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J Bacteriol. Oct 2000; 182(20): 5757–5764.
PMCID: PMC94697

Transport of C4-Dicarboxylates in Wolinella succinogenes

Abstract

C4-dicarboxylate transport is a prerequisite for anaerobic respiration with fumarate in Wolinella succinogenes, since the substrate site of fumarate reductase is oriented towards the cytoplasmic side of the membrane. W. succinogenes was found to transport C4-dicarboxylates (fumarate, succinate, malate, and aspartate) across the cytoplasmic membrane by antiport and uniport mechanisms. The electrogenic uniport resulted in dicarboxylate accumulation driven by anaerobic respiration. The molar ratio of internal to external dicarboxylate concentration was up to 103. The dicarboxylate antiport was either electrogenic or electroneutral. The electroneutral antiport required the presence of internal Na+, whereas the electrogenic antiport also operated in the absence of Na+. In the absence of Na+, no electrochemical proton potential (Δp) was measured across the membrane of cells catalyzing fumarate respiration. This suggests that the proton potential generated by fumarate respiration is dissipated by the concomitant electrogenic dicarboxylate antiport. Three gene loci (dcuA, dcuB, and dctPQM) encoding putative C4-dicarboxylate transporters were identified on the genome of W. succinogenes. The predicted gene products of dcuA and dcuB are similar to the Dcu transporters that are involved in the fumarate respiration of Escherichia coli with external C4-dicarboxylates. The genes dctP, -Q, and -M probably encode a binding-protein-dependent secondary uptake transporter for dicarboxylates. A mutant (DcuA DcuB) of W. succinogenes lacking the intact dcuA and dcuB genes grew by nitrate respiration with succinate as the carbon source but did not grow by fumarate respiration with fumarate, malate, or aspartate as substrates. The DcuA, DcuB, and DctQM mutants grew by fumarate respiration as well as by nitrate respiration with succinate as the carbon source. Cells of the DcuA DcuB mutant performed fumarate respiration without generating a proton potential even in the presence of Na+. This explains why the DcuA DcuB mutant does not grow by fumarate respiration. Growth by fumarate respiration appears to depend on the function of the Na+-dependent, electroneutral dicarboxylate antiport which is catalyzed exclusively by the Dcu transporters. Dicarboxylate transport via the electrogenic uniport is probably catalyzed by the DctPQM transporter and by a fourth, unknown transporter that may also operate as an electrogenic antiporter.

The rumen bacterium Wolinella succinogenes can grow by anaerobic respiration with fumarate as the terminal electron acceptor by the reaction HCO2 + fumarate + H+→ CO2 + succinate (reaction a) or with nitrate as the terminal electron acceptor by the reaction 4HCO2 + NO3 + 6H+→ 4CO2 + NH4+ + 3H2O (reaction b) (4, 7, 49). The electron donor formate can be replaced by H2. An electrochemical proton potential (Δp) is generated across the bacterial membrane by reaction a or b (4, 15, 34). The Δp drives ATP synthesis via an ATP synthase which is integrated in the membrane (5). The Δp generated by fumarate respiration (reaction a) was found to be 0.17 V, and the H+/e ratio was close to 1 (15, 34). Formate dehydrogenase (or hydrogenase) and fumarate reductase are constituents of the electron transport chain catalyzing fumarate respiration (17, 29, 47). The substrate sites of the dehydrogenases are oriented to the bacterial periplasm, whereas that of fumarate reductase faces the cytoplasm (18, 26). Therefore, transport of fumarate and of succinate is involved in fumarate respiration.

In W. succinogenes growing by fumarate respiration (reaction a), fumarate serves as the electron acceptor and as the carbon source (7). Fumarate can be replaced by malate or aspartate, which are converted to fumarate by fumarase or aspartase (35). In W. succinogenes growing by nitrate respiration, succinate, fumarate, malate, or aspartate may serve as the carbon source (4, 22).

