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Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands
SUMMARY INTRODUCTION FUNCTION Distribution and Phylogeny Transport Properties Physiological Function Malolactic fermentation. Genetic organization of malolactic fermentation genes. Oxidative malate decarboxylation pathway. Citrolactic fermentation. Citrate fermentation in the gamma subdivision of Proteobacteria. Distribution of citrate fermentation in the bacterial kingdom. Evolutionary states of energy coupling to oxaloacetate decarboxylases. PROTEINS Cloning and Expression Purification and Reconstitution STRUCTURE Primary Structure Superfamilies and structural classes. Sequence analysis. Domain structure. Membrane Topology Transmembrane segments. Amphipathic helix and membrane insertion. Pore-loop structure. Structural Model Quaternary Structure MECHANISMS Kinetics Symport and exchange. Antiport mechanism of CitS. Alternate access. Structure-Function Relationships The conserved Arg in TMS XI. Chimeras of CitP and MleP. Interaction of substrate and co-ions with the Xa region. Mutations in the Vb region. Mechanistic Model CONCLUSIONS ACKNOWLEDGMENTS REFERENCES
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Secondary transporters use the free energy stored in ion and/or solute gradients across the membrane to drive transport. The transporters are commonly classified in three groups based on their mode of energy coupling: (i) uniporters catalyze the translocation of a single solute across the membrane, (ii) symporters couple the translocation of a solute to the translocation of a co-ion(s) in the same direction, and (iii) antiporters couple the translocation of a solute and a co-ion(s) in opposite directions. Many antiporters couple the translocation of one solute to the translocation of another solute rather than a co-ion. They exchange a substrate at one side of the membrane for another substrate at the other side of the membrane. Symporters (and uniporters) can catalyze a similar reaction when they operate in the exchange mode of transport, a partial reaction. This exchange mode catalyzed by symporters differs from the antiport mechanism in that the translocations of the two substrates in the two directions are not obligatorily coupled. Symporters catalyzing exchange under physiological conditions are termed exchangers. The different modes of energy coupling enable transporters to play an important role in different aspects of the physiology of the cell. Thus, the symport and uniport mechanisms allow the cell to take up nutrients from the medium, while antiporters may function in the excretion of end products or in defense mechanisms by removing harmful compounds from the cell. Antiporters and exchangers may combine the uptake of a nutrient from the environment and the excretion of a metabolic end product.
As a rule, transport catalyzed by secondary transporters is a metabolic energy-requiring process. Symporters and antiporters couple the translocation of the substrate to the translocation of protons or sodium ions. The electrochemical gradients of H+ and Na+ across the membrane, or proton motive force (PMF) and sodium ion motive force, respectively, are directed inward. Both forces consist of a chemical gradient of the ions (
pH or pNa) and the membrane potential (
) that is common to both forces. The gradients are maintained across the membrane by the action of primary pumps that use the free energy released in chemical reactions to pump H+ and Na+ across the membrane. The free energy stored in the electrochemical gradients of the ions across the cytoplasmic membrane is used to concentrate the substrate in the compartment to which it is transported. Thus, the symporter accumulates the substrate inside the cell, while the antiporter depletes the substrate from the cell. Secondary transporters that catalyze exchange are not coupled to (net) proton or Na+ movement and are driven by the inward-directed substrate gradient and the outward-directed product gradient. Both gradients are maintained by metabolism of the substrate inside the cell, which lowers the internal substrate concentration and increases the internal product concentration. A small group of exchangers that is of particular importance to the transporter family that is the topic of this review use the free energy in the substrate and product gradients to generate metabolic energy in the form of a membrane potential. They are part of metabolic pathways that function as indirect proton pumps.
Secondary transporters are typical integral membrane proteins that fold as a bundle of hydrophobic 
-helices, which are oriented more or less perpendicular to the membrane. At the two sides of the membrane, the transmembrane segments (TMSs) are connected by hydrophilic loops of various lengths. Thus far, eight three-dimensional crystal structures of secondary transporters have been described. These structures have provided a first glimpse of the structural and mechanistic diversity that may be present in the many different families of secondary transporters. The structures of the drug transporter AcrB, the lactose transporter LacY, the glycerol-P/Pi exchanger GlpT, the Na+/H+ antiporter NhaA, and the Cl–/H+ antiporter ClC, all from Escherichia coli; of the glutamate transporter homolog GltPh from the archaeon Pyrococcus horikoshii; of the leucine transporter LeuT from Aquifex aeolicus; and of the mitochondrial ATP/ADP antiporter (2, 37, 47, 49, 98, 105, 153, 154) represent seven different structures and possibly as many different mechanisms (137).
Here we review the current knowledge about the 2-hydroxycarboxylate transporter (2HCT) family, a family of secondary transporters. The members are found exclusively in bacteria, and all transport substrates such as citrate, malate, and lactate. Well-studied members of the 2HCT family are the Na+-citrate symporter CitS of Klebsiella pneumoniae, the malate/lactate exchanger MleP and the citrate/lactate exchanger CitP found in lactic acid bacteria, and the citrate/malate H+-symporter CimH of Bacillus subtilis. The transport properties of the characterized 2HCT members will be discussed in the context of their physiological function. Based on sequence analysis and the genetic organization of the structural genes in the genomes, physiological functions are assigned to uncharacterized 2HCT members. No three-dimensional (3D) structure of any of the members of the 2HCT family is available, but a wealth of data obtained from experimental studies and computational analysis will be discussed, giving us models for the structures and translocation mechanisms of the transporters in this family.
