©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Membrane Potential-generating Transport of Citrate and Malate Catalyzed by CitP of Leuconostoc mesenteroides(*)

(Received for publication, June 5, 1995; and in revised form, August 17, 1995)

Claire Marty-Teysset (1) (2) Juke S. Lolkema (1)(§) Philippe Schmitt (2) Charles Divies (2) Wil N. Konings (1)

From the  (1)Department of Microbiology, Groningen Biotechnology and Biomolecular Sciences Institute, University of Groningen, 9751NN Haren, The Netherlands and the (2)Departement de Microbiologie-Biotechnologie, École Nationale Supérieure de Biologie Appliquee à la Nutrition et à l'Alimentation, Université de Bourgogne, Esplanade Erasme, Campus Universitaire Montmuzard, 21000 Dijon, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Citrate uptake in Leuconostoc mesenteroides subsp. mesenteroides 19D is catalyzed by a secondary citrate carrier (CitP). The kinetics and mechanism of CitP were investigated in membrane vesicles of L. mesenteroides. The transporter is induced by the presence of citrate in the medium and transports both citrate and malate. In spite of sequence homology to the Na-dependent citrate carrier of Klebsiella pneumoniae, CitP is not Na- dependent, nor is CitP Mg-dependent. The pH gradient (DeltapH) is a driving force for citrate and malate uptake into the membrane vesicles, whereas the membrane potential (Delta) counteracts transport. An inverted membrane potential (inside positive) generated by thiocyanide diffusion can drive citrate and malate uptake in membrane vesicles. Analysis of the forces involved showed that a single unit of negative charge is translocated during transport. Kinetic analysis of citrate counterflow at different pH values indicated that CitP transports the dianionic form of citrate (Hcit) with an affinity constant of 20 µM. It is concluded that CitP catalyzes Hcit/H symport. Translocation of negative charge into the cell during citrate metabolism results in the generation of a membrane potential that contributes to the protonmotive force across the cytoplasmic membrane, i.e. citrate metabolism in L. mesenteroides generates metabolic energy. Efficient exchange of citrate and D-lactate, a product of citrate/carbohydrate co-metabolism, is observed, suggesting that under physiological conditions, CitP may function as an electrogenic precursor/product exchanger rather than a symporter. The mechanism and energetic consequences of citrate uptake are similar to malate uptake in lactic acid bacteria.


INTRODUCTION

Citrate utilization in bacteria is mostly mediated by cation-dependent transport systems. Na-dependent citrate transport has been described in Salmonella typhimurium and Klebsiella pneumoniae. The primary sequences of the genes coding for the transport proteins in these two organisms, CitC and CitS, respectively, are highly identical (van der Rest et al., 1992a; Ishiguro et al., 1992). CitS of K. pneumoniae transports citrate in symport with two sodium ions and one proton (van der Rest et al., 1992b; Lolkema et al., 1994a). A second citrate carrier from K. pneumoniae coded by the citH gene, which is not homologous to the citS gene, catalyzes citrate transport in symport with protons (van der Rest et al., 1991). A similar claim has been made for the mechanism for citrate uptake in atypical Escherichia coli cit strains (Reynolds and Silver, 1983). In Bacillus subtilis, citrate transport was found to be coupled, in addition to protons, to divalent metal ions, with a preference for Mg (Bergsma and Konings, 1983). Citrate uptake by these secondary carriers is driven by the electrochemical cation gradients (i.e. the protonmotive force or sodium ion-motive force) that are maintained across the cytoplasmic membrane.