Anaerobic growth of Escherichia coli by fumarate respiration with external fumarate, malate, or aspartate was shown to be dependent on the function of at least one of its three Dcu transporters, which consist of only one subunit species (11, 12, 41, 50). Each Dcu transporter catalyzes the electrogenic uptake as well as the electroneutral antiport of C4-dicarboxylates (succinate, fumarate, malate, and aspartate). During aerobic growth of E. coli with C4-dicarboxylates, these compounds are taken up in an electrogenic process catalyzed by the DctA transporter (9, 19). Under the same growth conditions, the electrogenic uptake of C4-dicarboxylates by Rhodobacter capsulatus is catalyzed by the DctPQM transporter, which consists of three different subunits, including a periplasmic binding protein (13).

In this communication, we report on the properties of the C4-dicarboxylate transport catalyzed by W. succinogenes. Furthermore, we report on three gene loci on the W. succinogenes genome that are predicted to encode proteins resembling C4-dicarboxylate transporters of other bacteria, and the corresponding mutants are characterized.

MATERIALS AND METHODS

Bacterial strains, phage, plasmids, and growth conditions.

E. coli strains were grown aerobically at 37°C in NZYM medium (38). Ampicillin (100 mg liter−1), kanamycin (50 mg liter−1), or chloramphenicol (25 mg liter−1) was added to the medium as appropriate. Phage λ-EMBL3 (14) and its derivatives were propagated in E. coli Q358 (24). Plasmids were amplified in E. coli DH5α (Gibco BRL).

W. succinogenes (DSMZ 1740) and the mutants derived therefrom were grown with formate (0.1 M) and fumarate (90 mM) in the anoxic minimal medium described by Kröger et al. (27). For growth with malate or aspartate, fumarate was replaced by 0.1 M l-malate or 0.1 M l-aspartate. The anoxic medium for growth with formate, fumarate, and nitrate contained 50 mM fumarate and 20 mM nitrate. For growth with formate (80 mM) and nitrate (20 mM), the anoxic minimal medium contained 10 mM succinate (32).

Measurement of uptake of labeled succinate.

Cells of W. succinogenes grown in the medium containing nitrate and fumarate were harvested and washed with 50 mM HEPES buffer (pH 7.5). The washed cells were mixed with the same anoxic buffer at 37°C containing [2,3-14C]succinate (0.1 mM; 20 MBq mmol−1), 50 mM sodium formate, and 40 mM nitrate. After various incubation times, samples (0.1 ml) of the suspension (1 g of cell protein liter−1) were centrifuged for 3 min at 10,000 × g. The amount of labeled succinate taken up by the cells was calculated from the radioactivity remaining in the supernatant.

Measurement of the electrical proton potential across the bacterial membrane (ΔΨ).

W. succinogenes cells were washed with an anoxic buffer (50 mM HEPES adjusted to pH 7.5 with KOH) and suspended (0.5 g of cell protein liter−1) in the same buffer containing 1 μM tetraphenylphosphonium (TPP+) at 37°C. The buffer either was saturated with H2 or contained 10 mM formate. Fumarate respiration was initiated by the addition of 2 mM fumarate, and the external TPP+ concentration of the suspension was recorded using a TPP+ electrode. The ΔΨ was calculated from the amount of TPP+ taken up by the cells in the steady state of fumarate respiration and from the corresponding external TPP+ concentration as described by Geisler et al. (15). Wild-type cells were grown with formate and fumarate. The DcuA DcuB mutant was grown with formate and nitrate. The preparation of inverted membrane vesicles of W. succinogenes and the determination of ΔΨ with a tetraphenylboranate electrode was performed as described previously (15, 34). As a control, all of the experiments were also performed after treatment of the cells or vesicles with the protonophore TTFB (4,5,6,7-tetrachloro-2′-trifluoromethyl-benzimidazole) (77 μmol g of protein−1). Under these conditions, ΔΨ values of <10 mV were measured (not shown).

Determination of electron transport activities and cell protein.

Electron transport activities with formate or H2 as the electron donor and fumarate as the acceptor were recorded photometrically under anoxic conditions (47). One unit of enzyme activity was equivalent to the oxidation of 1 μmol of formate or H2 min−1. Cell protein was determined after protein precipitation with trichloroacetic acid using the biuret method with KCN (3).

Genetic techniques.