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The citrate transporter CitS of K. pneumoniae is by far the best-studied transporter in the 2HCT family. Nevertheless, the exact stoichiometry between substrate and co-ions has still not been resolved. Dimroth and Thomer (30) reported that citrate transport catalyzed by CitS was Na+ dependent and proposed that translocation involved symport of trivalent citrate (cit3–) with three Na+ ions (30). The proposal was based on the observed lack of charge translocation during turnover (electroneutral transport), which, however, was disputed in other studies (142, 143). Moreover, the latter studies reported that the proton gradient across the membrane was a driving force, suggesting that in addition to Na+ ions, protons were cotransported. Since at physiological conditions, the divalent form of citrate is most abundant, it was proposed that Hcit2– would be symported with at least three co-ions, including both Na+ and H+ (142, 143). The affinity of the transporter for total citrate was found to be about 10 µM at a pH range of between 5.5 and 6.5. Kinetic analysis of citrate uptake measured in E. coli cells expressing CitS revealed that translocation of citrate is coupled to translocation of two sodium ions (76), a result that was confirmed much later by uptake studies in right-side-out membrane vesicles (134). The relationship between the uptake rate and Na+ ion concentration was shown to be sigmoid, with approximately 3 mM Na+ yielding half of the maximal rate. The coupling stoichiometry of two Na+ ions was observed at the pH range of pH 6 to 8, suggesting that H+ does not compete with Na+ for the two binding sites (76). A strict coupling between citrate and Na+ ions is also supported by point mutations that lower the affinity for Na+ by an order of magnitude but leave the stoichiometry unaltered (135). The differing opinions on the coupling stoichiometry of CitS extend to the kinetic mechanism of the transporter (see "Kinetics" in "MECHANISMS" below) (109). Little is known about the stoichiometries of the other Na+ symporter, MaeN of B. subtilis, and of the H+ symporters, MalP of S. bovis, and CimH of B. subtilis (60, 63, 151). The latter transporter is believed to catalyze electroneutral transport (63).
The exchangers in the 2HCT family belong to a special group of secondary transporters that are involved in the generation of secondary metabolic energy (78). They are precursor/product exchangers that couple the uptake of the substrate (the precursor) to the excretion of the end product of a metabolic pathway (5, 78, 107). Membrane potential is generated during turnover, because of a charge difference between the two substrates. CitP of Leuconostoc mesenteroides and MleP of L. lactis exchange internal monovalent lactate (lac–) for external divalent citrate and malate (Hcit2– and mal2–, respectively), which results in a membrane potential of physiological polarity (positive outside). CitP was shown to be a proton symporter with affinity for both citrate and lactate. In the symport reaction, CitP couples the translocation of Hcit2– to a single H+, resulting in the translocation of one unit of negative charge per turnover. As a consequence, the symport reaction is driven by a pH gradient but is counteracted by the membrane potential (94). With lactate as the substrate, CitP catalyzes electroneutral symport of lac– and H+. In the exchange mode, the proton is believed to go back and forth during transport. The precursor/product exchangers are symporters that were optimized to catalyze exchange, which is their physiological function; exchange is much faster than symport (see also "Kinetics" in "MECHANISMS" below) (7, 94, 96, 107). Exchangers such as CitP and MleP recognize structurally related compounds; citrate and lactate, and malate and lactate, are pairs of 2-hydroxycarboxylates, HO-CR2-COO–, that differ in the R groups. The proteins are very specific towards the hydroxyl and carboxylate groups and, at the same time, very tolerant towards the two R groups of the molecules. As a consequence, both CitP and MleP were shown to translocate a wide range of nonphysiological substrates that differ in the R groups (7). The only restriction seems to be the size of the R group, which at the upper limit is set by the size of the physiological substrates. Thus, CitP accepts citrate, while the most bulky substrate for MleP is malate. At the lower limit, glycolate, HO-CH2-COO– is recognized and translocated by both transporters. Nonphysiological substrates with different R groups that were shown to be translocated include the dicarboxylates tartarate, citramalate, and 2-hydroxyglutarate and the monocarboxylates mandalate, 2-hydroxyisovalerate, 2-hydroxyisobutyrate, and glycolate (7, 9). Despite the broad specificity, both CitP and MleP were found to be highly stereoselective with a strong preference for the S over the R enantiomers. The stereoselectivity was observed only with dicarboxylate substrates, suggesting a specific interaction of the protein with a carboxylate of the R group in the S enantiomers (9). In agreement, the affinity of both MleP and CitP for the S enantiomers of dicarboxylate substrates is 1 to 2 orders of magnitude higher than that observed for monocarboxylates and for the R enantiomers of dicarboxylates (see also "Structure-Function Relationship" in "MECHANISMS" below).
CitW of K. pneumoniae was shown to be a citrate/acetate exchanger and the first member of the 2HCT family where the physiological substrate is not a 2-hydroxycarboxylate (58). Previously, it was demonstrated that CitP and MleP of lactic acid bacteria showed some activity with the 2-oxocarboxylates glyoxylate and oxaloacetate (7, 9). CitW differs from CitP and MleP in that it does not share the broad substrate specificity of the latter two exchangers. Only a low activity was observed with L-malate. The transported species was shown to be Hcit2–, suggesting electrogenic citrate/acetate exchange, but the electrogenicity was not demonstrated.
