To access the private area of this site, please log in.

Username: 

Password:  

 

C₄-Dicarboxylate Degradation in Aerobic and Anaerobic Growth

Gottfried Unden* and Alexandra Kleefeld

[SECTION EDITOR: AUGUST BÖCK]

Posted July 30, 2004

Institut für Mikrobiologie und Weinforschung, Johannes Gutenberg-Universität, Becherweg 15, 55099 Mainz, Germany

*Corresponding author: Phone: 49-6131-3923550, Fax: 49-6131-3923550, E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

C₄-dicarboxylates, like succinate, fumarate, malate, tartrate, and the C₄-dicarboxylic amino acid aspartate, support aerobic and anaerobic growth of Escherichia coli and related bacteria, and can serve as carbon and energy sources. In aerobic growth, the C₄-dicarboxylates are oxidized in the citric acid cycle. Due to the interruption of the citric acid cycle under anaerobic conditions, anaerobic metabolism of the C₄-dicarboxylates depends on fumarate reduction to succinate. In some related bacteria (e.g., Klebsiella), degradation of C₄-dicarboxylates, like tartrate, uses a different mechanism and pathway. It requires the functioning of an Na⁺-dependent and membrane-associated oxaloacetate decarboxylase. Due to the incomplete function of the citric acid cycle in anaerobic growth, succinate supports only aerobic growth of E. coli. This module will describe the pathways and differences of aerobic and anaerobic C₄-dicarboxylate metabolism and the physiological consequences. The citric acid cycle, fumarate respiration, and fumarate reductase will be covered in other articles in detail and will be discussed here only in the context of aerobic and anaerobic C₄-dicarboxylate metabolism. Some recent aspects of C₄-dicarboxylate metabolism, like transport and sensing-regulation by C₄-dicarboxylates, will be treated in more detail.

 

DEGRADATION OF SUCCINATE AND OTHER C4-DICARBOXYLATES IN AEROBIC GROWTH

The C₄-dicarboxylates succinate, malate, fumarate, tartrate, and the C₄-dicarboxylic amino acid aspartate support aerobic growth of E. coli and related bacteria. The enzymes and proteins that are required specifically for C₄-dicarboxylate metabolism under aerobic and anaerobic conditions are summarized in Table 1. Aerobic growth relies on the citric acid cycle and pathways that link the carboxylates to the citric acid cycle (Fig. 1). The C₄-dicarboxylates are oxidized to CO₂ (succinate + 3.5 O₂ → 4 CO₂ + 7 H₂O), which supplies 14 [H] (2 QH₂ and 5 NADH). The reducing equivalents are reoxidized in aerobic respiration. Therefore, for growth on C₄-dicarboxylates, aerobic respiration is essential, and mutants deficient in aerobic respiration or ATP synthase are incapable of growth on succinate (8).

Uptake of C₄-dicarboxylates is effected by the DctA carrier, which has a broad substrate specificity, including fumarate, succinate, L-malate, L-aspartate, tartrate, and orotate, an aromatic monocarboylate (2, 48). dctA mutants show poor growth on C₄-dicarboxylates, indicating that DctA is the main C₄-dicarboxylate carrier in aerobic growth. Earlier, it was suggested that dctB encodes an additional aerobic C₄-dicarboxylate carrier. There were also experimental indications of a periplasmic succinate binding protein (Cbt binding protein, encoded by cbt), suggesting the existence of a binding-protein-dependent transport system (58, 59). However, only the presence of DctA (encoded bythe dctA gene) was later confirmed, and the identity of the succinate binding protein is not clear (2, 14). E. coli contains in addition a gene cluster (orfQMP) with sequence similarity to the putative products of the Rhodobacter capsulatus binding protein-dependent succinate carrier. Gene inactivation showed that the orfQMP gene products do not contribute to C₄-dicarboxylate uptake (14). However, there have to be further carriers for the uptake of C₄-dicarboxylates, since the dctA mutant is able to grow on succinate at low pH (pH 5 to 6) (43). Although succinate can diffuse passively across the membrane in the monocarboxylic form (H_succ⁻) at low pH (45), the rates are not sufficient for growth (43).

In addition to the general C₄-dicarboxylate carrier DctA, E. coli contains the aspartate carrier GlpT (15, 47, 100). GlpT is a secondary (Na⁺-independent) carrier which is, however, of minor significance for aspartate catabolism.

Intracellular succinate, fumarate, or malate is then metabolized by the reactions of the citric acid cycle with the standard set of enzymes for aerobic conditions (Fig. 1). Thus, succinate dehydrogenase (SdhABCD), fumarase C (FumC), and to some extent fumarase A (FumA) are used. For malate dehydrogenase, two isoenzymes are present in E. coli, the cytosolic NADH-dependent malate dehydrogenase (Mdh, encoded by the mdh gene) and the membrane-associated malate-quinone oxidoreductase (Mqo) (97). Mdh and Mqo are active side by side. The synthesis and activity of the Mqo enzyme are regulated by the carbon source and the ArcA/ArcB two-component system. Deletion of mdh, but not of the mqo gene, caused severely decreased growth rates. Thus, the malate dehydrogenase Mdh is the major enzyme in aerobic growth, whereas Mqo plays only a minor role. In mdh mutants, Mqo can take over the role of Mdh in part. A major role for Mqo in anaerobic growth is also unlikely, since reduction of oxaloacetate (E₀' malate/oxaloacetate = −172 mV at pH 7) (malate and oxaloacetate represent a redox pair; E₀' means E₀ at pH 7) by menaquinol (E₀' = −80 mV) is endergonic in contrast to reduction of oxaloacetate by NADH (E₀' = −320 mV) via the Mdh enzyme. In other bacteria (Corynebacterium glutamicum; Helicobacter pylori), the Mqo enzyme can be the principal or even the sole malate dehydrogenase (46, 97).

