C₄-Dicarboxylate Utilization in Aerobic and Anaerobic Growth

Abstract

C₄-dicarboxylates and the C₄-dicarboxylic amino acid l-aspartate support aerobic and anaerobic growth of Escherichia coli and related bacteria. In aerobic growth, succinate, fumarate, D- and L-malate, L-aspartate, and L-tartrate are metabolized by the citric acid cycle and associated reactions. Because of the interruption of the citric acid cycle under anaerobic conditions, anaerobic metabolism of C₄-dicarboxylates depends on fumarate reduction to succinate (fumarate respiration). In some related bacteria (e.g., Klebsiella), utilization of C₄-dicarboxylates, such as tartrate, is independent of fumarate respiration and uses a Na⁺-dependent membrane-bound oxaloacetate decarboxylase. Uptake of the C₄-dicarboxylates into the bacteria (and anaerobic export of succinate) is achieved under aerobic and anaerobic conditions by different sets of secondary transporters. Expression of the genes for C₄-dicarboxylate metabolism is induced in the presence of external C₄-dicarboxylates by the membrane-bound DcuS-DcuR two-component system. Noncommon C₄-dicarboxylates like l-tartrate or D-malate are perceived by cytoplasmic one-component sensors/transcriptional regulators. This article describes the pathways of aerobic and anaerobic C₄-dicarboxylate metabolism and their regulation. The citric acid cycle, fumarate respiration, and fumarate reductase are covered in other articles and discussed here only in the context of C₄-dicarboxylate metabolism. Recent aspects of C₄-dicarboxylate metabolism like transport, sensing, and regulation will be treated in more detail. This article is an updated version of an article published in 2004 in EcoSal Plus. The update includes new literature, but, in particular, the sections on the metabolism of noncommon C₄-dicarboxylates and their regulation, on the DcuS-DcuR regulatory system, and on succinate production by engineered E. coli are largely revised or new.

INTRODUCTION

C₄-dicarboxylates play a central role in energy and C-metabolism of many bacteria because of their close link to the central metabolism. Various groups of proteobacteria, such as the nonglucophilic Pseudomonads and Ralstonia, use C₄-dicarboxylates even as the preferred substrate for growth, whereas the glucophilic E. coli and Salmonella use sugars as substrates in preference to C₄-dicarboxylates. Remarkably, C₄-dicarboxylate metabolism is completely different under aerobic and anaerobic conditions. Under aerobic conditions, the common C₄-dicarboxylates (succinate, fumarate, L-malate, and oxaloacetate), as well as L-aspartate, are fed to the citric acid cycle and oxidized (Fig. 1). Under anaerobic conditions, however, fumarate respiration represents the fundamental pathway for C₄-dicarboxylates in catabolism. In E. coli L-malate, L-aspartate, and citrate, as well as the noncommon C₄-dicarboxylates L-tartrate, D-tartrate, and D-malate, are converted to fumarate and then used in fumarate respiration (Fig. 1). The succinate produced cannot be oxidized because of the nonfunctional citric acid cycle and is therefore excreted. Aerobic and anaerobic catabolism of common and noncommon C₄-dicarboxylates, including enzymes, reactions, transmembrane transport, and regulation in response to the C₄-dicarboxylates, will be the major topic of this overview.

Figure 1.

Different strategies for aerobic (A) and anaerobic (B) utilization of C₄-dicarboxylates by E. coli using the tricarboxylic acid cycle (TCA) (A) and fumarate respiration (B). Anaerobic utilization of L-tartrate and citrate by Salmonella and Klebsiella occurs by a different route that involves conversion of OAA (oxaloacetate) to pyruvate by a Na⁺-dependent oxaloacetate decarboxylase (without fumarate respiration).

OXIDATION OF C₄-DICARBOXYLATES IN AEROBIC GROWTH

Succinate, L-Malate, Fumarate, and L-Aspartate as the Substrates

E. coli is able to use the C₄-dicarboxylates succinate, fumarate, L-malate, and L-aspartate for growth. The enzymes and proteins that are required for metabolizing externally supplied C₄-dicarboxylates under aerobic and anaerobic conditions are summarized in Table 1. Aerobic growth relies on C₄-dicarboxylate transporters, the citric acid cycle, and the phosphoenolpyruvate (PEP)/pyruvate bypass to produce acetyl-CoA for feeding the citric acid cycle (Fig. 2). Succinate oxidation (succinate + 3.5 O₂ → 4 CO₂ + 3 H₂O) supplies 14 [H] (2 QH₂ and 5 NADH), which are reoxidized in aerobic respiration. Therefore, aerobic respiration is essential for growth on C₄-dicarboxylates, and mutants deficient in aerobic respiration or ATP synthase are incapable of growth on succinate (1).

Table 1.