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The malolactic fermentation pathway is not general to lactic acid bacteria, but is observed only in certain strains of Lactococcus, Lactobacillus, Pediococcus, and Leuconostoc. The process is best studied in Oenococcus oeni, a bacterium which plays an important role in the nonalcoholic fermentation in wine production (73, 83). Conversion of malate into lactate is an important step in the deacidification of wine following the alcoholic fermentation. The pathway in O. oeni (previously known as Leuconostoc oenos) is slightly different from the one described above for L. lactis, and the transporter is not a member of the 2HCT family. Rather than malate2–/lactate– exchange, the transporter of O. oeni catalyzes uniport of monovalent malate (Hmal–) (120). The effect on the membrane potential is the same: net negative charge is moved into the cell. Inside the cell, malolactic enzyme converts Hmal– into lactic acid (Hlac), thereby consuming a proton and releasing carbon dioxide. Lactic acid leaves the cell by passive diffusion. The energetic consequences of the pathway are the same, but the pathway is better adapted to the acidic conditions of wine fermentation (119). The transporter that catalyzes the electrogenic uniport reaction is a member of the auxin efflux carrier (AEC) family (TC 2.A.69 in the transporter classification system [17, 117]), which contains many members from bacteria, archaea, and plants (70).
Decarboxylation pathways built from a precursor/product exchanger and a decarboxylase are not restricted to lactic acid bacteria or to malate as the substrate. One of the first pathways described was the decarboxylation of oxalate to formate in Oxalobacter formigenes. The conversion is the main energy source for ATP production in this obligate anaerobic bacterium (5). Electrogenic exchange between divalent oxalate and monovalent formate is catalyzed by OxlT, a transporter from the major facilitator superfamily (MFS) (TC 2.A.1) (1). Another group of decarboxylation pathways is formed by the decarboxylation of amino acids, yielding biogenic amines. Examples are histidine to histamine and tyrosine to tyramine (20, 86, 87, 97). The exchangers that couple the uptake of the amino acid to the excretion of the amine are members of the amino acid-polyamine-choline superfamily (TC 2.A.3). Apparently, nature has implemented analogous pathways by selecting the appropriate transporters, and also decarboxylases, from different gene families.
Genetic organization of malolactic fermentation genes. In L. lactis, the genes coding for the decarboxylase MleS and the transporter MleP are organized in an operon structure in the order mleS-mleP (M-T) (Table 1). Upstream of mleS, but separated by 27 other genes, the mleR gene codes for a positive regulator of expression of the malolactic operon that senses the presence of L-malate. MleR is a member of the LysR family of activators (114). The three proteins MleR, MleS, and MleP are characteristic of the malolactic fermentation pathway, and highly similar proteins are encoded in the genome of Leuconostoc mesenteroides (6, 26). Here, the mleR gene is found immediately upstream of the mleS-mleP pair but divergently transcribed. The operon organization in which the malic enzyme is followed by the transporter is also found in the genome of Enterococcus faecium. Moreover, the transporter, ZP000379, clusters with MleP of L. lactis and Leuconostoc mesenteroides on the phylogenetic tree of the transporters (Fig. 1, cluster I), strongly suggesting that the two genes code for a malolactic fermentation pathway. Similarly to the case for L. lactis, a homologous gene coding for the transcriptional regulator MleR is located distantly on the chromosome. The two transporters of the plant pathogen Onion yellows phytoplasma in cluster VI of the phylogenetic tree (Fig. 1) are loosely associated with the transporters in cluster I, and the gene coding for CitSp$on is preceded by a malic enzyme homolog (Table 1). However, the latter is quite distant from the malolactic enzymes associated with the transporters in cluster I, and since no mleR homolog is found in the genome, the two genes are not likely to form a malolactic operon.
The genetic organization of the malolactic operon in the wine bacterium O. oeni is identical to that observed in Leuconostoc mesenteroides; a divergently transcribed regulator gene precedes the malic enzyme/transporter pair (R<>M-T) (69). Strikingly, however, as mentioned above, the transporter MleP of O. oeni is a member of the AEC rather than the 2HCT transporter family. In fact, a search of 156 completed bacterial genomes available at the microbial genome site of the NCBI (http://www.ncbi.nlm.nih.gov/genomes/MICROBES/Complete.html; April 2005) for the combination of the three genes characteristic of malolactic fermentation, mleR, mleS, and mleP, showed that transporters of the AEC family are most frequently observed in conjunction with MleS (Table 2). The search showed that the malolactic fermentation pathway is quite rare in the bacterial kingdom and is only found in low-GC gram-positive organisms. However, even then, the capacity is not at all general, with only 5 out of 38 genomes from the phylum Firmicutes coding for the pathway (Table 2). In this group, the malic enzymes and transcriptional regulators represent highly conserved groups of proteins, with >50 and >30% sequence identity for MleS and MleR, respectively. In contrast, the divergence in the associated transporters is remarkable. Lactobacillus plantarum, Lactobacillus acidophilus, and Streptococcus mutants use a transporter of the AEC family for the uptake of malate, as was described for O. oeni above. L. lactis uses a transporter of the 2HCT family, and Clostridium acetobutylicum uses one of the [st303]AIT family. Members of the latter family are known to catalyze exchange; e.g., CitT of E. coli catalyzes citrate/succinate exchange, and SODiT1 of spinach chloroplasts catalyzes 2-oxoglutarate/malate exchange (110). It is likely that the transporter of C. acetobutylicum catalyzes malate/lactate exchange, as does MleP of L. lactis. Both the [st326]2HCT and [st303]AIT1 transporter families are found in structural class ST[3] in the MemGen classification (see "Primary Structure" in "STRUCTURE" below) and may well be distantly related.
