(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-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.
Citrate utilization in bacteria is mostly mediated by
cation-dependent transport systems. Na-dependent
citrate transport has been described in Salmonella typhimurium and Klebsiella pneumoniae. The primary sequences of the
genes coding for the transport proteins in these two organisms, CitC
and CitS, respectively, are highly identical (van der Rest et
al., 1992a; Ishiguro et al., 1992). CitS of K.
pneumoniae transports citrate in symport with two sodium ions and
one proton (van der Rest et al., 1992b; Lolkema et
al., 1994a). A second citrate carrier from K. pneumoniae coded by the citH gene, which is not homologous to the citS gene, catalyzes citrate transport in symport with protons
(van der Rest et al., 1991). A similar claim has been made for
the mechanism for citrate uptake in atypical Escherichia coli cit
strains (Reynolds and Silver, 1983). In Bacillus subtilis, citrate transport was found to be coupled,
in addition to protons, to divalent metal ions, with a preference for
Mg
(Bergsma and Konings, 1983). Citrate uptake by
these secondary carriers is driven by the electrochemical cation
gradients (i.e. the protonmotive force or sodium ion-motive
force) that are maintained across the cytoplasmic membrane.
In
lactic acid bacteria, the ability to transport citrate is
plasmid-encoded. In Leuconostoc mesenteroides subsp. mesenteroides, the gene coding for the citrate transporter (citP) is localized on a 22-kb ()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).
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 () (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.
Figure 1:
Inducibility and specificity of CitP
of L. mesenteroides 19D. Shown is citrate (A), malate (B), and leucine (C) uptake in membrane vesicles of L. mesenteroides fused with COVs. Membrane vesicles were
prepared from L. mesenteroides cit cells
grown in the presence (
) 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.
Figure 2:
Heterologous exchange catalyzed by CitP.
Shown is the exchange of [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).
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 (), 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
(
).
If
indeed (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,
).
Figure 4:
Leucine (A), SCN (B), and citrate (C) uptake driven by a pH
jump. Membrane vesicles fused with liposomes were equilibrated with 4.5
µM citrate, 1.6 µM leucine, 2.3 µM thiocyanate in 50 mM potassium phosphate, pH 7.0. Uptake
was initiated by addition of sulfuric acid (0.5 N), which
resulted in a drop of the external pH from 7 to 6, thereby generating
pH. Experiments were performed in the absence (
) or presence
(
) of 1 µM valinomycin.
, no acid was
added.
The
complementary experiment, in which an artificial thiocyanate diffusion
potential was generated, is shown in Fig. 5. Membrane vesicles
of L. mesenteroides fused with liposomes were loaded with
potassium thiocyanate. Thiocyanate is negatively charged and diffuses
passively out of the membranes upon dilution, generating an inverted
membrane potential (inside positive). In response to ,
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.
Figure 5:
Transport of citrate (A) and
malate (B) driven by an inverted membrane potential. Hybrid
membranes loaded with 100 mM SCN were
incubated for 10 min at 30 °C in the presence of nigericin (
)
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).
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 (). At
the arrow, 0.5 µM nigericin without (
;
efflux) or with (
; exchange) 1 mM unlabeled citrate was
added.
, not energized.
Figure 7:
Exchange of citrate and products of
citrate metabolism. Membrane vesicles fused with liposomes were
preloaded with 5 mM citrate and subsequently diluted 100-fold
into buffer containing 5 mM citrate (), 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.
citP genes coding for citrate transporters have been cloned from Lactococcus lactis, Leuconostoc lactis, and L. mesenteroides and were found to be virtually the same. The citP genes are located on different endogenous plasmids in the three organisms. Regulation of expression may differ among the organisms. The Lactococcus lactis citP gene is located on a 5.6-kb plasmid. Magni et al.(1994) demonstrated that the transcript of citP and citrate uptake are independent of the presence of citrate in the growth medium, i.e. CitP is constitutively expressed. In L. mesenteroides, citP is located on a much bigger plasmid of 22 kb. The present results show that citrate acts as an inducer of CitP expression. Membrane vesicles from cells of L. mesenteroides grown in the presence of citrate were significantly more active in citrate uptake than those from cells grown in the absence of citrate (Fig. 1).
The
primary sequence of CitP is 30% identical to the
Na-dependent citrate carrier of K. pneumoniae (CitS). Hydropathy profiling reveals an even stronger structural
similarity between the two proteins (van der Rest et al.,
1992a; Lolkema et al., 1994b). Translocation of citrate by
CitS is obligatory coupled to the translocation of Na
ions (Lolkema et al., 1994a). However, CitP is not
Na
ion-dependent. As observed in other families of
homologous secondary transporters, structural similarity does not
necessarily correlate with cation specificity. Surprisingly, CitP not
only catalyzes citrate transport, but also malate and lactate
transport. Malate transport by CitP could be demonstrated by the
correlation between the ability of membrane vesicles to accumulate
malate and the expression of CitP (Fig. 1) and by heterologous
exchange between citrate and malate (Fig. 2). Transport of
lactate via CitP was demonstrated by its accelerating effect on citrate
efflux, which is explained as citrate/lactate exchange (Fig. 7).
Both D- and L-lactate showed the accelerating effect,
indicating that the carrier is not stereoselective. Citrate (, i), malate (ii), and lactate (iii) are structurally
related, suggesting that CitP is a general carrier for
hydroxycarboxylic acids presenting the motive
R
R
COHCOOH.
The following results indicate that the citrate carrier of L. mesenteroides catalyzes translocation of net negative
charge. (a) Citrate uptake driven by the protonmotive force is
largely stimulated when the membrane potential component is dissipated; (b) the highest accumulation level of citrate is observed in
the presence of a pH gradient of physiological polarity and a membrane
potential of opposite polarity; and (c) an inverted membrane
potential by itself can drive the accumulation of citrate into the
membranes. The analysis of the forces (see Fig. 3and Table 1) shows that a single unit of charge is translocated per
catalytic cycle, i.e. Hcit
,
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.
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
Hcit
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. (
)