide are also effective inhibitors of transport. Specificity of dicarboxylic acid transport. Initial rates of L-malate, fumarate, and succinate transport as a function of the substrate concentration display simple Michaelis-Menten kinetics, and Lineweaver-Burk plots yield straight lines for all three dicarboxylic acids over a 20-fold concentration range (Fig. 2). Transport occurs via high-affinity system(s) with K,12 values of the order of 10-5 M and Vmax, values of 0.6 to 3.7 nmol!mg of membrane protein per min (Table 2). These K 12 values are the average values of five independent determinations. The transport system(s) has the highest affinity for succinate, a twofold-lower affinity for fumarate, and a threefold-lower affinity for L-malate. In Fig. 2 it is also shown that the transport of each dicarboxylic acid is inhibited competitively by the other two individual dicarboxylic acids (only one set of figures is presented). The TABLE 1. Effect of metabolic inhibitors on initial rates of C4 dicarboxylic acid transport in membrane vesicles from B. subtilis W23a Inhibio Inhibitor
Concn
(mM)
% Inhibition of initial transport rate of:
L-Malate Fumarate Succinate
Amytal HOQNO Rotenone KCN DNP PNP Sodium
0.1 10-2 0.1 10 0.1 0.5 1.0
57 82 43 49 98 99 90
60 88
10-3 1.0 0.5
100 79 40
100
37 47 97 92 91
58 77 35 45 96 99 96
azide CCCP
pCMB NEM
97 39
100 71
37
a The inhibitors were added prior to preincubation of the incubation mixture. 2-Heptyl-4-hydroxyquinoline-N-oxide (HOQNO), amytal, car-
bonyl cyanide-m-chlorophenyl hydrazone (CCCP), and rotenone were dissolved in dimethylsulfoxide. All values are stated with respect to controls having the same dimethyl sulfoxide concentration. DNP, Dinitrophenol; pCMB, p-chloromercuribenzoate; NEM, N-ethyl maleimide; PNP, p-nitrophenol.
J. BACTERIOL.
inhibition constants (Ki) calculated from these data are given in Table 2. Each value is the average of at least three experiments. The K, values are in the same order of magnitude as the K1/2 values, but in all cases the lowest values were obtained with succinate, slightly higher values with fumarate, and the highest values with L-malate as transported substrate. No explanation can be offered at this time for this observation. These observations suggest the existence of a common transport system for L-malate, fumarate, and succinate. Other lines of evidence support this contention. The effects of various structurally and metabolically related metabolites on the initial rates of transport are the same for all three dicarboxylic acids (Table 3). The strongest inhibitory effects were exerted by the dicarboxylic acids themselves (more than 80%); the dicarboxylic amino acids L-aspartate and L-glutamate were moderately inhibitory (50 to 60%), whereas other mono-, di-, and tricarboxylic acids had no significant effect on dicarboxylic acid transport. Similar observations were made with membrane vesicles from mutants of B. subtilis lacking succinic dehydrogenase (strain JH 402), lacking fumarase (strain JH 404), and lacking malic dehydrogenase (strain HS1A21), all grown on the same medium as strain W23 in Table 3, and with membrane vesicles of B. subtilis W23 grown on different carbon sources (data not shown). More evidence for the existence of a common transport system for the dicarboxylic acids was obtained from exchange experiments. In Fig. 3 it is shown that accumulated L-malate exchanges rapidly with external L-malate and also with fumarate and succinate, and that 85% of the radioactivity is lost in less than 1 min. Similar observations were made with the other C4 dicarboxylic acids (data not shown). Differences between dicarboxylic acid and dicarboxylic amino acid transport system. In Table 3 it is shown that the dicarboxylic amino acids L-glutamate and L-aspartate inhibit the transport of the dicarboxylic acids significantly. However, the two groups of metabolites do not share a common transport system because the dicarboxylic acids inhibit transport of the dicarboxylic amino acids non-competitively, as was shown previously (16). This is supported by the observation that membrane vesicles from a B. subtilis mutant defective in dicarboxylic amino acid transport do not transport the dicarboxylic amino acids while transport of the dicarboxylic acids is unimpaired (Fig. 4). Effect of the growth medium on dicarbox-
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DICARBOXYLIC ACID TRANSPORT IN B. SUBTILIS
617
l/[fumarateJ x 105 M-1 FIG. 2. Kinetics of competitive inhibition of C4 dicarboxylic acid transport by membrane vesicles from B. subtilis W23. The initial rates of transport were determined after a 1-min incubation with 0.047 mg of membrane protein. (A) Kinetics of L-malate transport in the presence of succinate. Concentrations of succinate used: *, 0 M; 0, 12.5 pM; A, 25 pM; x, 50 pM. (B) Kinetics of fumarate transport in the presence of Lmalate. Concentrations of L-malate used: 0, 0 p; 0, 25 p; A, 50 pM; x, 100 M. (C) Kinetics of succinate transport in the presence of fumarate. Concentrations offumarate used: 0, 0 p; 0, 10 W; A, 20 PM; x, 40
pM.