In lactic acid bacteria, the ability to transport citrate is plasmid-encoded. In Leuconostoc mesenteroides subsp. mesenteroides, the gene coding for the citrate transporter (citP) is localized on a 22-kb (^1)plasmid (Lin et al., 1991), and the base sequence is 99% identical to similar citP genes of Lactococcus lactis and Leuconostoc lactis (David et al., 1990). (^2)The CitP proteins are homologous to the sodium-dependent citrate transporter of K. pneumoniae (CitS) with 30% primary sequence identity (van der Rest et al., 1992a). Uptake studies in membrane vesicles derived from E. coli cells expressing CitP of Lactococcus lactis showed protonmotive force-driven uptake of citrate, suggesting an electrogenic proton symport mechanism (David et al., 1990). In contradiction to this, studies in whole cells of Lactococcus lactis demonstrated that citrate uptake was associated with the generation of a protonmotive force (Hugenholtz et al., 1993). It was suggested that divalent anionic citrate (Hcit) was taken up in exchange with one of the products of citrate metabolism, monovalent acetate or pyruvate. Moreover, in Leuconostoc oenos, citrate transport was shown to be catalyzed via a membrane potential-generating H(2)cit uniport mechanism (Ramos et al., 1994). The gene coding for the citrate permease in L. oenos is not known. The latter two observations place the citrate carriers of lactic acid bacteria in the class of secondary transporters that generate a membrane potential by translocating negative charge into the cell. Such transporters are involved in secondary metabolic energy-generating pathways that generate a protonmotive force as a result of an electrogenic transport step and proton consumption in cytoplasmic metabolic steps (for a recent review, see Poolman(1993) and Konings et al. (1995)). Malate fermentation in lactic acid bacteria is a well known example. Internalized malate is decarboxylated by malolactic enzyme to yield lactate and carbon dioxide. The reaction requires a proton, and consequently, a pH gradient is generated. In Lactococcus lactis, uptake of malate and excretion of lactate are catalyzed in a single step by a malate transporter (precursor/product exchange). Divalent malate is exchanged for monovalent lactate, and a membrane potential (inside negative) is formed (Poolman et al., 1991). In L. oenos, the membrane potential is generated by a monovalent malate uniport mechanism as described for citrate above. The product lactate leaves the cell by passive diffusion in its protonated state (Salema et al., 1994). Similar decarboxylation-driven pathways have been described for oxalate/formate exchange in Oxalobacter formigenes (Anatharam et al., 1989) and histidine/histamine exchange in Lactobacillus buchneri (Molenaar et al., 1993).

This report shows a detailed study of the mechanism of citrate transport catalyzed by CitP of L. mesenteroides. The citrate carrier transports both malate and citrate by a mechanism that translocates net negative charge across the membrane and is able to catalyze heterologous exchange between citrate and lactate. A model is presented for pmf generation by citrate metabolism based upon the results.


MATERIALS AND METHODS

Bacteria and Growth Conditions

L. mesenteroides subsp. mesenteroides 19D was obtained from the collection of Institut National de la Recherche Agronomique (Jouy en Josas, France). L. mesenteroides 19D cit and cit were selected as blue and white colonies, respectively, on Kempler and McKay plates (Kempler and McKay, 1980). Cells were grown at 28 °C in MRS broth, pH 6.4 (De Man et al., 1960), containing ammonium citrate (2 g/liter), glucose (20 g/liter), and acetate (5 g/liter). Cells were harvested at the end of the exponential growth phase (OD = 1), washed once, and resuspended in 50 mM potassium phosphate, pH 7.0, at an OD of 500 and subsequently rapidly frozen in liquid nitrogen until use.

Preparation of Right-side-out Membrane Vesicles

Membrane vesicles of L. mesenteroides were prepared by the osmotic shock lysis procedure essentially as described previously by Otto et al.(1982). Lysis was improved by treating the cells with 18 mg/ml lysozyme (muramidase, Merck) in 100 mM potassium phosphate, 10 mM MgSO(4), pH 7.0, for 1 h at 30 °C. Cells were lyzed by rapid addition of 0.75 M K(2)SO(4). Nucleic acids released from the cells were eliminated by treatment with 250 µg/ml DNase (deoxyribonuclease I from bovine pancreas, Sigma) and 250 µg/ml RNase (ribonuclease A from bovine pancreas, Sigma) in 100 mM potassium phosphate, 10 mM MgSO(4), pH 7.0, for 30 min at 30 °C. After incubation with 25 mM EDTA for 10 min at 30 °C and addition of 10 mM MgSO(4), lyzed cells were collected by centrifugation (48,200 times g for 30 min at 4 °C) and resuspended in 50 mM phosphate buffer, pH 7.0, without MgSO(4). Cells and debris were removed by a low spin (750 times g for 1 h), after which the vesicles were collected by spinning the supernatant at 48,200 times g for 30 min at 4 °C and subsequently resuspended in 50 mM potassium phosphate, pH 6.0. Residual whole cells in the vesicle preparation were eliminated by extrusion through a 400-nm pore size polycarbonate filter (Avestin, Inc.). Membrane vesicles were rapidly frozen and stored in liquid nitrogen. The protein concentration was determined by the method of Lowry et al. (1951).