Genes and proteins specific for C₄-dicarboxylate metabolism in E. coli^a

Enzyme or protein	Gene	Function or property (reaction)	Family
AspA	aspA	Aspartase (aspartate ammonia lyase)	Class II fumarase/aspartase family
CS	gltA	Citrate synthase	Citrate synthase family
DmlA	dmlA	D-Malate dehydratase	Isocitrate and isopropylmalate dehydrogenases family
FrdABCD (M)	frdABCD	Fumarate reductase (fumarate + MKH2 → succinate + MK)
FumA	fumA	Fumarase A (microaerobic); catalytic FeS cluster	Class I fumarase family
FumB	fumB	Fumarase B (anaerobic); catalytic FeS cluster	Class I fumarase family
FumC	fumC	Fumarase C (aerobic)	Class II fumarase/aspartase family
Mdh	mdh	NAD-dependent malate dehydrogenase	LDH/MDH superfamily
Mez1	maeB	Putative malic enzyme; NADP linked (malate + NADP+ → pyruvate + NADPH + CO2)	Malic enzymes family
Mez2	sfcA	Putative malic enzyme; NAD(P) linked (malate + NAD(P) → pyruvate + NAD(P)H + CO2)	Malic enzymes family
Mqo (M)	mqo	Malate dehydrogenase (Q dependent) (malate + Q → oxaloacetate + QH2)	MQO family
Pck	pck	PEP carboxykinase (oxalacetate + ATP → PEP + ADP + CO2)	Phosphoenolpyruvate carboxykinase [ATP] family
Ppc	ppc	PEP carboxylase (PEP + HCO3- → oxalacetate + Pi)	PEPCase type 1 family
SdhABCD (M)	sdhCDAB	Succinate dehydrogenase SdhABCD (succinate + Q → fumarate + QH2)
Sbm	sbm	Methylmalonyl CoA mutase
L-Ttd	ttdAB	L-Tartrate dehydratase (TtdAB-catalytic FeS) (L-tartrate → oxaloacetate + H2O)
YgfG/ScpB	ygfG/scpB	Methylmalonyl-CoA decarboxylase
YgfH/ScpC	ygfH/scpC	Propionyl-CoA-succinate CoA transferase	Acetyl-CoA hydrolase/transferase family
YgfD/ArgK	ygfK/argK	GTPase interacts with methylmalonyl-CoA mutase ScpA	SIMIBI class G3E GTPase family
DauA (M)	dauA	Aerobic succinate carrier (uptake) at pH 5	Sulfate permease (SulP) family(TC 2.A.53.3.11)
DctA (M)	dctA	Aerobic succinate carrier (uptake) at pH 7	Dicarboxylate/amino acid:cation (Na+ or H+) symporter (DAACS) family (TC 2.A.23.1.7)
DcuA (M)	dcuA	Fumarate carrier; anaerobically active	C4-dicarboxylate uptake (Dcu) family (TC 2.A.13.1.1)
DcuB (M)	dcuB	Fumarate-succinate antiporter	C4-dicarboxylate uptake (Dcu) family (TC 2.A.13.1.2)
DcuC (M)	dcuC	Succinate export carrier	C4-dicarboxylate uptake c (DcuC) family (TC 2.A.61.1.1)
DcuD (M)	dcuD	Carrier of DcuC family (silent?)	C4-dicarboxylate uptake c (DcuC) family (TC 2.A.61.1.2)
GltP	gltP	Proton-dependent glutamate/aspartate symporter	Dicarboxylate/amino acid:cation (Na+ or H+) symporter (DAACS) family (TC 2.A.23.1.1)
SatP	satP	Succinate/acetate proton symporter at pH 6	Acetate uptake transporter (AceTr) family (TC 2.A.96.1.1)
TtdT (M)	ygiR/ttdT	Tartrate-succinate antiporter	Divalent anion:sodium symporter(dass) family (TC 2.A.47.3.3)
DcuS (M) DcuR	dcuS dcuR	C4-DC sensor (DcuS) and response regulator (DcuR) of DcuSR two-component system
DmlR	dmlR/yeaT	DNA-binding transcriptional regulator
RyhB	ryhB	Regulatory RNA

^a Only enzymes or proteins specifically involved in C₄-dicarboxylate metabolism are shown. Most of the enzymes are designated according to genes names. (M), membranous location of a protein. For some enzymes, the reactions catalyzed are given.

Figure 2.