618
BISSCHOP, DODDEMA, AND KONINGS
J. BACTERIOL.
10
20
30
1/[succinate] x 105 M1 FIG. 2C TABLE 2. Kinetic constants for transport of C4 dicarboxylic acids in membrane vesicles from B. subtilis W23 substrate
K~ "' Ki(A)
L-Malate Fumarate Succinate
13.5 (+1.5) 7.5 (±0.8) 4.3 (±0.2)
Transported
Vmaz (nmol/nin
x
KK
mg of protein)
L-Malate
1.6 3.7 0.6
7.6 (±1.8) 5.3 (±0.3)
ylic acid transport. To investigate the inducibility of the C4 dicarboxylic transport system, we measured initial transport rates of L-malate, fumarate, and succinate in membrane vesicles from B. subtilis W23 grown under different nutrient conditions. In all cases, membrane vesicles were prepared by a standardized procedure, as described above, to avoid variations due to experimental conditions. However, variations in the stage of growth at which the cells were harvested for the membrane isolation were unavoidable. These variations, together with variations in the respiratory chain activity due to the different growth conditions, might account for slight differences in the transport activities as was shown previously (1). Membrane vesicles from cells grown on the media listed in Table 4 perform active transport of L-malate, fumarate, and succinate with AscPMS as the electron donor. In all cases the ratio between the initial rates of transport of L-mal-
of the competitive inhibitor
(jLM)
Fumarate
Succinate
8.5 (±1.8)
14.0 (±5.5) 9.0 (±2.6)
3.8 (±0.1)
ate, fumarate, and succinate is about 2:4:1. Membrane vesicles from cells grown in the presence of DL-malate or fumarate demonstrate the same transport activity as vesicles grown in the presence of L-glutamate or L-aspartate. The effect of succinate as the sole carbon source on the initial rates of C4 dicarboxylic acid transport could not be tested due to the low growth rate on this substrate. Membrane vesicles prepared from B. subtilis W23 grown on a rich medium containing tryptone transport C4 dicarboxylic acids at relatively high rates. Addition of dicarboxylic acids seems to cause repression of dicarboxylic acid transport; a strong decrease of transport is observed especially in the presence of succinate. Supplementation of yeast extract medium with L-malate or fumarate has just the opposite effect (Table 5). A threefold stimulation is observed in B. subtilis W23 and a 30-fold increase is observed in B. subtilis JH 402 (lacking suc-
DICARBOXYLIC ACID TRANSPORT IN B. SUBTILIS
VOL. 124, 1975
TABLE 3. Effect of structurally and metabolically related compounds on the initial rates oftransport of L-malate, fumarate, and succinate in membrane vesicles from B. subtilis W23
619
c
.UCL 10,0-
% Inhibition of the initial
Inhibitor (1.0 mM)
L-Malate Succinate Fumarate L-Glutamate L-Aspartate meso-Tartrate Maleate Malonate Oxaloacetate a-Ketoglutarate 1)-Lactate L-Lactate n-Butyrate Oxalate Citrate
rate of transport of: L-Malate Fumarate Succinate 94 93
78 89
86 87
93 52
89 48
87
54
50 15 0 2
3 13
2 19 0 9
56 60
'- 8,0 7.0,6.