Preparation of Hybrid Membranes

L-alpha-Phosphatidylethanolamine was purified from 1 g of E. coli extract (Avanti Polar Lipids, Inc.) by successive washing with acetone and diethyl ether. L-alpha-Phosphatidylethanolamine concentration was determined as described by Driessen et al.(1991). Cytochrome c oxidase isolated from beef heart mitochondria was reconstituted into liposomes by detergent dialysis. The liposomes consisted of purified E. coli lipids and egg phosphatidylcholine in a 3:1 ratio (Driessen et al., 1985). Proteoliposomes containing cytochrome c oxidase (COVs) were fused with membrane vesicles of L. mesenteroides (1 mg of protein/10 mg of lipids) by freezing in liquid nitrogen followed by slow thawing at room temperature (Driessen et al., 1985). The resulting hybrid membranes were made unilamellar by sonication (eight cycles of 15 s on and 45 s off, amplitude of 4-6 µm) or by extrusion using successively 400- and 200-nm pore size polycarbonate filters (Mayer et al., 1986). Membrane vesicles were fused with liposomes lacking cytochrome c oxidase by the same procedure. Hybrid membranes were concentrated by centrifugation (250,000 times g for 45 min at 4 °C).

Transport Assays

All transport studies were performed in 50 mM potassium phosphate, pH 6, at a membrane protein concentration of 0.25-0.3 mg/ml, unless otherwise stated, and at 30 °C. Valinomycin and nigericin were used at final concentrations of 1 and 0.5 µM, respectively.

pmf-driven Uptake

The experiments were performed under a flow of water-saturated air. Membrane vesicles fused with COVs were incubated for 10 min in the presence or absence of ionophores. The hybrid membranes were energized by addition of 200 µMN,N,N`,N`-tetramethyl-p-phenylenediamine (TMPD), 20 µM cytochrome c (from horse heart, Sigma), and 10 mM potassium ascorbate. After incubation for 1 min, the radiolabeled substrates were added. Samples (100 µl) were taken at subsequent time points, transferred into 2 ml of ice-cold 0.1 mM LiCl to stop the reaction, and filtered through 0.45-µm pore size cellulose-nitrate filters (Schleicher & Schuell). Filters were rinsed with 2 ml of ice-cold 0.1 mM LiCl and transferred to scintillation vials, and the internalized radioactivity was determined. [1,5-^14C]Citrate, L-[U-^14C]leucine, and L-[U-^14C]malate were used at final concentrations of 4.5, 1.6, and 7.8 µM, respectively. Control membranes were incubated with radiolabeled substrate in the absence of potassium ascorbate and without aeration.

Artificial Gradient-driven Uptake

Membrane vesicles fused with liposomes devoid of cytochrome c oxidase were preincubated at 30 °C for 10 min in 50 mM potassium phosphate, pH 7, containing ionophores and the radiolabeled substrates. An artificial pH gradient was generated by adding a small aliquot of 0.2 N H(2)SO(4), resulting in a pH drop of 1 unit. Uptake assays were performed as described above.

The generation of an inverted membrane potential (Delta) (positive inside) was obtained by a thiocyanate diffusion potential. Hybrid membranes were prepared as described above and concentrated in 50 mM potassium phosphate, pH 6, containing 100 mM potassium thiocyanate. At the zero time point, concentrated hybrid membranes were diluted 100-fold into the same buffer without SCN containing 4.5 µM [1,5-^14C]citrate or 7.8 µML-[U-^14C]malate.

Exchange Measurements

Hybrid membranes obtained by fusion of membrane vesicles of L. mesenteroides with liposomes lacking cytochrome c oxidase were loaded with 5 mM citrate as described above. Concentrated hybrid membranes were incubated with 5 µM [1,5-^14C]citrate for 30 min at 20 °C. After incubation with valinomycin and nigericin, hybrid membranes were diluted 100-fold into buffer containing 5 mM unlabeled substrates.