Standard genetic procedures as well as the isolation of phages and of phage DNA were performed as described by Sambrook et al. (38). DNA was isolated from W. succinogenes by the method of Kaiser and Murray (23). PCR was carried out using the Expand High Fidelity PCR System (Roche) with standard amplification protocols on a Hybaid OmniGene thermocycler. Southern blotting onto nylon membranes (Roche) was performed as described previously (31). Transfer of phage plaques to nitrocellulose membranes (type BA85; Schleicher and Schuell) as well as denaturation and fixation of phage DNA was performed as described previously (38). DNA probes were generated with a PCR DIG Probe Synthesis Kit (Roche), and hybrids were visualized using a DIG Luminescent Detection Kit (Roche). Plasmid DNA or PCR products were purified using Qiagen tips or a PCR purification kit (Qiagen) and were sequenced using BigDye terminator cycle sequencing (Applied Biosystems) with specifically synthesized oligonucleotide primers.

Identification and cloning of W. succinogenes gene loci.

A physical map of the W. succinogenes dcuA, dcuB, and dct loci is shown in Fig. Fig.1.1. The 3′ end of dcuA and the adjacent ansA gene were sequenced previously (33) (EMBL/GenBank/DDBJ accession number X89215). A dcuA probe was generated from the known sequence and used for screening a W. succinogenes genomic library in phage λ-EMBL3 (30). Restriction enzyme digests of the DNAs of several positive phages were cloned in pBR322 (6) for sequencing of the dcuA locus.

FIG. 1
Physical map of the gene loci dcuA (A), dcuB (B), and dct (C) on the genome of W. succinogenes. The plasmids depicted were used for mutant construction. The segments of the plasmids designated by light boxes are identical to the indicated genomic regions. ...

A fragment of W. succinogenes dcuB (662 bp) was amplified by PCR using genomic DNA as a template and two oligonucleotide primers identical to sequences within E. coli dcuB (41) [forward primer, 5′-(483)GTACGGGTCATGTGGTTTACACC-3′; reverse primer, 5′-(1127)TTTCTGCCATCCATGCGATACCG-3′ [position numbers in parentheses refer to the E. coli dcuB gene]). Screening of the W. succinogenes genomic library with a DNA probe hybridizing to this PCR fragment yielded a phage containing the dcuB gene. The sequence of the dcuB locus was obtained with various pBR322 derivatives containing cloned fragments of the phage insert.

Part of the W. succinogenes dctP gene (0.5 kb) was identified unintentionally by sequencing a PCR by-product obtained with genomic DNA. The residual sequence of the dct locus was amplified by inverse PCR, using templates that were obtained by digesting genomic DNA with NarI and religating with T4 DNA ligase. Sequencing of two PCR products completed the sequence of the dctP, -Q, and -M genes and part of the adjacent open reading frame orfN.

Construction of plasmids and of W. succinogenes mutants.

Mutants of W. succinogenes were constructed via double homologous recombination as outlined in Fig. Fig.1.1. Plasmid pdcuA::kan was constructed from a pBluescript SK(+) derivative containing the 1.3-kb HindIII fragment indicated in Fig. Fig.1A1A (33). This plasmid was linearized by NruI restriction and blunt-end ligated with the kanamycin resistance gene (kan) excised from pUC4K with HincII (37, 48). For the construction of plasmids pΔdcuBkan, pΔdcuBcat, and pΔdctQM, DNA fragments designated by the light boxes in Fig. Fig.1B1B and C were synthesized using PCR with primers that carried suitable restriction sites for cloning at their 5′ ends. Each of the two downstream fragments was inserted into pBR322 using the BamHI and SalI restriction sites. Subsequently, each of the upstream fragments was inserted using the EcoRI and BamHI restriction sites. The identity of the cloned PCR fragments was confirmed by sequencing. Finally, the kan gene from pUC4K or the cat gene from pDF4a (20) was inserted using the BamHI restriction site. The orientations of kan or cat in all plasmids shown in Fig. Fig.11 were confirmed by restriction analysis.