3 10
9
1 4
0
10
12
19
11
10
17
5
5 10 0
9
7 10
E 9.0
60-
5,0
5 9
4.0
0.5 1
2
3
4 *-
minues
FIG. 3. Transport of L-malate by membrane vesicles from B. subtilis W23 grown on a medium containing 0.3% yeast extract. At the time indicated by the arrow, 1 p1 ofunlabeled L-malate, fumarate, or succinate was added to a final concentration of 1.0 mM. The 100-pi incubation mixture contained 0.046 mg of membrane protein. Symbols: *, Uptake with 10 mM Asc and 100 p PMS; 0, uptake without electron donor; V, exchange upon the addition of -malate; O, exchange upon the addition offumarate; A, exchange upon the addition of succinate.
cinic dehydrogenase) upon the addition of DLmalate. Stimulation of the transport activity is also observed in the presence of fumarate, but this effect is only about one-quarter of that observed with malate.
5
minutes
FIG. 4. Transport of dicarboxylic acids and dicarboxylic amino acids by membrane vesicles from B. subtilis 60346. Open symbols, in the presence of 10 mM ascorbate plus 100 juM PMS; closed symbols, in the absence of energy source. (0) L-Malate; (A) fumarate; (O) succinate; (V) L-glutamate; (O) Laspartate. The 100-p incubation mixture contained 0.083 mg of membrane protein.
DISCUSSION The C4-dicarboxylic acids L-malate, fumarate, and succinate are actively transported by membrane vesicles of B. subtilis in the presence of the electron donors NADH or Asc-PMS. Calculated on the basis of an internal membrane vesicle volume of 3 p.1/mg of membrane protein (19), the internal concentrations of these dicarboxylic acids at steady-state levels are 6 to 45 times the initial external concentrations. These observations indicate that transpart of these dicarboxylic acids is coupled to the respiratory chain in a way similar to that dem-
620
BISSCHOP, DODDEMA, AND KONINGS
TABLE 4. Initial rates of L-malate, fumarate, and succinate transport by membrane vesicles from B. subtilis W23 grown on different media Additions to mineral salt medium
Initial rates of transport (nmol/min x mg of protein) L-Malate Fumarate Succinate
1% Glutamate 1% DL-Malate 1% Fumarate 0.5% Tryptone 0.5% Tryptone + 5 mM DL-malate 0.5% Tryptone + 5 mM fumarate 0.5% Tryptone + 5 mM succinate a
1.30 1.50 1.15 0.98 0.88
2.10 ND 1.40 2.76 2.46
NDa ND 0.70 0.90 0.45
0.54
1.40
0.30
0.44
1.35
0.27
ND, Not determined.
onstrated for the transport of amino acids (15, 16, 18, 19). The inhibitory effect of respiratory chain inhibitors on the transport of dicarboxylic acids supports this conclusion. No detectable metabolism of the dicarboxylic acids occurs during or after transport. This observation also holds for succinate despite the fact that the vesicles contain a relatively high activity of succinic dehydrogenase (14). This enzyme is, however, very loosely coupled to the respiratory chain. Moreover, electron flow from succinic dehydrogenase to this respiratory chain might be inhibited further by the electron flow supplied by the oxidation of NADH or AscPMS under the conditions of the transport assay.
The results described in this paper indicate that transport of the C4 dicarboxylic acids occurs by one specific transport system with a high affinity for all three dicarboxylic acids. Lineweaver-Burk plots demonstrate simple saturation kinetics over a 20-fold concentration range, indicating that, most probably, only one saturable site is involved. The affinity constants (K112) for L-malate, fumarate, and succinate are 13.5, 7.5, and 4.3 txM, respectively. These affinity constants are of the same order of magnitude as those observed for the amino acid transport systems in membrane vesicles of B. subtilis (16, 19). They are in sharp contrast, however, to the affinity constants determined by other investigators in whole cells of B. subtilis. Fournier et al. (5) obtained affinity constants of 400 ,uM for L-malate and of 700 ,uM for fumarate transport, and Ghei and Kay (7) obtained affinity constants of 100 ,uM for succinate in a succinic dehydrogenase-deficient mutant and for fumarate of 140 ,uM in a fumarasedeficient mutant. Willecke and Lange (31),