Acetyl-coenzyme A (CoA) for the citrate synthase reaction is produced by malic enzyme (Mez), which directly forms pyruvate. E. coli encodes NADP⁺ (encoded by the maeB gene)-, as well as NAD⁺ (encoded by the sfcA gene)-dependent malic enzymes. Alternatively, phosphoenolpyruvate (PEP) is produced from oxaloacetate by PEP carboxykinase (Pck, encoded by pck) (oxaloacetate + ATP → PEP + ADP + CO₂), and PEP is then converted into pyruvate by the reactions of glycolysis. During growth on C₄-dicarboxylates, both pathways are used (20, 26, 35). Acetyl-CoA is produced from pyruvate by pyruvate dehydrogenase.

The function of the citric acid cycle is decreased and interrupted under anaerobic conditions and by glucose, by transcriptional and posttranslational regulation, and by regulatory RNA (for reviews, see references 61, 64, 81, 83). Details on the functioning of the citric acid cycle are given in Chapter (Tricarboxylic Acid Cycle and Glyoxylate Bypass).

For growth on aspartate or tartrate, additional enzymes are required. Aspartate is converted into fumarate by aspartase (aspartate ammonia lyase, encoded by the aspA gene [29]) into fumarate. The pathway for aerobic tartrate degradation is mostly unknown. Tartrate can be taken up by DctA (48) or by an unknown tartrate carrier. Conversion of tartrate into an intermediate of the citric acid cycle is different from anaerobic tartrate metabolism and does not involve the tartrate dehydratase reaction (73). Alternative routes for tartrate degradation could be via glycerate using tartrate dehydrogenase and oxaloglycolate reductase or tartrate decarboxylase (22, 51, 52).

 

DEGRADATION OF FUMARATE AND RELATED C4-DICARBOXYLATES UNDER ANAEROBIC CONDITIONS (FUMARATE RESPIRATION)

The major route of anaerobic C₄-dicarboxylate catabolism in E. coli and related bacteria is fumarate respiration. In this pathway, fumarate is used as an electron acceptor for anaerobic respiration and is reduced to succinate. In most bacteria, growth by fumarate respiration requires a supply of an additional electron donor, like glycerol-3-P, H₂, or NADH (which is derived from glucose fermentation or other pathways). Malate, aspartate, and tartrate are converted to fumarate and then also metabolized by fumarate respiration. The enzymes and proteins specifically required for anaerobic C₄-dicarboxylate metabolism are summarized in Table 1.

 

Lack of Succinate Degradation under Anaerobic Conditions

Succinate is not degraded under anaerobic conditions, and succinate produced in fumarate respiration is excreted. The lack of succinate degradation is a consequence of a nonfunctional citric acid cycle under anoxic conditions and the lack of capacity for ubiquinol reoxidation. Succinate dehydrogenase is specific for ubiquinone as an electron acceptor, and the resulting ubiquinol cannot be reoxidized by anaerobic fumarate, nitrite, dimethyl sulfoxide, or trimethylamine–N-oxide respiration. The corresponding terminal reductases accept only menaquinol (MKH₂). The same applies to demethylmenaquinol or reduced menaquinone) or demethylmenaquinol (DMKH₂) as the electron donor (30, 90, 103, 104). Of the anaerobic terminal reductases, only nitrate reductase is able to use ubiquinol (as well as menaquinol) as an electron donor. E. coli, therefore, is able to degrade carbon sources, like lactate, glycerol, or succinate, oxidatively by a partially active citric acid cycle in the presence of nitrate (72, 108). Under these conditions, the citric acid cycle works with reduced activity but is not inactivated or interrupted due to the lack of glucose repression.

 

Anaerobic Growth with Fumarate by Fumarate Respiration

The metabolic pathways for anaerobic growth on fumarate, malate, and aspartate differ completely from the pathways for the same substrates during aerobic growth (Fig. 2). Fumarate reductase, which catalyzes the reverse reaction of succinate dehydrogenase, was shown at an early date to differ genetically from succinate dehydrogenase (36). E. coli requires an additional electron donor, like H₂, glycerol, or glucose, for anaerobic growth on fumarate (idealized fermentation balances: H₂ + fumarate → succinate, or glucose + 2 fumarate → 2 acetate + 2 succinate + 2 CO₂). Fumarate is taken up by a C₄-dicarboxylate (or fumarate-succinate) antiporter (DcuB) (16, 17, 80) and is then used as an electron acceptor for fumarate reductase. Succinate is excreted by the DcuB antiporter. Fumarate reductase is the terminal reductase of the fumarate respiratory chain, which uses H₂, NADH, or glycerol-3-P as the electron donor. Fumarate respiration is coupled to the generation of a proton gradient over the membrane, which can be used for the phosphorylation of ADP by ATP synthase or for other proton potential (Δp)-dependent reactions. Fumarate reductase and the fumarate-succinate antiporter DcuB are produced specifically during anaerobic fumarate respiration (24, 25, 32, 80, 83, 94, 111, 112). DcuA is a constitutively synthesized C₄-dicarboxylate carrier which has catalytic properties and activities similar to those of DcuB, and it can replace or support DcuB. In dcuA dcuB mutants, a third anaerobic C₄-dicarboxylate carrier (DcuC) can replace DcuB and DcuA in part (111). Antiport and anaerobic growth by fumarate respiration are completely lost, however, in the dcuA dcuB dcuC mutant, demonstrating that no further carrier for fumarate-succinate antiport is available.