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In B. subtilis, the Na+/malate symporter MaeN, which is closely related to the H+/malate symporter of S. bovis (Fig. 1, cluster IV), is essential for the uptake of malate from the medium during growth on malate as the sole carbon source. A transcriptome analysis showed that the transporter is induced by the presence of malate in the medium, which is mediated by a two-component signal transduction system, YufLM (140). The two-component system is similar to the MalSR system in S. bovis and is encoded upstream of the transporter gene, separated by four other genes (Table 1). The same organization is observed in the genome of Bacillus licheniformis. Transcriptional analysis revealed coinduction via the two-component system of YwkA, one of four malic enzyme homologs encoded in the genome of B. subtilis. YwkA was shown to be a malic enzyme of the oxidative malate decarboxylation type, strongly suggesting that the malate-to-pyruvate pathway is operative in B. subtilis (33). It should be noted that while the transporter MaeN was shown to be essential for growth on malate, the ywkA gene product was not. Instead, a constitutively expressed malic enzyme homolog of the oxidative type, encoded by the ytsJ gene, was essential (33). Moreover, the ywkA gene is transcribed in a single transcript with ywkB, coding for an AEC family transporter, which may encode a malate transporter as well (see previous section). Other transporters that are likely to play a role in malate metabolism in B. subtilis are YflS in the [st303]AIT family, a transporter which is also part of the YufLM regulon, and CimH in cluster V of the 2HCT family (Fig. 1). The presence of the gene coding for CimH correlates with the presence of the malSR-4/5-MaeN gene cluster in the genomes of B. subtilis and B. licheniformis (Table 1). CimH of B. subtilis transports citrate with a high affinity but a low maximal rate and transports malate with a low affinity but a high maximal rate (63). It has been suggested that CimH and MaeN would cover efficient uptake over a large range of malate concentrations in the medium (66). MaeN would take care of the lower concentrations and CimH of the higher concentrations. Unfortunately, no conditions under which the CimH protein is expressed have been identified (66, 140). The interplay between the different malate transporters and decarboxylases in B. subtilis needs to be investigated further.
The 2HCT members from several Bacillus species found in phylogenetic clusters II and IV (Fig. 1) are organized in gene clusters together with a malic enzyme homolog and a MalSR two-component signal transduction system, suggesting their involvement in oxidative malate decarboxylation pathways (Table 1). In contrast to the clusters in the genomes of the lactic acid bacteria, the genes coding for the two-component systems of Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus clausii (cluster IV) are transcribed in the same direction (malSR-T-M), while in Bacillus halodurans (139) and Oceanobacillus iheyensis (cluster II) the order of the transporter and malic enzyme is reversed. MaeN of Erwinia carotovora in cluster IV is the only transporter in the gamma subdivision of the phylum Proteobacteria which is clustered with a MalSR two-component system. As in B. subtilis and B. licheniformis, a malic enzyme homolog is missing and is probably located elsewhere in the genome. The remaining transporters in clusters II and IV, MaeN of Chromobacterium violaceum from the beta subdivision of the Proteobacteria and NP973298 of Treponema denticola from the phylum Spirochaetales, are associated neither with a malic enzyme nor with a two-component signal transduction system. The gene coding for MaeN of C. violaceum is clustered together with the genes for the two subunits of 3-isopropylmalate dehydratase, an enzyme in the leucine biosynthetic pathway. Possibly, the transporter is involved in the uptake of the intermediate 3-isopropylmalate rather than malate.
Citrolactic fermentation. Lactic acid bacteria in the phylum Firmicutes are facultative anaerobic bacteria that cannot grow on citrate as the sole source of carbon and energy, but many species are known to ferment citrate in cometabolism with a carbohydrate. The citrolactic fermentation pathway, like the malolactic fermentation pathway, is a secondary metabolic energy-generating route that produces proton motive force (96). The transporter involved in the uptake of citrate from the medium is the citrate/lactate exchanger, CitP, of the 2HCT transporter family. Exchange of divalent citrate for monovalent lactate by CitP yields a membrane potential of physiological polarity. Inside the cell, citrate is split by citrate lyase (CL), yielding oxaloacetate and acetate (Fig. 2B). The latter leaves the cell by passive diffusion. A cytoplasmic enzyme, oxaloacetate decarboxylase (CitM), converts oxaloacetate into pyruvate and carbon dioxide. This is the step in which a pH gradient is generated as the decarboxylation reaction consumes a cytoplasmic proton (77, 78). Therefore, like for malolactic fermentation, the proton motive force-generating system is built around a precursor/product exchanger (CitP) and a decarboxylase (CitM). The latter was recently shown to be a member of the malic enzyme family, to which malolactic enzyme (MleS) and the oxidative malate decarboxylase (MalS) also belong (129). The oxaloacetate decarboxylases are closely related to the MalS type of malic enzymes, which decarboxylate malate to pyruvate. Mechanistically, the conversion of malate to pyruvate by MalS proceeds via oxaloacetate (Fig. 3A). The electrons are donated to the cofactor NAD(P)+, while the subsequent decarboxylation of oxaloacetate to pyruvate is redox neutral. CitM is believed to skip the first step and to accept oxaloacetate directly as the substrate. NAD+/NADH was shown to be nonessential to the oxaloacetate decarboxylation activity. Nevertheless, the cofactor binding site appears to be conserved in the CitM proteins, and the ability to bind NAD+ and NADH was demonstrated by inhibition of enzyme activity in their presence (129).