J. BACTERIOL.
with the non-metabolizable L-malate analogue 3-fluoro-L-erythro-malate, obtained affinity constants for uninduced cells of 230 ,uM and in cells induced for L-malate transport affinity constants of 20 ,tM. Only this last value is of the same order of magnitude as those obtained in our experiments. At this time, no explanation can be offered for the discrepancy in affinity constants measured in whole cells and membrane vesicles. It is of interest, however, that affinity constants determined in whole cells of other organisms are of the same order of magnitude as our determinations. In P. putida the affinity constants for succinate transport are 11.6 ,M in uninduced cells and 12.5 ,uM in induced cells (3), and those for the transport of dicarboxylic acids in E. coli are all in the range of 15 to 30 ,uM (21). Similar values have been found in membrane vesicles from this same strain of E. coli (24), and in membrane vesicles of E. coli K-12 an affinity constant of 5 ,tM for succinate transport was found (23). The involvement of a common transport system for the three dicarboxylic acids is suggested by the observation that each of these C4 dicarboxylic acids competitively inhibits transport of the other two and that the affinity constants determined during the transport process (K+) are very similar to those determined during the inhibitory action (K). Moreover, after accumulation each dicarboxylic acid exchanges rapidly with external dicarboxylic acid. Furthermore, only the dicarboxylic amino acids inhibit transport of the dicarboxylic acids significantly; other mono-, di-, and tricarboxylic acids are only slightly inhibitory. However, transport of the dicarboxylic amino acids is mediated by a highly specific system, and the inhibition exerted by these amino acids on dicarboxylic acid transport is noncompetitive (16). The best evidence for the existence of separate transport systems for the dicarboxylic acids and the dicarboxylic amino acids comes from studies of memTABLE 5. Initial rates of L-malate transport by membrane vesicles from several B. subtilis strains grown on different media L-Malate transport rate Additions to salt medium
0.3% Yeast extract 0.3% Yeast extract + 5
(nmol/min x mg of protein) W23
168 168
JH402
0.45 1.20
1.90 2.66
0.17 5.20
NDa
ND
1.25
mM DL-malate 0.3% Yeast extract + 5 mM fumarate a
ND, Not determined.
DICARBOXYLIC ACID TRANSPORT IN B. SUBTILIS
VOL. 124, 1975
brane vesicles from B. subtilis 60346, a mutant lacking dicarboxylic amino acid transport. Membrane vesicles of this mutant transport the dicarboxylic acids at rates that are even higher than those observed in wild-type B. subtilis W23 (Fig. 4). Evidence for the presence of one common transport system for C4 dicarboxylic acids was also presented for P. putida (3) and E. coli (21, 24). In contrast to these observations, Ghei and Kay reported competitive inhibition of dicarboxylic acid transport by the dicarboxylic amino acid L-aspartate in whole cells of B. subtilis 168 (6, 7) and inferred the existence of one system predominant in L-malate-grown cells, whose affinity is L-malate > fumarate > succinate > aspartate. Moreover, they also presented evidence for a second transport system in citrate-grown cells which transports fumarate more efficiently than L-malate (6, 7). C4 dicarboxylic acid transport activity seems to be present constitutively in B. subtilis W23, but the presence of the dicarboxylic acids in the growth medium influences this transport activity. In agreement with the observations made with intact cells (5), an increase in transport activity was observed upon the supplementation of yeast extract with DL-malate or fumarate. Supplementation of tryptone with these dicarboxylic acids results, however, in a decreased transport activity. Ghei and Kay (7) reported an induction of dicarboxylic acid transport in the presence of the dicarboxylic acids or precursors of these dicarboxylic acids. Malate was found to be the best dicarboxylic acid transport inducer in mutants lacking a functional tricarboxylic acid cycle. Our experiments cannot exclude the fact that the dicarboxylic acids are indeed the inducers for their own transport. The apparently conflicting observations for vesicles from cells grown on yeast extract or tryptone might possibly be explained by the low vitamin content of tryptone. Preliminary results indicate that supplementation of minimal media with vitamins results in vesicles with higher transport activities. Such a role of vitamins in the activity of amino acid transport has already been reported for Lactobacillus plantarum (8). ACKNOWLEDGMENTS This study was supported by the Netherlands Organization for Advancement of Pure Scientific Research (ZWO). We are grateful to M. Diesterhaft, G. Venema, J. A. Hoch, and R. S. Hanson for sending us the mutant strains of Bacillus subtilis. LITERATURE CITED 1. Bisschop, A., L. de Jong, M. E. Lima Costa, and W. N. Konings. 1975. Relation between reduced nicotinamide adenine dinucleotide oxidation and amino acid