Measurement of Delta and DeltapH

The membrane potential was determined from the distribution of the lipophilic cation tetraphenylphosphonium using a tetraphenylphosphonium-selective electrode (Lolkema et al., 1982). The membrane potential was calculated from the Nernst equation after correction for concentration-dependent binding of the probe to the membrane. The estimated binding constant was 195. The specific internal volume of the hybrid membranes was assumed to be 8 µl/mg of protein (Driessen et al., 1985). The transmembrane pH gradient was measured with the fluorescent membrane-impermeable dye 8-hydroxy-1,3,6-pyrene trisulfonate (pyranine) as described previously by Damiano et al.(1984). Conditions for the measurements were similar to those described for the transport experiment.

Chemicals

[1,5-^14C]Citrate (110 mCi/mmol), L-[U-^14C]leucine (319 mCi/mmol), [^14C]potassium thiocyanate (55 mCi/mmol), and L-[U-^14C]malate (48 mCi/mmol) were obtained from Amersham International (Buckinghamshire, United Kingdom). Pyranine was obtained from Eastman Kodak Co.


RESULTS

Substrate Specificity

Citrate (Fig. 1A) and malate (Fig. 1B) transport was studied in membrane vesicles derived from L. mesenteroides cit cells grown in the presence or absence of citrate and from L. mesenteroides cit cells lacking the 22-kb plasmid containing the citP gene, grown in the presence of citrate. Membrane vesicles were fused with proteoliposomes containing cytochrome c oxidase as a protonmotive force-generating system. In these hybrid membranes, a pmf (inside negative and alkaline relative to the outside) is generated in the presence of the electron donor system (cytochrome c/TMPD/potassium ascorbate). The highest level of citrate and malate accumulation was observed in hybrid membranes derived from cit cells grown in the presence of citrate. For both substrates, the rates of uptake were significantly lower in membranes derived from cit cells grown in the absence of citrate. As expected, the L. mesenteroides cit strain did not take up citrate, but also malate uptake was not observed. The integrity of the different membrane preparations was checked by the accumulation of leucine (Fig. 1C), which is catalyzed by a secondary pmf-driven transport system (Winters et al., 1991). These results show that both citrate and malate uptake systems are encoded by a gene located on the 22-kb plasmid and are induced by citrate, which is consistent with a single carrier for both substrates. This suggestion was substantiated by demonstrating heterologous citrate/malate exchange. Internally accumulated [^14C]citrate (Fig. 2A) and [^14C]malate (Fig. 2B) could be chased by addition of an excess of both unlabeled citrate and unlabeled malate. To exclude the possibility that efflux of the radiolabeled substrates in these experiments would be caused by dissipation of the pmf instead of heterologous exchange, the same concentrations of citrate and malate were added to membrane vesicles that had accumulated [^14C]leucine. No release of leucine was observed (Fig. 2C). Furthermore, uptake of citrate was inhibited in the presence of 5 mM unlabeled malate, whereas leucine uptake was not affected by 5 mM citrate or malate (data not shown). Taken together, these results show that the citrate carrier of L. mesenteroides transports both citrate and malate.


Figure 1: Inducibility and specificity of CitP of L. mesenteroides 19D. Shown is citrate (A), malate (B), and leucine (C) uptake in membrane vesicles of L. mesenteroides fused with COVs. Membrane vesicles were prepared from L. mesenteroides cit cells grown in the presence (down triangle) or absence (circle) of citrate and from L. mesenteroides cit cells grown in the presence of citrate (bullet). Transport assays were performed in the presence of 1 µM valinomycin. Concentrations were 4.5 µM citrate (A), 7.8 µM malate (B), and 1.6 µM leucine (C). , not energized.




Figure 2: Heterologous exchange catalyzed by CitP. Shown is the exchange of [^14C]citrate (A) and [^14C]malate (B) with unlabeled citrate and malate. A control experiment with leucine is also shown (C). Membranes of L. mesenteroides fused with COVs were allowed to accumulate [^14C]citrate (4.5 µM), [^14C]malate (7.8 µM), and [^14C]leucine (1.6 µM) in the presence (circle) or absence (box) of the electron donor system cytochrome c/TMPD/ascorbate and 1 µM valinomycin. At the arrows, unlabeled citrate (down triangle) or malate (bullet) was added. Citrate was added at a concentration of 1 mM, and malate at 5 mM (A and C) or 1 mM (B).