In contrast to E. coli and Salmonella, which undergo anaerobic growth on fumarate only in the presence of hydrogen donors, like H₂ or glycerol, Proteus (Providencia) rettgeri is able to grow anaerobically with fumarate as the sole energy substrate (7 fumarate → 6 succinate + 4 CO₂) (55). Part of the fumarate is oxidized using the citric acid cycle, and the malic enzyme-pyruvate dehydrogenase bypass (2 fumarate → 1 succinate + 4 CO₂ + 10 [H]). The major portion of the fumarate is reduced to succinate by fumarate respiration (5 fumarate + 10 [H] → 5 succinate) by reoxidation of the excess [H] from the first reaction. Fumarate disproportionation by this reaction sequence requires that the citric acid cycle from fumarate to succinate is functional under anaerobic conditions in the oxidative direction, which is in contrast to the situation in E. coli and other enteric bacteria.

 

Growth with Malate or Aspartate by Fumarate Respiration

For growth on malate or aspartate, only a minor complementation of fumarate respiration is required (Fig. 2). DcuB (and DcuA) catalyze the uptake of malate or aspartate in exchange for succinate with rates similar to those for fumarate-succinate antiport. In the cytoplasm, malate is converted into fumarate by fumarase. E. coli contains three different fumarases, FumA, FumB, and FumC (5, 28, 63, 107). FumA and FumB require a catalytic iron-sulfur cluster for dehydration, whereas FumC requires no iron for activity (18, 91, 101). FumB is specifically induced during fumarate respiration under anoxic conditions (5, 68, 89). FumC is produced only under aerobic conditions, and its synthesis is increased by Fe limitation. FumA, on the other hand, is found under anaerobic and microaerobic conditions, with maximal activities under the latter conditions. The expression of the fumarase genes, in addition, is transcriptionally regulated by the C source and the iron supply. Since the fumarate respiratory system of E. coli is synthesized under anaerobic and microaerobic conditions (up to 3 to 5% air saturation) (4, 77, 95), FumB, and potentially also FumA, is used for malate dehydration during fumarate respiration. The conversion of aspartate to fumarate is catalyzed by L-aspartase (encoded by the aspA gene) (86, 106). From aspartase, only one isoenzyme is found (AspA), which has a remarkable sequence similarity to FumC (106).

The anaerobic conversion of malate to succinate represents a reversion of a segment of the citric acid cycle, which is catalyzed in aerobic metabolism by succinate dehydrogenase (Sdh) and fumarase (FumC) (63, 81, 82). The replacement of FumC by FumB, and of Sdh by Frd, is required because of the repression and interruption of the citric acid cycle under anaerobic conditions and the low activity of succinate dehydrogenase in the reverse reaction.

 

Formation of Succinate from Endogenous Fumarate (Glucose Fermentation)

Glucose can be fermented by a mixed acid fermentation with acetate, ethanol, and formate as the fermentation products (reaction 1), or by a mixed acid fermentation combined with succinate formation (reaction 2) (Fig. 3).

1 Glucose → 1 acetate + 1 ethanol + 2 formate (1)

(ΔG₀' = −218 kJ/mol of glucose)

1 Glucose + 1 HCO₃⁻ → 1 acetate + 1 succinate + 1 formate + 3 H⁺ (2)

(ΔG₀' = −260 kJ/mol of glucose)

Mostly, fermentation occurs via a mixture of both reactions, resulting in up to 0.2 mol of succinate/mol of glucose. For succinate production, PEP from the glycolytic pathway is carboxylated by PEP-carboxylase, yielding oxaloacetate, which is converted by the reductive branch of the citric acid cycle, including Mdh, fumarase (FumB), and fumarate reductase (FrdABCD), to succinate. Succinate is not further metabolized, due to the repression of the citric acid cycle and lack of capacity for ubiquinol reoxidation. Succinate is excreted by DcuC, a succinate exporter (43, 110, 112) in an electrogenic mode, presumably by symport of 3 H⁺ with succinate²⁻. DcuC is produced under anaerobic conditions in the presence of glucose. When dcuC is deleted, DcuB and DcuA, and other unknown carriers, take over the function of DcuC (43, 110). In addition, diffusion of H_suc⁻ presumably may contribute to some extent to the export of succinate (43, 45).

 

Fumarate Respiration and Energetics of Fumarate Respiration

The electron transport chain of fumarate respiration consists of various dehydrogenases and fumarate reductase, which are linked by menaquinone (MK) or by demethylmenaquinone (DMK). Of the large number of electron donors used by E. coli, NADH, H₂, and glycerol-3-P have been shown to be useful in fumarate respiration (Fig. 4), whereas formate is only a poor electron donor. Reduced menaquinone (MKH₂) (see above) (E₀' MK/MKH₂ = −80 mV) (shill separates the two partners of the redox pair), as well as reduced demethylmenaquinone (DMKH₂) (E₀' DMK/DMKH₂ = +36 mV), serves as the electron donor for fumarate reductase, whereas ubiquinol (QH₂) is not suitable (30, 57) due to the unfavorable redox potential (E₀' Q/QH₂ = +110 mV) compared to fumarate (E₀' fumarate/succinate = +30 mV). MKH₂ is the preferred electron donor for fumarate respiration and the major quinone synthesized during growth by fumarate respiration (92, 103, 104).