Cells of W. succinogenes were transformed with plasmid DNA as described previously (40). Transformants were selected on agar plates containing formate and nitrate as energy substrates with 2.6% (wt/vol) brain heart infusion agar (Gibco BRL). The agar medium was supplemented with kanamycin (25 mg liter−1) and/or chloramphenicol (12.5 mg liter−1) as appropriate. In each case, the genomes of several transformants were checked for the intended recombination events by means of Southern blot analysis. The identity of each desired mutant genotype (Fig. (Fig.1)1) was confirmed using SacI digestion of the genomic DNA and hybridization to appropriate DNA probes. The results of the Southern blot analysis excluded integration or replication of the respective plasmid. In the DcuA mutant, the kan gene was inserted in dcuA so that 37% of the gene at the 3′ end was separated from the rest (Fig. (Fig.1A).1A). The DcuB mutant retained only the start and stop codons of the dcuB gene (Fig. (Fig.1B).1B). The genome of the DctQM mutant lacks 34 bp of dctP at its 3′ end, the entire dctQ gene, and 64% of dctM (Fig. (Fig.11C).

Computer analysis.

Database searches made use of the program BLAST (1). Multiple-sequence alignments were performed using the program CLUSTAL W (44). The programs Signal P (36) and TMpred (21) were used for the prediction of signal peptides or membrane-spanning helices.

RESULTS

Dicarboxylate accumulation and release.

In the experiment with the results shown in Fig. Fig.2,2, 14C-labeled succinate (0.1 mM) was added to a suspension of washed cells of wild-type W. succinogenes in the presence of formate and nitrate. The resulting electron transport from formate to nitrate and nitrite was maintained for more than 10 min. After various incubation times, samples of the suspension were centrifuged, and the amount of succinate taken up by the cells was determined from the remaining radioactivity in the supernatants. After 3 min, 60% of the added radioactivity was found to have been taken up by the bacteria. It was calculated that the concentration of succinate in the bacterial cytoplasm was 3 orders of magnitude above the external concentration (1 g of cell protein liter−1 in the cell suspension and 1.2 ml [cytoplasmic volume] g of cell protein−1). No accumulation of [14C]succinate was observed when formate or nitrate was left out of the incubation medium or when the bacterial membrane was depolarized with the protonophore TTFB (data not shown). These results suggest that succinate accumulation is driven by the Δp generated by anaerobic respiration.

FIG. 2
Uptake and release of [2,3-14C]succinate by W. succinogenes cells in the steady state of anaerobic respiration with nitrate and nitrite. Labeled succinate (0.1 mM) was added at incubation time zero (●). Unlabeled succinate (10 ...

The accumulation of labeled succinate was not observed when a 10 mM concentration of either succinate, fumarate, malate, or aspartate was added to the bacterial suspension prior to the labeled succinate. In contrast, malonate, citrate, or pyruvate did not affect the accumulation of labeled succinate (not shown). When 10 mM unlabeled succinate was added after 3 min of incubation with labeled succinate (0.1 mM), the cells lost 80% of the label within 30 s, suggesting that rapid import and export of succinate occur in the steady state of electron transport (Fig. (Fig.2).2). The release of labeled succinate was also observed when either fumarate, malate, or aspartate (10 mM each) was added instead of the unlabeled succinate (data not shown). In contrast, less than 10% of the radioactivity was released when the same amount of malonate, citrate, or pyruvate was added. It is concluded that cells of W. succinogenes in the steady state of respiration specifically accumulate succinate, fumarate, malate, or aspartate. Furthermore, these C4-dicarboxylates appear to be taken up in exchange for internal succinate.

Properties of the dicarboxylate transport.

Dicarboxylate transport was too fast to be resolved by the centrifugation method used in the experiment with the results shown in Fig. Fig.2.2. Therefore, the consumption of fumarate by intact cells was recorded photometrically in order to characterize the dicarboxylate transport of W. succinogenes. It has been shown that in the presence of formate, W. succinogenes converts more than 90% of the added fumarate to succinate and malate (7). Fumarate conversion to malate is catalyzed by fumarase, which is located in the cytoplasm of W. succinogenes (46), as is the substrate site of fumarate reductase. Fumarate consumption was recorded at 270 nm, where succinate and malate do not absorb light.