The common decarboxylation step catalyzed by enzymes from the same family and the 50% sequence identity shared by the exchangers CitP and MleP (7) in cluster I on the phylogenetic tree (Fig. 1) indicate that the citrolactic and malolactic fermentation pathways are evolutionarily related. In Leuconostoc mesenteroides and Weissella paramesenteroides, all the genes involved in the citrolactic fermentation pathway are organized in a single operon that is located on a 22-kb plasmid. A transcriptional regulator (citR) and the citrate/lactate exchanger (citP) are downstream of the citDEFXG genes, coding for the citrate lyase subunits and accessory proteins, while the oxaloacetate decarboxylase gene (citM) is upstream (Table 1) (12, 13, 91). In L. lactis, the pathway is encoded in two separate operons; the transcriptional regulator is encoded together with the transporter on an 8-kb plasmid, while the remaining metabolic enzymes are encoded on the chromosome (35, 84, 88, 149). The only 2HCT transporter found in a Clostridium species, CitN in Clostridium perfringens, is also clustered with the citrate lyase genes and a malic enzyme homolog, suggesting its involvement in citrate degradation (Table 1). The order of the genes differs from the one in the lactobacilli; the malic enzyme is in between the citrate lyase genes and the transporter, while the citR and citG genes are upstream. CitN is closely related to the citrate transporters found in the gamma subdivision of the Proteobacteria (Fig. 1, cluster III).
The link between citrolactic fermentation and carbohydrate metabolism is evident, since lactate is a product of carbohydrate metabolism rather than citrate degradation. The pathways for citrate breakdown and carbohydrate breakdown merge at pyruvate, which subsequently, is reduced by lactate dehydrogenase to yield lactate, the substrate of the exchanger CitP (Fig. 2B). The redox equivalents necessary for the reduction are produced in glycolysis. Therefore, citrate fermentation is dependent on glycolysis. The coupling between the two pathways and the effect of citrate on growth are different for homofermentative and heterofermentative species (8, 48, 121). Homofermentative Lactococcus species produce 2 moles of pyruvate and, subsequently, of lactate per mole of glucose. The 2 moles of NADH needed for the reduction of pyruvate are produced in the earlier steps of the glycolytic pathway, which makes the pathway redox neutral as a whole. The end product, lactate, is used by CitP to take up citrate from the medium. The surplus of pyruvate fed into the pyruvate pool by citrate degradation is converted in a redox-neutral manner into aroma compounds such as diacetyl, a typical product of citrate/carbohydrate cometabolism. Heterofermentative Leuconostoc species yield 1 mol of ATP per mole of glucose, producing 1 mol of pyruvate and 1 mol of acetylphosphate (acetyl-P), which are reduced to 1 mol of lactate and 1 mol of ethanol, respectively, to balance the redox equivalents produced upstream in glycolysis. The more advantageous conversion of acetyl-P to acetate by acetate kinase, which would yield one more mole of ATP, would result in detrimental accumulation of redox equivalents and inhibition of growth. Consequently, the yield of ATP per mole of glucose is only 1 for a heterofermentative bacterium and 2 for a homofermentative bacterium. However, in the presence of citrate, pyruvate produced from citrate provides an alternative redox sink, allowing the conversion of acetyl-P to acetate. Citrate induces a metabolic shift from ethanol to acetate production with concomitant formation of additional ATP. The yield increases from 1 to 2 mol of ATP per mole of glucose. In Leuconostoc mesenteroides, lactate excreted by CitP is a more direct product of citrate fermentation, needing only the redox equivalents from glycolysis (89).
The proton motive force generated in the citrate degradation pathway is in addition to the proton gradient generated by F0F1-ATPase at the expense of ATP produced by substrate-level phosphorylation in glycolysis. Since the hydrolysis of one ATP is coupled to the pumping of three or four H+ ions across the membrane, the relative contribution of the metabolic energy generated by the secondary mechanism in the citrate degradation pathway is likely to be small when the fluxes through the two pathways are coupled. Assuming that energy is growth rate limiting, this is supported by the similar growth rates observed for L. lactis on glucose and on glucose/citrate (18, 138). In Leuconostoc mesenteroides, the energetic consequence of citrate cometabolism is more pronounced, but mainly because the ATP yield of glycolysis increases when metabolism shifts from ethanol to acetate production. Accordingly, a significant increase of growth rate is observed in the presence of citrate (19, 121, 123).
The lack of effect on the growth of L. lactis raises the question of the physiological benefit of the pathway in this organism. The answer is provided by the different induction profiles of the citrate metabolic pathways in Leuconostoc and Lactococcus species. In Leuconostoc mesenteroides, the enzymes of the pathway are induced by the presence of citrate in the medium, while in L. lactis, the induction additionally requires a low pH in the medium, suggesting a role in acid stress (40, 85, 89, 92, 93, 95). The conversion of carbohydrate to lactic acid results in acidification of the medium, which eventually inhibits growth. Proton consumption in the decarboxylation of oxaloacetate in the citrate degradation pathway initially results in alkalinization of the cytoplasm, but in the steady state of growth, when the pH gradient has developed, the alkalinizing effect is in the medium and counteracts the acidifying effect of glycolysis. Hence, the pH of the medium is buffered and the exponential phase of growth is prolonged during glucose/citrate cometabolism. Additionally, Magni et al. (89) demonstrated that the presence of citrate in the medium protects L. lactis against the toxicity exerted by lactate, which is produced in high concentrations in the medium (>10 mM) at the end of growth. Since lactic acid is a weak acid, it accumulates in the cell in response to a pH gradient across the cytoplasmic membrane. Cometabolism with citrate effectively lowers the cytoplasmic concentration of lactate by the action of the citrate/lactate exchanger CitP (89).