Co-ion Specificity

In various bacteria, citrate is transported in symport with cations such as H, Na, or Mg. The citP gene is homologous to citS of K. pneumoniae, which is a Na/citrate symporter (van der Rest et al., 1992a). The nature of ions symported with citrate by CitP was studied in membrane vesicles fused with proteoliposomes. Citrate uptake was measured in the presence of varying concentrations of sodium or magnesium ions. Precautions were taken to avoid Na ion contaminations by using special potassium phosphate buffers that contained low concentrations of sodium. Uptake of citrate was not affected by the absence or presence of Na up to 25 mM. Magnesium ions form a soluble complex with citrate. Uptake of citrate was increasingly inhibited by increasing magnesium concentrations, showing that citrate is not transported in a complex with magnesium (data not shown). It is concluded that neither Mg nor Na is cotransported with citrate by CitP of L. mesenteroides.

Driving Force for Uptake of Citrate and Malate

The protonmotive force generated by cytochrome c oxidation is composed of a membrane potential (Delta) and a pH gradient (DeltapH). Uptake of citrate and malate (Fig. 3) and the magnitude of DeltapH and Delta (Table 1) were measured under the same experimental conditions. The role of each component of the pmf in driving citrate uptake was investigated in more detail by manipulating DeltapH and Delta with the ionophores nigericin and valinomycin. In the absence of ionophores, when the pmf is highest (-125 mV), a low but significant citrate accumulation was observed. In the presence of nigericin, when the pmf consists solely of a membrane potential of -90 mV, no uptake of citrate occurred, indicating that the membrane potential is not a driving force for citrate transport. On the other hand, in the presence of valinomycin, when the pmf is only composed of a pH gradient of -70 mV, a strong stimulation of citrate uptake was observed. In the lower portion of Table 1, the driving force on citrate transport was calculated from the membrane potential and pH gradient assuming different proton/citrate transport stoichiometries (first column). The forces correlate with the equilibrium accumulation levels for citrate that are obtained with the different transport mechanisms. The final accumulation levels of citrate in Fig. 3A correlate with forces of about -30 mV in the absence of ionophores, -100 mV in the presence of valinomycin and a zero or positive force in the presence of nigericin. These steady-state values fit qualitatively best with the Hcit/H mechanism. The translocation of net negative charge by the carrier explains the poor uptake in the absence of ionophores when the driving force is low due to the counteracting membrane potential. Only after dissipation of the membrane potential was a high citrate uptake observed. Uptake of malate catalyzed by CitP was found to be qualitatively similar to citrate (Fig. 3B). Malate transport via CitP also is driven by DeltapH, while Delta acts as a counteractive force. The effect of Delta on citrate and malate transport was further investigated with artificial gradients.


Figure 3: Effect of ionophores on citrate (A) and malate (B) uptake in the presence of a protonmotive force. Citrate (4.5 µM) and malate (7.8 µM) uptake by membrane vesicles fused with COVs was assayed in the presence of no ionophores (circle), valinomycin (bullet), and nigericin (down triangle). The electron donor system cytochrome c/TMPD/potassium ascorbate was present in all samples. Control membranes were incubated with labeled substrates in the absence of potassium ascorbate and without aeration with water-saturated air ().





If indeed Delta (inside negative) acts as a counterforce for citrate uptake, Delta of opposite polarity (inside positive) should be able to drive citrate uptake. An inverted membrane potential was generated in membrane vesicles of L. mesenteroides fused with liposomes lacking cytochrome c oxidase by the ``pH jump'' technique (Maloney and Hansen, 1982). The external pH was rapidly dropped from 7.0 to 6.0 by adding sulfuric acid, which created a pH gradient (inside alkaline). Since the membrane is less permeable to sulfate ions than to protons, the diffusion of protons through the membrane generates a diffusion potential (positive inside). At equilibrium, the pmf equals zero. The formation of inverted Delta is evidenced by uptake of the permeable anion SCN (Fig. 4B, circle). Delta = 62 mV could be estimated from the level of SCN accumulation, which is in agreement with a pH jump of 1 unit. Under these conditions, no leucine uptake was observed, which is consistent with a zero pmf (Fig. 4A, circle). Only after quenching of the inverted membrane potential by valinomycin was DeltapH-driven leucine accumulation observed (Fig. 4A, bullet). The highest level of citrate accumulation was observed when both a pH gradient (inside alkaline) and an inverted membrane potential were present (Fig. 4C, circle). Dissipation of the inverted membrane potential by valinomycin results in a strong reduction of citrate uptake (Fig. 4C, bullet).