The cells were depleted of Na+ and dicarboxylates by washing with an anoxic buffer at pH 7.6 and were incubated in the same buffer at pH 7.1 (Fig. (Fig.3).3). As seen from the decrease in absorbance recorded after fumarate had been added, the bacteria took up fumarate in the absence of formate (Fig. (Fig.3A).3A). Upon the addition of formate, the velocity of fumarate consumption was increased fivefold. In the presence of formate, fumarate is reduced to succinate (reaction a [see the introduction]), and a Δp is generated across the membrane by fumarate respiration, which in turn drives fumarate uptake (Fig. (Fig.2).2). Most of the succinate produced by fumarate respiration must be exported. Otherwise, after 4 s the internal succinate concentration would exceed 50 mM, which is the maximum internal succinate concentration observed in the steady state of electron transport (Fig. (Fig.2).2). Hence, under the experimental conditions of Fig. Fig.3A,3A, fumarate was taken up initially according to the uniport mechanism and then according to the antiport mechanism. The first process was electrogenic, since fumarate consumption in the absence or presence of formate did not occur with cells pretreated with the membrane-depolarizing protonophore TTFB (Fig. (Fig.3B).3B). Also, fumarate uptake according to the antiport mechanism was electrogenic under the experimental conditions of Fig. Fig.3A,3A, since cells preincubated with succinate did not take up fumarate after treatment with TTFB (Fig. (Fig.3D),3D), and it is expected that succinate is taken up during preincubation, as is fumarate (Fig. (Fig.3A).3A).

FIG. 3
Photometric recording of fumarate consumption by cells of W. succinogenes. A dual-wavelength spectrophotometer was used. Bacteria grown with formate and fumarate to the early stationary phase were washed three times with an anoxic buffer containing 0.5 ...

Electroneutral fumarate uptake was observed if the bacteria were incubated with succinate and NaCl prior to the addition of the protonophore (Fig. (Fig.3C).3C). Since succinate is probably taken up during preincubation, as is fumarate (Fig. (Fig.3A),3A), the electroneutral uptake of fumarate (Fig. (Fig.3C)3C) appears to depend on the presence of internal succinate. Hence, the electroneutral fumarate uptake operates according to the antiport mechanism. The specific activity of fumarate consumption in the presence of formate (Fig. (Fig.3C)3C) was 0.7 U mg of cell protein−1 at 22°C. This activity was consistent with that of fumarate respiration (reaction a) in a culture growing with formate and fumarate (2 U mg of cell protein−1) at 37°C. The latter activity was calculated from the doubling time (1.3 h) and the growth yield (4 g of cell protein mol of formate−1).

While fumarate is taken up in the absence of Na+ and TTFB (Fig. (Fig.3A),3A), the electroneutral antiport did not operate after preincubation with succinate alone (Fig. (Fig.3D)3D) but required preincubation with succinate and Na+ (Fig. (Fig.3C).3C). The addition of NaCl after incubation with succinate and TTFB treatment had no effect (Fig. (Fig.3D).3D). The simplest explanation is that internal Na+ is required for operation of the electroneutral antiport and that Na+ uptake is associated with succinate import during preincubation (Fig. (Fig.3C).3C). In agreement with this explanation, a 20-fold accumulation of labeled succinate was observed upon the addition of 100 mM NaCl to a cell suspension lacking Na+ (data not shown). It is likely that the electroneutral dicarboxylate antiport (Fig. (Fig.3C)3C) requires the presence of both internal and external Na+, although the requirement for external Na+ is not demonstrated by the experiment with the results shown in Fig. Fig.3.3. The effect of preincubation (Fig. (Fig.3C)3C) was maximal with 1 mM succinate, while less than 50% of the activity was recorded with 0.1 or 10 mM succinate. NaCl had the same effect at 10 and 100 mM, while less than half of the activity was recorded with 1 mM NaCl. Replacement of NaCl (10 mM) by Na2SO4 (5 mM) led to similar results.

Δp generation by fumarate respiration.