Citrate fermentation in the gamma subdivision of Proteobacteria.
K. pneumoniae is one of the best-studied organisms with respect to citrate fermentation (for a review, see reference 14). The organism is a gram-negative bacterium that under anaerobic conditions can grow on citrate as the sole carbon and energy source. Two transporters in the 2-hydroxycarboxylate transporter family are involved in the breakdown route, CitS in cluster III of the phylogenetic tree and CitW in cluster V (Fig. 1). CitS is responsible for the uptake of citrate from the medium. The first metabolic steps in the degradation of internalized citrate are the same as those observed in lactic acid bacteria, but the energetics of the pathway are different (Fig. 2D). Citrate is converted into acetate and oxaloacetate by CL, and oxaloacetate is converted to pyruvate and carbon dioxide by an oxaloacetate decarboxylase (OAD) which differs from the malic enzyme homologs (CitM) found in gram-positive bacteria. OAD is an integral membrane protein that uses the free energy released in the decarboxylation of oxaloacetate to pump Na+ across the membrane (29, 31). The activity of OAD recycles the sodium ions that have entered the cell in the symport reaction catalyzed by CitS. OAD consists of two integral membrane subunits, ß and 
(encoded by oadB and oadG), and one membrane-associated subunit, (encoded by oadAD) (152). Subunit is the actual decarboxylase and consists of two domains, and 
. Domain , the biotin acceptor domain, contains a biotin prosthetic group that accepts the carboxyl moiety of oxaloacetate in a reaction catalyzed by domain . Subsequently, decarboxylation of the carboxybiotin group is coupled to Na+ translocation by the ß subunit (Fig. 3B). The pyruvate product is converted via acetyl-P into acetate. The latter reaction is catalyzed by acetate kinase and yields ATP, which is the main product of the citrate fermentation pathway in K. pneumoniae. It is believed that the exchanger, CitW, provides an additional uptake mechanism for citrate by coupling the transport of citrate into the cell to the excretion of the acetate end product produced by citrate lyase and acetate kinase (58). It is not known if the exchange is energy conserving.
The citrate fermentation pathway in K. pneumoniae is induced under anaerobic conditions in the presence of citrate (15, 16). The genes coding for the enzymes are clustered in the genome in two operons that are transcribed in opposite directions (Table 1). The organization in the genome is typical for the pathway in the gamma subdivision of the Proteobacteria and is also found in the Salmonella enterica, Photobacterium profundum, and Vibrio cholerae genomes. The transporters in these organisms share a high degree of similarity with CitS of K. pneumoniae (Fig. 1, cluster III; Table 1). The gene coding for the CitS transporter is followed by the three genes coding for the three subunits of oxaloacetate decarboxylase, OadG, OadAD, and OadB (Fig. 3B), and the CitA and CitB pair, which forms a two-component signal transduction system that senses the presence of citrate in the medium. In the opposite direction and upstream of the citS gene are the genes for the biosynthesis of the citrate lyase complex in the same order as observed in the genomes of the lactic acid bacteria discussed above (CDEFXG). In the K. pneumoniae cluster, the citX gene is missing, but it is located distantly in the genome next to the gene coding for the citrate/acetate exchanger CitW (125). The location of citX correlates with the presence of citW in the genome. Apparently CitW is not essential to the pathway, since the genomes of S. enterica, P. profundum, and V. cholerae do not contain the citW gene. A close homolog of CitW is found in the genome of E. carotovora (76% sequence identity) immediately upstream of the citrate lyase gene cluster. The CitAB two-component system is divergently transcribed, but the OAD oxaloacetate decarboxylase genes are missing.
Distribution of citrate fermentation in the bacterial kingdom.
The enzyme CL, which splits citrate into oxaloacetate and acetate, is typical for bacteria and is involved in all known anaerobic bacterial citrate fermentation pathways. The presence of genes involved in the biosynthesis of CL is a diagnostic tool for the presence of the anaerobic citrate degradation pathway in the bacterium. The CL complex is built from three different subunits, , ß, and , which are encoded by the citF, citE, and citD genes, respectively (Fig. 3C). Subunit is an acyl carrier protein that carries the acetyl group via a thioester bond on a coenzyme A (CoA)-derived prosthetic group covalently linked to the protein at a serine residue. The acetyl-loaded subunit is an intermediate during catalysis. Subunit replaces the acetyl group with a citryl group with the release of acetate. Subsequently, subunit ß regenerates the acetyl-loaded state of the cofactor by releasing oxaloacetate from the citryl group. Three additional gene products are involved in the maturation of the CL complex. CitG is involved in the modification of the CoA cofactor, CitX in the attachment of the prosthetic group to the subunit, and CitC in the activation of the group by catalyzing the initial acetylation (124).
The citrate lyase genes are encoded in the genomes of only 19 of the 156 bacteria listed in Table 2 and for which the complete genome sequences are available (Table 3). Each organism listed in Table 3 contains a complete set of the three structural genes citDEF and the three accessory genes citC, citX, and citG, indicating that active CL complexes are produced. Salmonella enterica and Salmonella enterica serovar Typhimurium each contain two sets of the genes, while Clostridium tetani contains an extra set of the structural genes, but not of the accessory genes. The CL genes are found mainly in the Bacillales and Clostridia of the phylum Firmicutes and in the gamma subdivision of the Proteobacteria. In part this may be due to the low number of sequenced genomes of organisms from many of the other phyla. It seems safe to conclude that citrate fermentation is not a trait present in the phylum Actinobacteria, the Mollicutes in the Firmicutes, and the alpha and beta subdivisions of the Proteobacteria (see Table 2 for the number of finished genomes). In the gamma subdivision, the genes are remarkably well-conserved in the order CDEFXG. In Haemophilus influenzae and Haemophilus ducreyi, the CitX and CitG proteins are combined as two domains in a single polypeptide chain. In the Firmicutes, the order of the structural genes citDEF is also conserved, but the positions of the accessory genes are more variable (Table 3). A noteworthy difference between the pathways in Proteobacteria and Firmicutes is that in the former regulation of expression seems to involve a two-component signal transduction system (CitAB), while in the latter a transcriptional regulator, CitR, is involved.