The function of fumarate reductase (MKH₂ + fumarate → MK + succinate) is not electrogenic, and fumarate reduction serves only as an electron sink for the dehydrogenase reactions, many of which seem to couple the redox reaction with proton translocation. Many details of fumarate reductase have been studied, and a high-resolution three-dimensional structure of the protein is available (12, 13, 31, 38). The active site for menaquinol oxidation is located in the membrane (subunits FrdCD), the active site for fumarate reduction in the cytoplasm. Electron transfer between the two sites is effected by FeS clusters in FrdB (one each [2Fe–2S], [4Fe–4S], [3Fe–4S] cluster) and a FAD residue (FrdA) at the fumarate site. Release (at the MKH₂ site) and consumption (at the fumarate site) of the protons occur on the same (cytoplasmic) side of the membrane and do not contribute to the generation of a proton potential. Details of fumarate reductase function and fumarate respiration are given elsewhere in this website.

The free energy of fumarate respiration is sufficient for generation of a proton potential and for ADP phosphorylation (H₂ + fumarate → succinate; ΔG₀' = −87 kJ/mol). E. coli is able to grow by fumarate respiration with H₂ as the electron donor, which demonstrates that fumarate respiration is coupled to the generation of a proton potential and ATP synthesis. From growth yields, it can be concluded that <0.5 ATP-fumarate is formed (6), which is in accordance with the growth and ATP yields determined for fumarate respiration by other bacteria (56). In fumarate respiration, a proton potential (▵p ≈ −160 mV) similar to the potential for nitrate or aerobic respiration is found (88). Formation of the potential depends on the reaction of the dehydrogenases, i.e., hydrogenase and NADH dehydrogenase, as indicated in Fig. 4. For NADH → fumarate respiration, the H⁺ or Na⁺/e⁻ ratio can be as high as 2, which would allow an ATP/fumarate ratio as high as 1.

 

The Dehydrogenases of the Fumarate Respiratory Chain

For NADH dehydrogenase, glycerol-3-P dehydrogenase, and hydrogenase, which play important roles in fumarate respiration, isoenzymes are present in E. coli. The isoenzymes catalyze the same redox reaction with different bioenergetic properties (for a review, see reference 93). In fumarate respiration, generally only one of the isoenzymes is used, whereas the other functions in aerobic or other electron transfer chains. The energy-conserving NADH dehydrogenase I (encoded by the nuoA to -N genes) is essential and highly expressed in anaerobic respiration with fumarate or dimethylsulfoxide (7, 87, 99). The noncoupling NADH dehydrogenase II (encoded by the ndh gene) is the major enzyme in aerobic growth and is repressed during anaerobic growth by fumarate respiration (82). Thus, in NADH-fumarate respiration, NADH dehydrogenase I, which is responsible for energy conservation, is used (Fig. 4).

NADH dehydrogenases I from E. coli and Klebsiella pneumoniae, which are highly homologous, have been supposed to couple NADH oxidation to the translocation of 2 to 4 Na⁺/NADH (54, 84). The use of Na⁺ as a coupling ion is in contrast to previous assumptions. Thus, the NADH-dependent reduction of the quinones by membranes is stimulated by sodium, and upon NADH oxidation, Na⁺ ions are transported across the membrane . The translocation of the Na⁺ ions was either resistant to or stimulated by an uncoupler. These properties suggest that the enzyme is a redox-driven primary Na⁺ pump. The enzyme complex apparently has evolved, however, from a redox-driven proton pump (hydrogenase), and there are also indications that the enzyme functions as a redox-driven proton pump (21). Future experiments must settle whether NADH dehydrogenase is a proton or a sodium pump or whether it can pump either ion depending on the conditions.

Isoenzymes for the oxidation of H₂ and glycerol-3-P are also found in E. coli, and only one of the isoenzymes is used in fumarate respiration in each case. Glycerol-3-P dehydrogenase (GlpD) of aerobic respiration is a noncoupling enzyme similar to Ndh. In fumarate respiration, anaerobic glycerol-3-P dehydrogenase (GlpABC) is used. Because of the midpoint potential of glycerol-3-P (E₀' Gly3P/dihydroxyacetone phosphate = −190 mV), which is close to that of menaquinone (ΔE₀' = −110 mV), it is unlikely that menaquinone-dependent oxidation of glycerol-3-P is used for H⁺ translocation, considering the Δp value of ~−150 to −160 mV over the membrane during fumarate respiration (88). The reason for the use of GlpABC in fumarate respiration, therefore, is not known.

Of the alternative hydrogenases, hydrogenase 2, or HybAB (encoded by the hybAB genes), is used in fumarate respiration (62, 75). H₂-fumarate respiration has to generate a proton potential, which is used for ADP phosphorylation and growth. The proton potential could stem from a redox half-loop, as suggested in Fig. 4, but additional mechanisms for generating a proton potential could be included. Thus, the dehydrogenases of fumarate respiration (or most of them) conserve redox energy in a proton or sodium gradient, whereas many of the dehydrogenases functioning in aerobic respiration do not (93). However, the functions of most of the respiratory dehydrogenases of anaerobic respiration have not been studied in detail.