In W. succinogenes, the specific activity of fumarate respiration with formate or H2 was nearly the same both in the absence and in the presence of Na+ (data not shown). However, fumarate respiration generated an electrical proton potential (ΔΨ) across the membrane of cells only if Na+ was present (Table (Table1).1). The ΔΨ was nearly equal to the electrochemical proton potential (Δp = ΔΨ + ΔpH · R · T · F−1), since the ΔpH across the membrane was 0.5 or less. With inverted membrane vesicles prepared from cells, the ΔΨ generated by fumarate respiration with H2 was the same in the presence and absence of Na+ and was as high as that measured with cells in the presence of Na+ (Table (Table1).1). Since dicarboxylate transport is involved only in the fumarate respiration of cells, it appears that the transport is affected by Na+. As shown by the experiment with the results shown in Fig. Fig.3,3, fumarate uptake is electroneutral only in the presence of Na+, whereas it is electrogenic in the absence of Na+. Therefore, the Δp generated by the fumarate respiration of cells appears to be dissipated by the concomitant electrogenic transport of fumarate and succinate operating in the absence of Na+. Inverted vesicles do not catalyze fumarate respiration with formate, since formate dehydrogenase is oriented towards the inside of the vesicles and therefore is not accessible to its substrate.

TABLE 1
ΔΨ generation by fumarate respiration in the presence and absence of Na+ at 37°C

The dcu genes.

On the genome of W. succinogenes, two open reading frames, dcuA and dcuB, were discovered (Fig. (Fig.1A1A and B) (for details, see Materials and Methods). The predicted gene products of dcuA (433 residues) and dcuB (452 residues) share 40% identical residues and high hydropathy indices (+1.1 and +0.8 [28]). DcuA (62% identity) and DcuB (71% identity) are highly similar to the corresponding C4-dicarboxylate transporters of E. coli (41). DcuA of E. coli was demonstrated to form 10 membrane-spanning helices (16). Those authors concluded from sequence comparison that the same number of membrane traversions may be valid for other bacterial C4-dicarboxylate transporters, like DcuB of E. coli and DcuA of W. succinogenes.

The gene aspA upstream of dcuA probably encodes the aspartase (AspA) of W. succinogenes (Fig. (Fig.1A).1A). AspA (471 residues) is predicted to share 57% identical residues with E. coli aspartase (43). The ansA gene downstream of dcuA is known to encode the well-characterized asparaginase of W. succinogenes (33). The gene products predicted by fumBα (281 residues) and fumBβ (185 residues) upstream of dcuB probably are the two subunits of a hetero-oligomeric fumarase (Fig. (Fig.1B).1B). FumBα is similar to the N-terminal parts of E. coli fumarases A and B, and FumBβ resembles the C-terminal parts of these enzymes (2, 45). The cysteine residues ligating the tetranuclear iron-sulfur centers in E. coli FumA and FumB are conserved in FumBα of W. succinogenes. Fumarases consisting of two different subunits with sizes similar to FumBα and FumBβ are predicted to occur in Aquifex aeolicus and in several archaea (8, 10, 25, 42). These putative enzymes are up to 56% identical (A. aeolicus) with FumBα and FumBβ of W. succinogenes. The open reading frame orfB1 downstream of dcuB possibly encodes an iron-sulfur protein belonging to an uncharacterized protein family (designated UPF 0004 in the PROSITE data bank).

The dct genes.

Three adjacent open reading frames (dctP, -Q, and -M) on the genome of W. succinogenes (Fig. (Fig.1C)1C) possibly encode a secondary dicarboxylate uptake system belonging to the tripartite ATP-independent periplasmic (TRAP) transporter family (13). This is suggested by the similarity of the predicted gene products to those of the dctP, -Q, and -M genes of R. capsulatus. The predicted DctP protein of W. succinogenes is 44% identical to the DctP of R. capsulatus, which is a periplasmic binding protein for C4-dicarboxylates (39). The primary dctP gene product of W. succinogenes is predicted to carry a sec-dependent signal peptide (19 residues) which is similar to that encoded by R. capsulatus dctP. DctQ (170 residues) and DctM (415 residues) of W. succinogenes share 22 and 49% identical residues with the corresponding proteins of R. capsulatus. DctQ and DctM are predicted to form 4 and 12 membrane traversions, respectively.