621
transport in membrane vesicles from Bacillus subtilis. J. Bacteriol. 121:807-813. 2. Carls, R. A., and R. S. Hanson. 1971. Isolation and characterization of tricarboxylic acid cycle mutants of Bacillus subtilis. J. Bacteriol. 106:848-855. 3. Dubler, R. E., W. A. Toscano, Jr., and R. A. Hartline. 1974. Transport of succinate by Pseudomonas putida. Arch. Biochem. Biophys. 160:422-429. 4. Eagon, R. G., and L. S. Wilkerson. 1972. A potassiumdependent citric acid transport system in Aerobacter aerogenes. Biochem. Biophys. Res. Commun. 46:1944-1950. 5. Fournier, R. E., M. N. McKillen, A. B. Pardee, and K. Willecke. 1972. Transport of dicarboxylic acids in Bacillus subtilis. Inducible uptake of L-malate. J. Biol. Chem. 247:5587-5595. 6. Ghei, Om. K., and W. W. Kay. 1972. A dicarboxylic acid transport system in B. subtilis. FEBS Lett. 20:137140. 7. Ghei, Om. K., and W. W. Kay. 1973. Properties of an inducible C4-dicarboxylic acid transport system in Bacillus subtilis. J. Bacteriol. 114:65-79. 8. Holden, J. T., 0. Hild, Y. L. W. Leung, and G. Rouser. 1970. Reduced lipid content as the basis for defective amino accumulation capacity in pantothenate- and biotin-deficient Lactobacillus plantarum. Biochem. Biophys. Res. Commun. 40:123-128. 9. Imai, K., T. Iijima, and T. Hasegawa. 1973. Transport of dicarboxylic acids in Salmonella typhimurium. J. Bacteriol. 114:961-965. 10. Kaback, H. R. 1970. Preparation and characterization of bacterial membranes, p. 99-120. In W. B. Jakoby (ed.), Methods in enzymology, vol. 22. Academic Press Inc., New York. 11. Kaback, H. R. 1974. Transport in bacterial membrane vesicles. Science 186:882-892. 12. Kaback, H. R., and J. S. Hong. 1973. Membranes and transport. C.R.C. Crit. Rev. Microbiol. 2:333-376. 13. Kay, W. W., and H. L. Kornberg. 1971. The uptake of C4-dicarboxylic acids by Escherichia coli. Eur. J. Biochem. 18:274-281. 14. Konings, W. N. 1975. Localization of membrane proteins in membrane vesicles ofBacillus subtilis. Arch. Biochem. Biophys. 167:570-575. 15. Konings, W. N., E. M. Barnes, Jr., 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. 16. 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. 17. 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. 18. Konings, W. N., and E. Freese. 1971. L-serine transport in membrane vesicles of B. subtilis energized by NADH or reduced phenazine methosulfate. FEBS Lett. 14:65-68. 19. Konings, W. N., and E. Freese. 1972. Amino acid transport in membrane vesicles ofBacillus subtilis. J. Biol. Chem. 247:2408-2418. 20. Lawford, H. G., and G. R. Williams. 1971. The transport of citrate and other tricarboxylic acids in two species of Pseudomonas. Biochem. J. 123:571-577. 21. Lo, T. C. Y., M. K. Rayman, and B. D. Sanwal. 1972. Transport of succinate in Escherichia coli. Biochemical and genetic studies of transport in whole cells. J. Biol. Chem. 247:6323-6331. 22. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.
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J. BACTERIOL. Bacteriol. 104:826-833. 28. Sachan, D. S., and J. R. Stern. 1971. Studies of citrate transport in Aerobacter aerogenes: binding of citrate by a membrane bound oxalo acetate dicarboxylase. Biochem. Biophys. Res. Commun. 45:402-408. 29. Thomas, C. L., H. Weissbach, and H. R. Kaback. 1973. Further studies on metabolism of phosphatidic acid of isolated E. coli membrane vesicles. Arch. Biochem. Biophys. 150:797-806. 30. Ting, J. P., and W. M. Dugger, Jr., 1965. Separation and detection of organic acids on silicagel. Anal. Biochem. 12:571-578. 31. Willecke, K., and R. Lange. 1973. C4-dicarboxylate transport in Bacillus subtilis studied with 3-fluoro-Lerythro-malate as a substrate. J. Bacteriol. 117:373378. 32. Willecke, K., and A. B. Pardee. 1971. Inducible transport of citrate in a Gram-positive organism Bacillus subtilis.J. Biol. Chem. 246:1032-1040.