 

ANAEROBIC TARTRATE DEGRADATION

Anaerobic tartrate metabolism differs significantly from the metabolism of the other C₄-dicarboxylates. Tartrate can be degraded by two different pathways involving either fumarate respiration (E. coli) or oxaloacetate decarboxylation (Salmonella and Klebsiella) as the key reaction.

 

Tartrate Degradation and Fumarate Respiration (E.coli)

E. coli is able to degrade L-, D-, and meso-tartaric acid under aerobic and anaerobic conditions (98). In anaerobic growth, tartrate is converted to fumarate via oxaloacetate and is used as an electron acceptor for fumarate respiration (Fig. 2). The dehydration of tartrate to oxaloacetate is catalyzed by stereospecific dehydratases (73), similar to a route present in other bacteria (3, 53, 79). L-Tartrate dehydratase (L-Ttd) is induced during anaerobic growth on L- and meso-tartrate, whereas D-tartrate dehydratase was found on anaerobic growth with L-, D-, and meso-tartaric acids. Of the tartrate isoenzymes, L-tartrate dehydratase has the highest activity. L-Ttd is a tetramer of two subunits (TtdA and TtdB), which are encoded by the ttdAB operon (65, 73). The enzyme is oxygen labile, and the TtdA subunit is similar in sequence and other properties to the iron-sulfur-containing class I fumarases (FumA and FumB) of E. coli. L-Ttd, as well as class I fumarases, contains catalytic (nonredox) iron-sulfur centers, like aconitase (73). The tartrate carrier has not been identified. A putative tartrate-succinate antiporter (YgjE or TtdT) is encoded by the ygjE or ttdT gene, which is located downstream of ttdAB in a probable operon with the ttdAB genes. YgjE/TtdT is similar to the citrate-succinate antiporter CitT of E. coli and other di- and tricarboxylate carriers (71). The presence of the putative TtdT carrier suggests the uptake of tartrate in exchange for succinate, which is the end product of anaerobic tartrate degradation. There have to be further (unknown) tartrate dehydratases specific for D- and meso-tartrate in anaerobic growth.

 

Tartrate Degradation Involving Na+-Dependent Oxaloacetate Decarboxylase

Salmonella enterica serovar Typhimurium strain LT2 and K. pneumoniae are able to grow by tartrate fermentation on L- or D-tartrate as the sole carbon and energy source (60, 66, 105). In these bacteria, growth on tartrate is Na⁺ dependent and induces the synthesis of an Na⁺-activated oxaloacetate decarboxylase, which is located in the membrane (see Chapter Molecular Basis for Bacterial Growth on Citrate or Malonate). The oxaloacetate decarboxylases couple decarboxylation to the export of sodium ions and the generation of a sodium motive force (102). Pyruvate formed by oxaloacetate decarboxylation enables fermentative metabolism and ADP phosphorylation by acetate production without the need for fumarate respiration and an external electron donor, as in E. coli.

 

CONVERSION OF SUCCINATE TO PROPIONATE BY E. COLI

Recently, a new pathway for the conversion of succinate into propionate (succinate → propionate + CO₂) was identified in E. coli (34). First indications of the pathway came from the identification of genes for paralogues of the crotonase (enoyl CoA hydratase) family with unknown function on the E. coli genome. The gene for one of the paralogues (YgfG) is part of a four-gene operon. To identify the functions of the genes, the gene products were overproduced, and their enzyme activities were analyzed. The YgfG protein proved to have methylmalonyl CoA decarboxylase activity. The other genes encoded propionyl CoA-succinate CoA transferase (YgfH), methylmalonyl CoA mutase (Sbm), and a putative protein kinase (YgfD/ArgK). The enzyme reactions can be linked to a metabolic cycle for the decarboxylation of succinate to propionate. The metabolic context of the cycle and the conditions for the synthesis of the enzymes and functioning of the pathway are unknown. The pathway could be used for continued decarboxylation and degradation of succinate, and therefore it is unlikely that this pathway finally utilizes succinyl-CoA or ketoglutarate from the citric acid cycle, which already provides succinyl-CoA. The function of the kinase YgfD/ArgK is not known, but it does not function as a succinate or propionate CoA ligase. It was suggested, therefore, that YgfD/ArgK represents a protein kinase which regulates the activities of other enzymes in E. coli that utilize succinyl-CoA, propionyl-CoA, propionate, or succinate. Although there is clear evidence that the enzymes catalyze the suggested reactions, the metabolic function of the suggested pathway remains unknown.

 

TRANSCRIPTIONAL REGULATION OF C4-DICARBOXYLATE METABOLISM BY ELECTRON ACCEPTORS

E. coli and related bacteria use electron acceptors in catabolism in a specific order, or hierarchy (31, 32, 83, 93, 94). O₂ is the preferred electron acceptor and represses other respiratory pathways and fermentation, whereas nitrate represses other anaerobic respiratory pathways, including fumarate respiration, and fermentation. Thus, fumarate respiration functions only in the absence of nitrate and O₂. The ΔG values, H⁺/e⁻ ratios, and ATP yields are highest in aerobic respiration, intermediate in nitrate respiration, and lowest in fumarate respiration (32, 93). Therefore, the hierarchical control maximizes ATP yields. In addition, the growth rates during aerobic growth are much higher and exceed those for growth by fumarate respiration (μ = 0.21 h⁻¹) by a factor of ~6 (with glycerol as the electron donor).

