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Journal of Bacteriology, October 2000, p. 5757-5764, Vol. 182, No. 20
Institut für Mikrobiologie, Johann
Wolfgang Goethe-Universität, D-60439 Frankfurt am
Main,1 and Institut für
Mikrobiologie und Weinforschung, Johannes Gutenberg-Universität,
D-55099 Mainz,2 Germany
Received 2 May 2000/Accepted 24 July 2000
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 ( The rumen bacterium Wolinella
succinogenes can grow by anaerobic respiration with fumarate as
the terminal electron acceptor by the reaction
HCO2 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.
Bacterial strains, phage, plasmids, and growth conditions.
E. coli strains were grown aerobically at 37°C in NZYM
medium (38). Ampicillin (100 mg liter
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Transport of C4-Dicarboxylates in
Wolinella succinogenes
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
p)
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.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
+ 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 (p) is generated across the bacterial membrane by reaction
a or b (4, 15, 34). The p drives ATP synthesis via an ATP
synthase which is integrated in the membrane (5). The p
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.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
1),
kanamycin (50 mg liter1), or chloramphenicol (25 mg
liter1) was added to the medium as appropriate. Phage
-EMBL3 (14) and its derivatives were propagated in
E. coli Q358 (24). Plasmids were amplified in
E. coli DH5
(Gibco BRL).
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
mmol1), 50 mM sodium formate, and 40 mM nitrate. After
various incubation times, samples (0.1 ml) of the suspension (1 g of
cell protein liter1) 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 (
).
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 liter1) 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 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 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
protein1). Under these conditions, 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 min1. 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.
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 -EMBL3 (30).
Restriction enzyme digests of the DNAs of several positive phages were
cloned in pBR322 (6) for sequencing of the dcuA
locus.
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Construction of plasmids and of W. succinogenes
mutants.
Mutants of W. succinogenes were constructed
via double homologous recombination as outlined in Fig. 1. Plasmid
pdcuA::kan was constructed from a pBluescript SK(+)
derivative containing the 1.3-kb HindIII fragment
indicated in Fig. 1A (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
pdcuBkan, pdcuBcat, and pdctQM, DNA fragments designated by
the light boxes in Fig. 1B 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. 1 were confirmed by restriction analysis.
1) and/or
chloramphenicol (12.5 mg liter1) 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. 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. 1A). The DcuB mutant retained only
the start and stop codons of the dcuB gene (Fig. 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. 1C).
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.
Nucleotide sequence accession numbers. The nucleotide sequences reported here have been deposited in the EMBL, GenBank, and DDBJ databases under accession numbers AJ002933 (dcuA locus), AJ131242 (dcuB locus), and AJ132740 (dct locus).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Dicarboxylate accumulation and release.
In the experiment with
the results shown in Fig. 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 liter1 in the cell suspension and 1.2 ml [cytoplasmic volume] g of cell protein1). 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 p
generated by anaerobic respiration.
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). Unlabeled succinate (10 mM) was
added to part of the cell suspension 3 min after the addition of
labeled 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. 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. 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. 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 ap is generated across
the membrane by fumarate respiration, which in turn drives fumarate
uptake (Fig. 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. 2). Hence, under the experimental conditions of Fig.
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. 3B). Also, fumarate uptake according to the antiport mechanism was electrogenic under the
experimental conditions of Fig. 3A, since cells preincubated with
succinate did not take up fumarate after treatment with TTFB (Fig. 3D),
and it is expected that succinate is taken up during preincubation, as
is fumarate (Fig. 3A).
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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 protein1) 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 formate1).
While fumarate is taken up in the absence of Na+ and TTFB
(Fig. 3A), the electroneutral antiport did not operate after
preincubation with succinate alone (Fig. 3D) but required preincubation
with succinate and Na+ (Fig. 3C). The addition of NaCl
after incubation with succinate and TTFB treatment had no effect (Fig.
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. 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. 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. 3. The
effect of preincubation (Fig. 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.
p 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 () across
the membrane of cells only if Na+ was present (Table
1). The was nearly equal to the
electrochemical proton potential (p = + pH
· R · T · F1), since the pH across the membrane was
0.5 or less. With inverted membrane vesicles prepared from cells, the
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 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. 3, fumarate uptake is electroneutral only in the presence of
Na+, whereas it is electrogenic in the absence of
Na+. Therefore, the p 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.
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The dcu genes. On the genome of W. succinogenes, two open reading frames, dcuA and dcuB, were discovered (Fig. 1A 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. 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 fumB (281 residues) and
fumB
(185 residues) upstream of dcuB probably
are the two subunits of a hetero-oligomeric fumarase (Fig. 1B). FumB
is similar to the N-terminal parts of E. coli fumarases A
and B, and FumB 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 FumB of W. succinogenes. Fumarases consisting of two
different subunits with sizes similar to FumB and FumB 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 FumB and FumB 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. 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. 1A). The DcuB (Fig. 1B) and DctQM
(Fig. 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. 1B.
DcuB mutant did not grow by
fumarate respiration with fumarate as the substrate (Table
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 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.
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and DcuB mutants grew by fumarate
respiration with each of the three dicarboxylates (Table 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. 2 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. 3 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. 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
protein1 at 37°C). However, only a very small
was measured across the membrane of the mutant cells in the steady
state of fumarate respiration in the presence of Na+ (Table
1). This result is in agreement with the finding that the
DcuA DcuB mutant did not grow by fumarate
respiration (Table 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.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In W. succinogenes, growth and p 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. 4C) as does fumarate
respiration (Fig. 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 p
generated by fumarate respiration is dissipated by the concomitant
dicarboxylate transport in the DcuA DcuB
mutant (Table 1).
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The experiment with the results shown in Fig. 3 indicated that the
electroneutral dicarboxylate antiport operates only in the presence of
Na+ (Fig. 4B). This conclusion is confirmed by the finding
that wild-type cells catalyzing fumarate respiration do not generate a
p in the absence of Na+ (Table 1). It is obvious that
the dicarboxylate transport is affected by Na+, since the
p 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. 4C); this dissipates the p generated by fumarate
respiration in the same way as described above for the
DcuA DcuB mutant. The failure to generate a
p 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. 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. 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. 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.
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. 4C). This process is shown to occur by the
experiment with the results shown in Fig. 3 and is used to explain the
results given in Table 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.
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ACKNOWLEDGMENTS |
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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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FOOTNOTES |
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* Corresponding author. Mailing address: Institut für Mikrobiologie, Johann Wolfgang Goethe-Universität, Marie-Curie-Str. 9, D-60439 Frankfurt am Main, Germany. Phone: 49-69-79829507. Fax: 49-69-79829527. E-mail: A.Kroeger{at}em.uni-frankfurt.de.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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