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The CL gene clusters lacking an oxaloacetate decarboxylase gene are found mainly in the gamma subdivision of the Proteobacteria. In E. coli, the citT gene coding for the citrate/succinate exchanger (AIT transporter family) is located downstream of the CL genes, while the CitAB two-component signal transduction system is located upstream, on the opposite strand (Table 3). One of the two CL clusters in the genomes of S. enterica and S. enterica serovar Typhimurium have exactly the same configuration, indicating that they also code for the citrate-to-succinate pathway. Similarly, the AIT transporter gene follows the CL cluster in Haemophilus influenzae, Haemophilus ducreyi, and Shigella flexneri, but a two-component system is absent. On the H. ducreyi genome, the transporter is followed by a malic enzyme, the only one found in combination with the CL genes in the Proteobacteria. The genome of E. carotovora in the gamma subdivision does not contain a decarboxylase clustered with the CL gene, but the associated transporter is from the 2HCT family. The transporter gene is inserted between the CL genes and the genes coding for the CitAB two-component signal transduction system. The transporter is a close homolog of CitW of K. pneumoniae (
76% sequence identity; Fig. 1, cluster V), which catalyzes citrate/acetate exchange. Possibly, in this organism the CitW homolog functions as a citrate/succinate, rather than as a citrate/acetate exchanger, and has taken over the role of CitT. The only CL cluster outside the phylum Proteobacteria that is devoid of a decarboxylase is found in L. acidophilus, a Lactobacillus species in the phylum Firmicutes. This transporter is from the AIT family, which suggests that the citrate-to-succinate pathway is operative in this gram-positive bacterium. The genes coding for malate dehydrogenase, fumarase, and fumarate reductase are located immediately upstream of the CL genes. L. plantarum and other lactobacilli have been reported to produce succinate from citrate (23, 72).
CL clusters containing the OAD type of oxaloacetate decarboxylase genes are as widely distributed as the CL genes, but the actual number of the four genes, oadABDG, found in genomes of the different phyla varies. The two divergently transcribed operons coding for the OAD-dependent citrate fermentation pathway in the gamma subdivision of the Proteobacteria are found in P. profundum, Salmonella enterica, Salmonella enterica serovar Typhimurium, and V. cholerae (44). The operons are extremely well conserved and contain a complete complement of the OAD genes. The associated transporter is the Na+-citrate symporter in cluster III of the 2HCT transporter family (Fig. 1). A second group of OAD-dependent pathways is found in the Firmicutes. The genomes of Streptococcus mutans, Streptococcus pyogenes, and E. faecalis contain the genes oadA, oadB, and oadD, coding for the , ß, and subunits of oxaloacetate decarboxylase. In the Proteobacteria, the carboxyl transferase and the biotin-containing carboxyl-accepting are two domains of one protein, but in the gram-positive bacteria they form separate proteins. A gene coding for the subunit is missing in the gene cluster and is not found elsewhere on the chromosome. The E. faecalis cluster contains both an OAD type and a malic enzyme type of decarboxylase. The transporter associated with the pathway in the Firmicutes is from the [st301]CITM transporter family in structural class ST[3] in the MemGen classification (TC 2.A.11 CitMHS). Characterized members from this family transport complexes of citrate and divalent metal ions. CitM from B. subtilis transports citrate in complex with Mg2+, while CitH transports the complex of citrate and Ca2+. Both transporters are H+ symporters (65, 66). The citrate fermentation-associated genes in the spirochete T. denticola are fragmented into three parts. As in the Firmicutes, the OAD cluster contains the genes coding for the and subunits (in one protein) and for the ß subunit, but not that coding for the subunit. However, no [st301]CITM transporter homolog is encoded on the chromosome. A 2HCT member which is a close relative of BH0400 of B. halodurans (Fig. 1, cluster II) is distantly located. Finally, the fusobacterium Fusobacterium nucleatum only contains the oadA gene in a cluster with a 2HCT transporter.
CL clusters with the gene coding for the malic enzyme (CitM) decarboxylase are found mainly in the phylum Firmicutes. The transporters in these clusters are from four different families. The pathways in L. lactis and C. perfringens involve a 2HCT transporter. L. plantarum utilizes a transporter from the AIT family, E. faecalis uses a transporter from the CITM family, and C. tetani uses a transporter of the [st313]AITC family, which is also found in structural class ST[3]. The substrate specificity of the [st313]AITC transporters is not known. It is unlikely that all these gene clusters represent citrolactic fermentation pathways as present in L. lactis. Rather, other metabolic pathways yielding the initial pyruvate product may exist, as is observed for the malate fermentation pathways. On the other hand, the related malolactic fermentation pathway also involves transporters from different families (Table 2). The cluster in L. plantarum resembles the cluster in L. acidophilus, which does not contain a malic enzyme homolog and is believed to catalyze the conversion of citrate to succinate (see above). Both clusters contain the necessary enzymes for the latter conversion, suggesting that in L. plantarum oxaloacetate may be converted to succinate or to pyruvate. If so, the AIT transporter in the cluster is likely to be involved in the pathway yielding succinate. The existence of two parallel pathways following the action of citrate lyase in the anaerobic breakdown of citrate is observed in other bacteria as well. E. faecalis contains both the OAD and CitM-dependent pathways, H. ducreyi contains both the CitM-dependent pathway and the citrate-to-succinate pathway, and the Salmonella species contain the OAD-dependent and the citrate-to-succinate pathways. The two pathways may not be operative under the same conditions.