The hierarchical control is achieved by transcriptional regulators responding to electron acceptors. Thus, many of the genes of C₄-dicarboxylate metabolism are regulated by overriding gene regulators responding to O₂ and nitrate and by a specific regulator responding to C₄-dicarboxylates. Transcriptional regulation by O₂ is effected by the FNR (fumarate nitrate reductase regulator) and ArcA (aerobic respiratory control) proteins. FNR is a cytoplasmic sensor and gene regulator with a helix-turn-helix DNA binding domain. The protein contains a sensory domain with a [4Fe4S] cluster which senses the presence of O₂ in the cytoplasm (4) and controls the functional state of FNR as a gene activator (27, 50, 83). ArcA is the response regulator of the ArcB-ArcA two-component system, which responds to the presence of O₂ via the functional state of the aerobic respiratory chain (37). Nitrate regulation is effected by two two-component systems, NarX-NarL and NarP-NarQ, which sense the presence of external nitrate (11, 85). The C₄-dicarboxylate-specific regulation is effected by the DcuS-DcuR two-component system (25, 39, 112). The transcriptional regulators bind in various combinations and modes to the promoter regions of the genes of fumarate metabolism (Table 2).

From the genes of anaerobic C₄-dicarboxylate metabolism (Table 2), the frdABC and dcuB fumB operons are transcriptionally activated by FNR under anaerobic conditions and repressed by NarL in the presence of nitrate. In addition, expression of both operons is induced by C₄-dicarboxylates via the DcuS-DcuR two-component system. fumB is cotranscribed with dcuB from the dcuB promoter.

From the genes and proteins of aerobic C₄-dicarboxylate metabolism (Table 2), the expression of dctA is subject to anaerobic repression by ArcA and to weak induction by DcuS-DcuR and succinate. Expression of the succinate dehydrogenase genes sdhCDAB occurs under aerobic conditions by negative repression of the sdhCDAB genes by ArcA and FNR during anaerobic growth. C₄-dicarboxylates have no DcuS-DcuR-dependent effect on sdhCDAB expression. Synthesis of α-ketoglutarate dehydrogenase (sucAB genes) and of succinyl CoA synthetase (sucCD genes) is also controlled from the sdhC promoter in the same way. Expression of sdhC is additionally regulated by Fe ions and the ferric uptake regulator Fur. The effect is exerted by a small regulatory RNA, RhyB, the synthesis of which is regulated by Fur (61). RhyB contains sequences which are complementary to sequences within sdhC, suggesting that RhyB acts as an antisense RNA. This regulation is responsible for the lack of growth of fur mutants on succinate.

The expressions of the fumA, fumB, and fumC genes encoding the alternative fumarases differ in their responses to electron acceptors. Fumarase A contains a catalytic FeS cluster, whereas fumarase C does not require iron for its function. Fumarase C is produced under aerobic conditions and repressed under anaerobic conditions by ArcA. Fumarase C is important for aerobic growth under Fe limitation. The fumA gene, encoding fumarase A, on the other hand is repressed under anaerobic conditions by ArcA and FNR. Activity of fumarase A is found under aerobic and anaerobic conditions, but the maximal activity is produced under microaerobic conditions.

 

C4-DICARBOXYLATE CARRIERS

E. coli contains a large number of carriers for C₄-dicarboxylates to meet the requirements for different catabolic conditions. The C₄-dicarboxylate carriers can be grouped in four families, the DctA, DcuA/B, DcuC, and CitT families (Fig. 5). E. coli contains in addition a putative C₄-dicarboxylate carrier of the TRAP (tripartite ATP-independent periplasmic transporter) family (19, 49).

 

Aerobic DctA Carrier

DctA from E. coli is one of the DctA carriers that are found in many gram-negative and gram-positive bacteria (Fig. 5) (14, 41). The DctA carrier is predicted to be a secondary carrier composed of 10 to 12 transmembrane helices. DctA catalyzes the uptake of succinate, fumarate, malate, aspartate, tartrate, and orotate (2, 48). The K_ms for the C₄-dicarboxylates are ~10 to 30 μM, and the V_max is ~25 to 50 μmol/min/g (dry weight). The transport depends on the presence of a proton potential (Fig. 6), and about two H⁺ ions enter the cell with each C₄-dicarboxylate (33) (reaction 3):

Succinate²⁻_out + 2 H⁺_out → succinate²⁻_in + 2 H⁺_in (3)

The DctA carrier is used in aerobic growth of E. coli (14); accordingly, dctA mutants show only poor growth on C₄-dicarboxylates. At more acidic pH (pH < 6), the dctA mutant regains growth on succinate, but not on fumarate, presumably by the transport of succinate as a monoanion (H_succinate⁻) by a monocarboxylate carrier (43). Carriers of the DctA type are found in many aerobic bacteria, but not in anaerobic bacteria, and DctA from E. coli belongs to the subfamily of DctA carriers which is found in gram-negative bacteria and gram-positive bacteria with low G+C contents (39).

 

The DcuA/DcuB Carriers

The DcuA and DcuB carriers form an independent family (Fig. 5) that is present only in anaerobic and facultatively anaerobic bacteria capable of fumarate respiration (39). DcuA and DcuB carriers are capable of C₄-dicarboxylate exchange, uptake, and efflux but operate preferentially as exchange carriers (Fig. 6). The DcuAB family, which was also called the Dcu family (76), can be subdivided into the DcuA and DcuB subgroups. The DcuAB carriers are required for growth by fumarate respiration and catalyze mainly fumarate-succinate antiport. The carriers also catalyze uptake or efflux, which might represent partial reactions of antiport (17, 41, 80, 112). DcuB is produced during fumarate respiration (24, 25, 111). The fumB gene, encoding anaerobic fumarase B, is located downstream of dcuB and cotranscribed with it (24, 80). dcuA, on the other hand, is located adjacent to aspA, encoding aspartase (24, 80). The dcuA and aspA genes are expressed constitutively (24).

