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(Received for publication, June 5,
1995; and in revised form, August 17, 1995) From the
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
Citrate utilization in bacteria is mostly mediated by
cation-dependent transport systems. Na 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 ( 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.
The
generation of an inverted membrane potential (
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
Figure 2:
Heterologous exchange catalyzed by CitP.
Shown is the exchange of [
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 (
If
indeed
Figure 4:
Leucine (A), SCN
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
Figure 5:
Transport of citrate (A) and
malate (B) driven by an inverted membrane potential. Hybrid
membranes loaded with 100 mM SCN
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 (
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 (
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
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 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
Volume 270,
Number 43,
Issue of October 27, 1995 pp. 25370-25376
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-dependent citrate carrier of Klebsiella
pneumoniae, CitP is not Na
- dependent, nor is
CitP Mg
-dependent. The pH gradient (
pH) is a
driving force for citrate and malate uptake into the membrane vesicles,
whereas the membrane potential () 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.
-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.
)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). (
)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
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).
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
, pH
7.0, for 1 h at 30 °C. Cells were lyzed by rapid addition of 0.75 M KSO. 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, 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, lyzed
cells were collected by centrifugation (48,200 
g for
30 min at 4 °C) and resuspended in 50 mM phosphate buffer,
pH 7.0, without MgSO. Cells and debris were removed by a
low spin (750 g for 1 h), after which the vesicles
were collected by spinning the supernatant at 48,200 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-
-Phosphatidylethanolamine was purified from 1
g of E. coli extract (Avanti Polar Lipids, Inc.) by successive
washing with acetone and diethyl ether. L--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 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-
C]Citrate, L-[U-C]leucine, and L-[U-C]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 HSO, resulting in a pH drop of 1
unit. Uptake assays were performed as described above.) (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-
C]citrate or 7.8 µML-[U-C]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-C]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
The membrane potential was determined from the distribution
of the lipophilic cation tetraphenylphosphonium and
pH 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-C]Citrate (110 mCi/mmol), L-[U-C]leucine (319 mCi/mmol),
[C]potassium thiocyanate (55 mCi/mmol), and L-[U-C]malate (48 mCi/mmol) were
obtained from Amersham International (Buckinghamshire, United Kingdom).
Pyranine was obtained from Eastman Kodak Co.
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
[
C]citrate (Fig. 2A) and
[C]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
[C]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.
cells
grown in the presence (
) or absence (
) of citrate and from L. mesenteroides cit cells grown in the
presence of citrate (
). 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.
C]citrate (A)
and [C]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 [C]citrate (4.5
µM), [C]malate (7.8
µM), and [C]leucine (1.6
µM) in the presence () or absence (
) of the
electron donor system cytochrome c/TMPD/ascorbate and 1
µM valinomycin. At the arrows, unlabeled citrate
() or malate () 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 () and a pH gradient
(
pH). Uptake of citrate and malate (Fig. 3) and the
magnitude of pH and (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
pH and 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
pH, while acts as
a counteractive force. The effect of
on citrate and malate
transport was further investigated with artificial gradients.
), valinomycin (),
and nigericin (). 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
().
(inside negative) acts as a counterforce for citrate
uptake,
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
is evidenced by uptake of the permeable anion SCN
(Fig. 4B,
). = 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,
). Only after
quenching of the inverted membrane potential by valinomycin was
pH-driven leucine accumulation observed (Fig. 4A,
). The highest level of citrate accumulation was observed when
both a pH gradient (inside alkaline) and an inverted membrane potential
were present (Fig. 4C, ). Dissipation of the
inverted membrane potential by valinomycin results in a strong
reduction of citrate uptake (Fig. 4C, ).
(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
pH. Experiments were performed in the absence () or presence
() of 1 µM valinomycin. , no acid was
added.
,
pH (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 and
pH of normal
polarity (Fig. 5A, ). The same result was obtained
with malate as the substrate (Fig. 5B, ). 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, ). 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.
were
incubated for 10 min at 30 °C in the presence of nigericin (
)
and nigericin and valinomycin () or without further addition
(). 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
= 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
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
cit and H
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 [C]citrate followed by dilution into medium
containing the weak acids. Ionophores were present to prevent the
generation of pH 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 and
). 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.
). At
the arrow, 0.5 µM nigericin without (;
efflux) or with (; exchange) 1 mM unlabeled citrate was
added. , not energized.
), malate
(), L-lactate (), D-lactate (
),
acetate (), and no further additions (
). Valinomycin and
nigericin were present at 1 and 0.5 µM, respectively. The
final protein concentration in the assay mixture was 112
µg/ml.
-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
RCOHCOOH.
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
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
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.
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. (
)
), divalent anionic citrate;
H
cit, monovalent anionic citrate; pmf,
protonmotive force; COVs, proteoliposomes containing cytochrome c oxidase; TMPD, N,N,N`,N`-tetramethyl-p-phenylenediamine.
))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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