Construction and properties of Dcu and Dct mutants.

The DcuA mutant was constructed by inserting the kan gene into dcuA of W. succinogenes (Fig. (Fig.1A).1A). The DcuB (Fig. (Fig.1B)1B) and DctQM (Fig. (Fig.1C)1C) mutants were obtained by replacing the respective genes by kan. The DcuA DcuB mutant was obtained from the DcuA strain upon replacement of dcuB by the cat gene, as shown in Fig. Fig.11B.

The DcuA DcuB mutant did not grow by fumarate respiration with fumarate as the substrate (Table (Table2).2). There was also no growth of this mutant in a complex medium or when fumarate was replaced by malate or aspartate. However, the mutant grew by nitrate respiration in a minimal medium with succinate as the sole carbon source, with a doubling time (Table (Table2)2) and a growth yield (data not shown) close to the values obtained with the wild-type strain. These results suggest that the inactivation of both dcuA and dcuB specifically prevents growth by fumarate respiration, whereas the uptake of succinate as carbon source is not affected.

TABLE 2
Growth of the W. succinogenes mutants by anaerobic respiration with formate as the electron donor at 37°C

The DcuA and DcuB mutants grew by fumarate respiration with each of the three dicarboxylates (Table (Table2).2). The doubling times of the mutants exceeded that of the wild-type strain during growth in the minimal medium with fumarate and were close to the values for the wild-type strain in the supplemented media. The growth yields did not significantly deviate from those of the wild-type strain (data not shown). Growth in the complex media was sustained exclusively by fumarate respiration, since no growth was observed in a complex medium containing formate but lacking fumarate, malate, or aspartate (not shown). The DcuA and DcuB mutants showed no apparent preference for a specific C4-dicarboxylate, despite the facts that the aspartase gene aspA is located adjacent to dcuA and that the fumarase genes are located adjacent to dcuB. It is concluded that DcuA and DcuB can each function alone in fumarate respiration with fumarate, malate, or aspartate. Since the DctQM mutant can grow by nitrate respiration with succinate as the carbon source, there must be an alternative for the DctPQM transporter in its anabolic function. However, the DctPQM transporter appears to be the major anabolic transporter, since the doubling time of the DctQM mutant is approximately 50% greater than those of the wild-type strain and of the dcu mutants growing with nitrate and succinate.

With the DctQM mutant, the extent of internal dicarboxylate accumulation under the conditions of the experiment with the results shown in Fig. Fig.22 was only 50-fold. With the DcuA DcuB mutant, the ratio of internal to external succinate concentration was 103, as with wild-type cells. When the experiment with the results shown in Fig. Fig.33 was performed with cells of the DctQM mutant, the results were similar to those obtained with wild-type cells (data not shown). Significantly, the mutant cells were found to take up fumarate in the absence of Na+, and the velocity of fumarate conversion was increased fivefold upon formate addition, just as was seen with wild-type cells (Fig. (Fig.3A).3A). These results confirm that the electrogenic uptake of fumarate according to the uniport mechanism is not exclusively catalyzed by the DctPQM transporter.

Cells of the DcuA DcuB mutant were found to be incapable of taking up fumarate in the presence of TTFB, even after preincubation with succinate and Na+ (data not shown). Cells of the DcuA DcuB mutant catalyzed fumarate respiration with formate (0.6 U mg of cell protein−1 at 37°C). However, only a very small ΔΨ was measured across the membrane of the mutant cells in the steady state of fumarate respiration in the presence of Na+ (Table (Table1).1). This result is in agreement with the finding that the DcuA DcuB mutant did not grow by fumarate respiration (Table (Table2).2). The results suggest that the mutant can perform only the electrogenic fumarate transport and that the electroneutral transport is catalyzed exclusively by the Dcu transporters.