The protein sequence suggests 12 transmembrane helices for DcuA and DcuB. Experimentally, 10 transmembrane helices were determined for DcuA (23). In this model, the protein contains a large cytoplasmic loop between transmembrane helices 5 and 6, and the N- and C-terminal ends are located in the periplasm. DcuB is the most important fumarate-succinate antiporter of E. coli and is ~2.3 times as active as DcuA (112). The K_m values for fumarate and succinate are ~100 μM for the exchange or uptake reactions. The DcuAB carriers catalyze electroneutral antiport (reaction 4), which exceeds uptake or efflux activities by factors of ~3. At neutral pH, the substrates are transported as divalent anions.

Succinate²⁻_in + fumarate²⁻_out ↔ succinate²⁻_out + fumarate²⁻_in (4)

Net uptake of C₄-dicarboxcylates is observed when no internal countersubstrate is present (17, 41, 112). The uptake is an electrogenic H⁺-fumarate symport that requires a proton potential over the membrane (reaction 5). By this transport process, an accumulation of C₄-dicarboxylates by a factor of ≥60 was found.

Fumarate²⁻_out + 3 H⁺_out → fumarate²⁻_in + 3 H⁺_in (5)

DcuA and DcuB are also able to catalyze an efflux of C₄-dicarboxylates The efflux (reaction 6) of succinate generates a membrane potential (ΔΨ), and it was assumed to represent the reversal of the uptake, i.e, an electrogenic efflux of 3 H⁺ with succinate²⁻. In this way, the reaction would contribute to the formation of an electrochemical proton potential by the excretion of the fermentation product (17, 39).

Succinate²⁻_in + 3 H⁺_in → succinate²⁻_out + 3 H⁺_out (6)

 

DcuC/DcuD Carriers

DcuC is similar to the DcuA/DcuB carriers in many respects. DcuC catalyzes the exchange, uptake, and efflux of C₄-dicarboxylates, but the major role is in efflux (39, 110, 112) (Fig. 6). It is assumed that efflux catalyzed by DcuC is electrogenic, as described in reaction 6. The DcuC carriers form a distinct family (Fig. 5) different from the DcuAB family (39, 112). Carriers of the DcuC family are found in bacteria capable of hexose fermentation and succinate production. DcuD from E. coli is a member of the same family. dcuD encodes an intact protein, but the gene appears to be silent, and the function of DcuD from E. coli and other bacteria is unknown (39, 40, 43).

The activity of DcuC is distinctly lower than those of DcuA and DcuB. Thus, only DcuA and DcuB have sufficient activity to maintain growth by fumarate respiration, whereas DcuC allows only decreased growth by fumarate respiration in the corresponding mutant strains. DcuC synthesis is not repressed by glucose, in contrast to DcuB. For these reasons, the specific role of DcuC apparently lies in succinate efflux during glucose fermentation. DcuA and DcuB, however, can fully replace DcuC in dcuC deletion strains.

 

TtdT and CitT Carriers

The putative anaerobic tartrate-succinate antiporter TtdT (or YgjE) and the citrate-succinate antiporter CitT of E. coli are members of the carboxylate–C₄-dicarboxlyate antiporter family. In E. coli, only the citrate-succinate antiporter CitT has been characterized (71), whereas most of the carriers of this type are found in mitochondria and chloroplasts. CitT is required for citrate fermentation, where citrate is converted to succinate and acetate. CitT is responsible for citrate uptake and succinate efflux and cannot be replaced by DcuA, DcuB, or DcuC, suggesting that CitT catalyzes coupled citrate-succinate antiport. The ttdT gene, which is located downstream of the tartrate dehydrogenase genes ttdAB, presumably is responsible for tartrate-succinate antiport in a similar way.

 

Regulation of C4-Dicarboxylate Carrier Synthesis

The expression of the dctA, dcuA, dcuB, and dcuC genes is subject to transcriptional regulation in response to electron acceptors, the carbon source, and other factors, like the growth phase (Table 2). Synthesis of DctA is maximal in the stationary growth phase during aerobic growth on succinate or other C₄-dicarboxylates (14). Under anaerobic conditions, dctA expression is strongly repressed by the ArcA/ArcB two-component system. C₄-dicarboxylates cause an ~2-fold induction via the DcuS/DcuR two-component fumarate regulatory system (15, 111). Glucose represses DctA ~30-fold by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. CRP is also responsible for stationary-growth-phase induction (14).

The synthesis and activities of the Dcu carriers are regulated at the transcriptional and posttranslational levels. Posttranslational inactivation is caused by electron acceptors like O₂ and is reversed by reducing agents (16). The basis for posttranscriptional regulation is not known. The expression of the dcuB and dcuC genes is highly regulated, whereas that of dcuA is constitutive and shows only a slight induction (>2-fold) under anaerobic conditions (24, 25, 110, 111). Expression of dcuB is activated under anaerobic conditions in the presence of fumarate by the O₂ sensor FNR and the C₄-dicarboxylate-regulatory DcuS/DcuR two-component system. Nitrate repression depends on the nitrate-responsive NarXL two-component system, and catabolite repression is effected by CRP. Altogether, 150-fold induction takes place during growth by fumarate respiration compared to aerobic growth.