Figure 4: Leucine (A), SCN (B), and citrate (C) uptake driven by a pH jump. Membrane vesicles fused with liposomes were equilibrated with 4.5 µM citrate, 1.6 µM leucine, 2.3 µM thiocyanate in 50 mM potassium phosphate, pH 7.0. Uptake was initiated by addition of sulfuric acid (0.5 N), which resulted in a drop of the external pH from 7 to 6, thereby generating DeltapH. Experiments were performed in the absence (circle) or presence (bullet) of 1 µM valinomycin. down triangle, no acid was added.



The complementary experiment, in which an artificial thiocyanate diffusion potential was generated, is shown in Fig. 5. Membrane vesicles of L. mesenteroides fused with liposomes were loaded with potassium thiocyanate. Thiocyanate is negatively charged and diffuses passively out of the membranes upon dilution, generating an inverted membrane potential (inside positive). In response to Delta, DeltapH (inside alkaline) will develop by proton diffusion out of the hybrid membranes. Again, the highest level of citrate uptake was observed in the presence of inverted Delta and DeltapH of normal polarity (Fig. 5A, circle). The same result was obtained with malate as the substrate (Fig. 5B, circle). When the pH gradient was dissipated by addition of nigericin, still significant uptake of citrate and malate occurred, showing that both substrates are accumulated when an inverted membrane potential is the only gradient across the membrane (Fig. 5, bullet). These results confirm that CitP translocates citrate and malate with net negative charge across the membrane. Under physiological conditions, the membrane potential is a counteractive force for citrate and malate accumulation.


Figure 5: Transport of citrate (A) and malate (B) driven by an inverted membrane potential. Hybrid membranes loaded with 100 mM SCN were incubated for 10 min at 30 °C in the presence of nigericin (bullet) and nigericin and valinomycin (down triangle) or without further addition (circle). Uptake was initiated by 100-fold dilution of the membranes into buffer without thiocyanate containing citrate (4.5 µM; A) and malate (7.8 µM; B).



Kinetic Characterization of Citrate Transport

Counterflow experiments were used to kinetically characterize citrate uptake. In the pH range 5-6.5, the maximal rate is rather constant at 40 nmol min mg. Also, the affinity constant of CitP for citrate did not change a lot and appears to be quite high, e.g. at pH 5.0, K(m) = 42 µM (Table 2). To determine the species of citrate transported by CitP, affinity constants for citrate were calculated at the different pH values for the different citrate species (Table 2). The K(m) of the transport system was found to be almost constant in the pH range 5-6.5 for the monoprotonated form of citrate (Hcit). In contrast, the affinity constants calculated for cit increase and for H(2)cit and H(3)cit decrease drastically and systematically in this pH range. Assuming one of the ionic species is transported by the carrier, these results indicate that Hcit is recognized and transported by the citrate carrier of L. mesenteroides.