DISCUSSION

In W. succinogenes, growth and Δp generation by fumarate respiration appear to depend on the electroneutral dicarboxylate antiport, which is catalyzed exclusively by the two Dcu transporters. In the DcuA DcuB mutant, the dicarboxylate antiport is apparently electrogenic. The electrogenic antiport seems to operate with the same H+/fumarate ratio of 2 (Fig. (Fig.4C)4C) as does fumarate respiration (Fig. (Fig.4A).4A). The two external protons generated by fumarate respiration are simultaneously imported in symport with fumarate by the electrogenic dicarboxylate antiport. As a consequence, the Δp generated by fumarate respiration is dissipated by the concomitant dicarboxylate transport in the DcuA DcuB mutant (Table (Table1).1).

FIG. 4
Hypothetical mechanism of Δp generation by fumarate respiration with H2 or formate (A) and hypothetical C4-dicarboxylate transport mechanisms in W. succinogenes (B to D). The sites of H2 and formate oxidation are exposed to the periplasmic side ...

The experiment with the results shown in Fig. Fig.33 indicated that the electroneutral dicarboxylate antiport operates only in the presence of Na+ (Fig. (Fig.4B).4B). This conclusion is confirmed by the finding that wild-type cells catalyzing fumarate respiration do not generate a Δp in the absence of Na+ (Table (Table1).1). It is obvious that the dicarboxylate transport is affected by Na+, since the Δp generation with inverted vesicles does not depend on the presence of Na+. In the absence of Na+, fumarate respiration of cells is dependent on the electrogenic dicarboxylate antiport (Fig. (Fig.4C);4C); this dissipates the Δp generated by fumarate respiration in the same way as described above for the DcuA DcuB mutant. The failure to generate a Δp in the absence of Na+ explains why W. succinogenes does not grow by fumarate respiration in the absence of Na+ (34). The maximal growth yield was observed with Na+ concentrations exceeding 5 mM, and half the maximal growth yield required approximately 1 mM Na+.

Each of the Dcu transporters is thought to catalyze the electroneutral dicarboxylate antiport according to the mechanism depicted in Fig. Fig.4B,4B, since the DcuA and DcuB mutants grow by fumarate respiration, in contrast to the DcuA DcuB mutant. The exchange of dicarboxylates is assumed to be coupled to the import and export of Na+. The electroneutral antiport was shown to require internal Na+ (Fig. (Fig.3),3), and it is unlikely that fumarate is imported in symport with protons while an equivalent amount of Na+ is exported with succinate. The dicarboxylates are thought to be transported as dianions at pHs of >7, as in E. coli (12), although transport of the monoprotonated dicarboxylates cannot be excluded. The electroneutral dicarboxylate antiport in E. coli was found to operate also in the absence of Na+ (12).

Electrogenic dicarboxylate uptake according to the uniport mechanism (Fig. (Fig.4D)4D) is catalyzed by the Dct transporter, as in R. capsulatus (13). This conclusion is based on the finding that the accumulation of internal dicarboxylate is lower in the DctQM mutant than in wild-type cells. However, a second uptake transporter has to be postulated to explain the 50-fold dicarboxylate accumulation by the DctQM mutant and to explain how the mutant can grow with dicarboxylate as the carbon source. It is not expected that one of the Dcu transporters would facilitate dicarboxylate accumulation. This process was observed to occur in the absence of Na+ in cells of the wild type (Fig. (Fig.3)3) and of the DctQM mutant, whereas the Dcu transporters appeared to function only in the presence of Na+. Therefore, it is postulated that a fourth, hitherto unknown, transporter exists in W. succinogenes. This transporter may also catalyze dicarboxylate transport according to the electrogenic antiport mechanism (Fig. (Fig.4C).4C). This process is shown to occur by the experiment with the results shown in Fig. Fig.33 and is used to explain the results given in Table Table1.1. The existence of the fourth transporter should be tested using a mutant lacking the two Dcu transporters and the DctPQM transporter. Such a mutant could not be constructed, since a third marker gene is not yet available.

ACKNOWLEDGMENTS

C. Derst and K. H. Röhm (Marburg) kindly provided plasmids carrying ansA and part of dcuA. Critical discussion of the manuscript with R. Krämer (Köln) is gratefully acknowledged.

This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 472) and from the Fonds der Chemischen Industrie to A.K.

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