Expression of dcuC also requires activation by FNR under anaerobic conditions. The expression of dcuC is slightly stimulated by fumarate (factor 2) and repressed by nitrate (110). Thus dcuC, in contrast to dcuB, is expressed to a high level in glucose fermentation.

The dcuA gene is expressed constitutively and shows only a slight increase in expression during anaerobiosis (24). DcuA seems to be a general back-up carrier. Despite the expression of DcuA under aerobic conditions, it does not support substantial growth on succinate under aerobic conditions (14, 43) which could be due to posttranslational inactivation by O₂ (16).

 

REGULATION BY C4-DICARBOXYLATES

 

The DcuS-DcuR Two-Component System

In E. coli, the expression of the dcuB, frdABCD, fumB, and dctA genes is stimulated by the presence of fumarate, succinate, or other C₄-dicarboxylates. Other genes, like sdhCDAB, dcuC, dcuA, or aspA, are not or only slightly transcriptionally regulated by the addition of C₄-dicarboxylates to the medium. A regulatory system (dcuS dcuR genes) responding to the presence of external C₄-dicarboxylates was identified upstream of the dcuB gene (25, 111). The dcuS dcuR genes encode a C₄-dicarboxylate sensor-regulator pair consisting of a sensory histidine kinase (DcuS) and a response regulator (DcuR), typical for two-component systems. The fumarate sensor is a member of the CitA family of histidine protein kinases and shows close similarity to the citrate sensor CitA from E. coli and Klebsiella (10, 41). The supposed topology and structural elements of the DcuS-DcuR system are shown in Fig. 7. DcuS contains a periplasmic sensory domain that is enclosed by two transmembrane helices. The second transmembrane helix is followed by a cytoplasmic PAS domain and the transmitter domain, with a conserved His residue and a kinase domain. The function of the cytoplasmic PAS domain is not known (1). The periplasmic sensory domain enables signal reception from the outside of the cytoplasmic membrane without uptake of the signal molecule (25, 111).

DcuS is able to use any of the physiological and nonphysiological C₄-dicarboxylates (fumarate, succinate, malate, aspartate, tartrate, or maleate) as a regulatory signal, whereas monocarboxylates (butyrate) and C₃- or C₅-dicarboxylates are only poor effectors. Obviously, DcuS recognizes stimuli or ligands containing two carboxylate groups in a spacing of 3.1 to 3.9 Å (44). The structure of the water-soluble periplasmic domain of DcuS has been determined by nuclear magnetic resonance spectroscopy in solution (67) (Fig. 8). The binding domain constitutes a novel fold (α₃βαβ₃α), and the nearest structural neighbor is the PAS domain of the photoactive yellow protein (PYP) from Halorhodospira halophila (9). The core of the binding domain is a β-sheet structure which is surrounded by α-helices. A cluster of positively charged or polar amino acid residues (Arg107, His110, Phe120, Arg147, and Phe149), which are situated in the core of the domain, is required for C₄-dicarboxylate binding. The C- and N-terminal ends of the periplasmic domain are formed by long α-helical structures that are closely linked to transmembrane helices 1 and 2 of DcuS. This type of linking could be important for signal transfer into and across the membrane. The structure of the DcuS binding domain is very similar to that of the citrate binding domain of the related CitA sensor (74). The structure of the binding domain of the aspartate sensor Tar, which belongs to the family of methyl-accepting chemotaxis proteins, however, is distinctly different. The periplasmic binding domain of Tar is constituted from a four-helix bundle structure per monomer, and Asp is bound at the interface between two monomers of the dimer (109).

The intact DcuS sensor protein can be solubilized in detergent. After reconstitution in liposomes, DcuS is capable of autophosphorylation, and the autophosphorylation responds to the presence of C₄-dicarboxylates. Thus the isolated protein has the same specificity as in vivo (41). The phosphoryl group can be transferred to the response regulator DcuR, which is phosphorylated at a conserved Asp residue (42). Phosphorylated DcuR binds specifically to promoters of target genes, like dcuB, frdA, and dctA (1, 42), and activates transcription of the target genes.

 

Other Regulators Specific for C4-Dicarboxylate Metabolism

In addition to DcuSR, other regulators have been suggested to play a role in the regulation of C₄-dicarboxylate metabolism. Thus, the succinate stimulation of dctA expression is lost only in part in dcuSR mutants, suggesting that further regulators are involved (25, 111). The gene regulator YhiF, which is a member of the LuxR family of helix-turn-helix transcriptional regulators, has been suggested to regulate succinate metabolism (8) and could be involved in DcuSR-independent succinate regulation. Furthermore, tRNAs have been implicated in expression control of the C₄-dicarboxylate metabolism of S. enterica serovar Typhimurium (70, 96). tRNAs from all organisms contain modified nucleotides, some of which improve reading frame maintenance. The ability of S. enterica serovar Typhimurium to grow on succinate, fumarate, or malate depends on the hydroxylation of a modified adenosine residue of tRNAs at position 37 (ms₂io₆A₃₇). The hydroxylation of A37 is effected by a hydroxylase, the miaE gene product. Only S. enterica serovar Typhimurium containing the hydroxylated form of tRNA is able to grow on the C₄-dicarboxylates. It is not known whether this effect can be regarded as a specific regulation or whether genes for succinate metabolism (and others) respond more sensitively to the loss of modification.

 

ACKNOWLEDGMENTS

Work in the author’s laboratory was supported by grants from Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.