Exchange with Metabolic Products

Transporters catalyzing translocation of net negative charge into the cell play a crucial role in secondary metabolic energy generation. They function either as uniporters or as exchangers that couple the uptake of a substrate to the excretion of a metabolic end product (precursor/product exchange). Typically, the latter class of transporters catalyzes exchange faster than unidirectional transport (Salema et al., 1994). Hybrid membranes were allowed to accumulate citrate driven by a pH gradient, after which nigericin (efflux) or nigericin plus unlabeled citrate (exchange) were added. Under these conditions, CitP performed homologous exchange faster than efflux (Fig. 6), suggesting that under physiological conditions, the carrier may function as an exchanger rather than a unidirectional transporter. The same technique cannot be used to show exchange with acetate or lactate since addition of these substrates at micromolar concentrations collapses the pH gradient by rapid diffusion of the protonated species across the membrane. To demonstrate heterologous exchange with metabolic end products, hybrid membranes were preloaded with [^14C]citrate followed by dilution into medium containing the weak acids. Ionophores were present to prevent the generation of DeltapH by diffusion of protonated lactate or acetate. The experiments were performed at 20 °C to slow down the processes. An additional advantage of the lower temperature is that it affects efflux more strongly than exchange (Fig. 7, compare circle and bullet). A slow release of citrate was observed in the absence of a counter-substrate (efflux) and in the presence of external acetate. Interestingly, exchange between citrate and lactate was observed. D-Lactate consistently resulted in faster exchange than L-lactate, suggesting higher affinity of the carrier for D-lactate than for L-lactate. These results show that, in addition to citrate and malate, CitP transports L- and D-lactate.


Figure 6: Efflux versus homologous exchange. Membrane vesicles fused with COVs were allowed to accumulate citrate in the presence of the electron donor system cytochrome c/TMPD/ascorbate and 1 µM valinomycin (circle). At the arrow, 0.5 µM nigericin without (bullet; efflux) or with (down triangle; exchange) 1 mM unlabeled citrate was added. , not energized.




Figure 7: Exchange of citrate and products of citrate metabolism. Membrane vesicles fused with liposomes were preloaded with 5 mM citrate and subsequently diluted 100-fold into buffer containing 5 mM citrate (bullet), malate (box), L-lactate (), D-lactate (down triangle), acetate (), and no further additions (circle). Valinomycin and nigericin were present at 1 and 0.5 µM, respectively. The final protein concentration in the assay mixture was 112 µg/ml.




DISCUSSION

citP genes coding for citrate transporters have been cloned from Lactococcus lactis, Leuconostoc lactis, and L. mesenteroides and were found to be virtually the same. The citP genes are located on different endogenous plasmids in the three organisms. Regulation of expression may differ among the organisms. The Lactococcus lactis citP gene is located on a 5.6-kb plasmid. Magni et al.(1994) demonstrated that the transcript of citP and citrate uptake are independent of the presence of citrate in the growth medium, i.e. CitP is constitutively expressed. In L. mesenteroides, citP is located on a much bigger plasmid of 22 kb. The present results show that citrate acts as an inducer of CitP expression. Membrane vesicles from cells of L. mesenteroides grown in the presence of citrate were significantly more active in citrate uptake than those from cells grown in the absence of citrate (Fig. 1).

The primary sequence of CitP is 30% identical to the Na-dependent citrate carrier of K. pneumoniae (CitS). Hydropathy profiling reveals an even stronger structural similarity between the two proteins (van der Rest et al., 1992a; Lolkema et al., 1994b). Translocation of citrate by CitS is obligatory coupled to the translocation of Na ions (Lolkema et al., 1994a). However, CitP is not Na ion-dependent. As observed in other families of homologous secondary transporters, structural similarity does not necessarily correlate with cation specificity. Surprisingly, CitP not only catalyzes citrate transport, but also malate and lactate transport. Malate transport by CitP could be demonstrated by the correlation between the ability of membrane vesicles to accumulate malate and the expression of CitP (Fig. 1) and by heterologous exchange between citrate and malate (Fig. 2). Transport of lactate via CitP was demonstrated by its accelerating effect on citrate efflux, which is explained as citrate/lactate exchange (Fig. 7). Both D- and L-lactate showed the accelerating effect, indicating that the carrier is not stereoselective. Citrate (, i), malate (ii), and lactate (iii) are structurally related, suggesting that CitP is a general carrier for hydroxycarboxylic acids presenting the motive R(1)R(2)COHCOOH.