Evolutionary states of energy coupling to oxaloacetate decarboxylases. The conversion of citrate to pyruvate is common to the anaerobic breakdown of citrate in bacteria. The conversion involves the splitting of citrate into oxaloacetate and acetate catalyzed by citrate lyase and the decarboxylation of oxaloacetate to pyruvate and carbon dioxide catalyzed by oxaloacetate decarboxylase. The free energy change associated with the conversion is mainly in the latter reaction. While the standard free energy of the reaction catalyzed by citrate lyase is around zero, the standard free energy of the decarboxylation reaction is about –32 kJ/mol, which is comparable to the value for the ATP hydrolysis reaction. In mitochondria, the reverse reaction, the carboxylation of pyruvate, is coupled to ATP hydrolysis by pyruvate carboxylase. It is therefore no surprise that nature has selected the oxaloacetate decarboxylase reaction as the target site for free energy conservation in the pathway from citrate to pyruvate. The oxaloacetate decarboxylases of the CitM type and the OAD type that are involved in the citrate fermentation pathways in bacteria are completely different enzymes, and the two types represent completely different mechanisms of conserving the free energy released in the decarboxylation reaction. The malic enzymes are single-gene products that do not themselves conserve the free energy. Instead, energy conservation is achieved indirectly by "metabolic" coupling to a PMF-generating secondary transporter that takes up the substrate and expels the product of the pathway. In contrast, the OAD complex is a multisubunit complex that directly conserves the free energy by pumping Na+ across the membrane, thereby generating a sodium ion motive force. It is likely that during evolution the function of energy conservation was added to the decarboxylation reaction catalyzed by a hypothetical primordial oxaloacetate decarboxylase. Different states in evolution may still be recognized in different organisms (Fig. 4).
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subunit of the Na+-pumping oxaloacetate decarboxylase found in Proteobacteria, is present. The subunit of the OAD complex is responsible for the transfer of the carboxyl group from oxaloacetate to the biotin cofactor on the subunit (encoded by oadD) (Fig. 3B). Since the latter is not present, it is likely that carbon dioxide is released freely by the subunit in F. nucleatum. Moreover, in the OAD complex the subunit is peripherally bound to the membrane through a specific interaction mediated by the subunit (oadG), an integral membrane protein (124, 127). It follows that in F. nucleatum, OadA is a cytoplasmic enzyme that does not conserve the free energy released upon decarboxylation of oxaloacetate, in much the same way as the malic enzyme in C. perfringens (Fig. 4A and C). The pathways in F. nucleatum and C. perfringens share a similar transporter for the uptake of citrate (cluster III) and a single-subunit enzyme, but of different origin, for the decarboxylation of oxaloacetate.
Two intermediate states between the single-subunit oxaloacetate decarboxylase in F. nucleatum (Fig. 4C) and the energy-conserving OAD complex in the Proteobacteria (Fig. 4F) are found in S. mutans in the phylum Firmicutes (Fig. 4D) and in T. denticola in the phylum Spirochaetales (Fig. 4E). Energy coupling is achieved by coupling the decarboxylation reaction catalyzed by OadA to a primary transporter that transports Na+ across the membrane. Na+ pumping is driven by the decarboxylation of a carboxybiotin group that is carried by a proteinaceous substrate. Essential to the coupling mechanism is that the carboxyl group released from oxaloacetate by OadA is transferred to the carboxyl-carrier protein rather than being released freely. The gene clusters in S. mutans and T. denticola lack the gene for the subunit, leaving the protein in the cytoplasm. In S. mutans, the carboxyl-carrier protein is likely to shuttle back and forth between the oxaloacetate decarboxylase subunit in the cytoplasm and the Na+ pump ß in the membrane. A separate acquisition of the gene coding for the subunit and for the ß- pair is evident from their positions in the CL cluster. In T. denticola, the genes coding for the and subunits have fused, making and two domains of one protein that shuttles between the cytoplasm and the ß subunit in the membrane. Subunit was added to the enzyme system, linking the - protein to the transporter in the membrane, resulting in the complex found in K. pneumoniae.
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Heterologous expression systems for E. coli are abundantly available, and the NICE system, based on the nisin-inducible promoter, has provided a reliable system for expression in L. lactis (27, 67). Together, gram-negative E. coli and gram-positive L. lactis may serve as suitable hosts for genes originating from a wide range of bacteria. E. coli is especially suitable for expression of the citrate transporters in the 2HCT family, since the inability to grow on citrate as the sole carbon source under aerobic conditions is due to lack of a citrate transport system in the membrane. Functional expression of a citrate transporter from a recombinant plasmid is immediately evident from growth on citrate or the phenotype on citrate metabolism indicator plates (Simmons agar) (131). Moreover, mutants deficient in malate uptake systems are available that do not grow or grow only poorly on malate, the other main substrate of the transporters of the 2HCT family (25, 75). The genera Klebsiella and Salmonella are closely related to the genus Escherichia, and CitS and CitW of K. pneumoniae (5