The following results indicate that the citrate carrier of L. mesenteroides catalyzes translocation of net negative charge. (a) Citrate uptake driven by the protonmotive force is largely stimulated when the membrane potential component is dissipated; (b) the highest accumulation level of citrate is observed in the presence of a pH gradient of physiological polarity and a membrane potential of opposite polarity; and (c) an inverted membrane potential by itself can drive the accumulation of citrate into the membranes. The analysis of the forces (see Fig. 3and Table 1) shows that a single unit of charge is translocated per catalytic cycle, i.e. H(2)cit, Hcit/H, or cit/2H. Translocation of 2 units of charge would result in an outwardly directed force on citrate in the absence of ionophores, which is not observed. Kinetic analysis of citrate transport in membrane vesicles of L. mesenteroides shows that the dianionic form of citrate (Hcit) is recognized by CitP (Table 2). Therefore, CitP catalyzes symport of Hcit with 1H. The characteristics of malate transport were similar to those observed for citrate, i.e. CitP catalyzes malate/H symport. Divalent anionic citrate is also the species translocated by the Na- and H-dependent citrate carriers of K. pneumoniae. However, these carriers translocate positive charge across the membrane, i.e. CitH catalyzes Hcit/3H symport, and CitS catalyzes Hcit/2Na/H symport. In L. oenos, citrate transport is catalyzed by a uniporter that recognizes H(2)cit (Ramos et al., 1994). These observations suggest that the form of citrate that is recognized by the transporter is determined by the pH of the medium at which the organism normally grows. L. oenos grows in wine at low pH, where H(2)cit is the predominant species, whereas L. mesenteroides and K. pneumoniae grow at more neutral pH values, where Hcit is the most abundant protonation state.

Transport of citrate by CitP results in the generation of a membrane potential (inside negative) in L. mesenteroides. Inside the cell, citrate is split in oxalacetate and acetate by citrate lyase. Subsequently, oxalacetate is decarboxylated, yielding pyruvate and carbon dioxide, in a reaction that consumes a scalar proton from the cytoplasm. The overall result of citrate metabolism is the generation of a protonmotive force in a similar way to that observed during malate fermentation in lactic acid bacteria. There appears to be a remarkable parallel between citrate and malate uptake in these bacteria. In L. oenos, both substrates are transported by a uniport mechanism translocating net negative charge into the cell as H(2)cit and monovalent malate (Ramos et al., 1994; Salema et al., 1994). In Lactococcus lactis, the malate transporter catalyzes in vitro negative charge translocation, most likely as malate/H symport. CitP of L. mesenteroides catalyzes symport of both Hcit and malate with one proton. However, in vivo, the malate carrier of Lactococcus lactis catalyzes exchange of divalent malate with monovalent lactate, which is the product of malate decarboxylation (precursor/product exchange). Exchange experiments showed that CitP has affinity for lactate as well (Fig. 7), and therefore, we hypothesize that in vivo CitP also functions as an electrogenic Hcit/lactate exchanger. In Leuconostoc species, lactate is a product of citrate/carbohydrate co-metabolism. Pyruvate formed from oxalacetate decarboxylation functions as an electron sink for the reducing equivalents generated by glycolysis and is stoichiometrically reduced to D-lactate (Starrenburg and Hugenholtz, 1991; Schmitt et al., 1992). Currently, we are investigating the in vivo mechanism of protonmotive force generation by citrate metabolism in L. mesenteroides. Electrogenic citrate/lactate exchange catalyzed by CitP would make the analogy with malate metabolism complete. Already, it is clear that the citrate and malate carriers in lactic acid bacteria form a closely related family of secondary transporters. Recently, the mal operon of Lactococcus lactis containing the structural gene coding for the malate permease (malP) was sequenced. The primary sequences of MalP and CitP were found to be 50% identical. (^3)


FOOTNOTES

*
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§
To whom correspondence should be addressed: Dept. of Microbiology, University of Groningen, Kerklaan 30, 9751NN Haren, The Netherlands. Tel.: 31-50-632155; Fax: 31-50-632154; j.s.lolkema@biol.rug.nl.

(^1)
The abbreviations used are: kb, kilobase(s); cit, citrate; Hcit, divalent anionic citrate; H(2)cit, monovalent anionic citrate; pmf, protonmotive force; COVs, proteoliposomes containing cytochrome c oxidase; TMPD, N,N,N`,N`-tetramethyl-p-phenylenediamine.

(^2)
M. E. Lhotte, J. Guzzo, V. Dartois, H. Prévost, and C. Divies, submitted for publication.

(^3)
S. Dequin, personal communication.


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