Pathways of aerobic C₄-dicarboxylate utilization by E. coli. The scheme gives the sequence of enzyme reactions for the utilization of succinate (Succ) and other C₄-dicarboxylates in aerobic growth (1 succinate + 3.5 O₂→ 4 CO₂ + 3 H₂O). For simplicity, not all intermediates for the conversion of citrate to succinate, and of PEP to pyruvate (Pyr), are shown. Membrane-associated or integral enzymes are indicated by their locations. DctA, SatP, and DauA are transporters for the uptake of the common C₄-dicarboxylates at pH > 6, pH 6, and pH 5, respectively. DctA catalyzes also the uptake of the noncommon C₄-dicarboxylates D-malate and L-tartrate under aerobic conditions. DcuA is produced constitutively and transports C₄-dicarboxylates and particularly L-aspartate (L-Asp). The pathway for aerobic oxidation of L-tartrate is not known. AcCoA, acetyl-CoA; CS, citrate synthase; DctA, aerobic C₄-dicarboxylate transporter; DmlA, D-malate dehydrogenase; Sdh, succinate dehydrogenase SdhABCD; FumA and FumC (aerobic) fumarase; Mal, malate; Mdh, cytosolic (NADH-dependent) malate dehydrogenase; Mqo, membrane-associated malate-quinone oxidoreductase; MaeB and SfcA, NAD(P)H-dependent malic enzymes; OAA, oxaloacetic acid; Pck, PEP carboxykinase; PDH, pyruvate dehydrogenase; TCA, tricarboxylic acid cycle. For details, see text.

Uptake of C₄-dicarboxylates is effected at neutral pH by the DctA transporter (dctA gene), and dctA mutants show poor growth on C₄-dicarboxylates (2, 3, 4), although they retain efficient growth on succinate at low pH (pH 5 to 6) (5). Succinate transport at acidic pH 5 is brought about by the DauA transporter, which is a member of the SLC26 or SulP family of secondary anion transporters (Table 1) (6). Details on the transporters and their function are presented in the section “C₄-Dicarboxylate Transporters” below.

The presence of aerobic C₄-dicarboxylate transporters in addition to DctA was suggested by previous genetic and physiological studies (dctB and cbt genes) (7, 8). The identity of the genes and their relation to the aerobic transporter genes (dauA, satP, dcuA, and gltP) is not clear. E. coli contains a gene cluster (orfQMP) encoding proteins with sequence similarity to the Rhodobacter capsulatus binding protein-dependent succinate carrier DctQMP (9). Gene inactivation indicated, however, that orfQMP is not involved in the transport of C₄-dicarboxylates (10), and the genes have been postulated to contribute to the transport of L-xylose or other pentoses (10, 11).

After uptake, the “common” C₄-dicarboxylates that are part of the citric acid cycle (succinate, fumarate, and L-malate) are metabolized by the reactions of the citric acid cycle (Fig. 2). Aspartate is deaminated by aspartase AspA (or aspartate ammonia lyase), which is encoded by aspA to fumarate (12). For dehydration of L-malate to fumarate, alternative fumarases can be produced. The class I fumarase FumA is the most abundant enzyme under aerobic growth conditions. The oxygen-stable FumC is a Fe-independent class II fumarase and is more important in aerobic growth with iron limitation and aerobic stress, which is characterized by superoxide radical accumulation and NADPH limitation (13, 14, 15). FumB, like FumA, is a class I fumarase with an oxygen-sensitive iron-sulfur cluster that is repressed by the presence of oxygen and glucose, so it is produced only under anaerobic conditions (15, 16). 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) (17). Mdh and Mqo are active side by side. 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, causes severely decreased growth rates. This indicates that Mdh is the major enzyme in aerobic growth even though oxidation of malate (E^′₀ oxaloacetate/malate = −172 mV) by NAD (E^′₀ NAD/NADH₂ = −320 mV) is very unfavorable and endergonic (ΔG^′ = +28.6 kJ/mol). On the other hand, ubiquinone (E^′ Q/QH = +110 mV), which is used by Mqo, represents a suitable electron acceptor for malate oxidation and renders the reaction exergonic. In mdh mutants, Mqo can take over the role of Mdh in part. In other bacteria like Corynebacterium glutamicum and Helicobacter pylori, the Mqo enzyme can be the principal or even the sole malate dehydrogenase (17, 18).

The acetyl coenzyme A (acetyl-CoA) for feeding the citric acid cycle from the C₄-dicarboxylates is produced by the PEP/pyruvate bypass (Fig. 2). E. coli encodes NADP⁺ (MaeB, maeB gene), as well as NAD⁺ (SfcA, sfcA gene) dependent malic enzymes, and pyruvate can be formed by both malic enzymes, MaeB and SfcA. Alternatively, PEP is produced from oxaloacetate by PEP carboxykinase (Pck, pck gene) (oxaloacetate + ATP → PEP + ADP + CO₂). PEP is then converted to pyruvate through glycolysis. During growth on C₄-dicarboxylates, both pathways are used (19, 20, 21). Under aerobic conditions, pyruvate is decarboxylated to acetyl-CoA by pyruvate dehydrogenase.

The function of the citric acid cycle is decreased and interrupted under anaerobic conditions. In addition, excess glucose and iron limitation impede expression of genes coding for Fe-containing citric acid cycle enzymes (SdhABC, FumA, aconitases AcnA and AcnB) and for respiratory enzymes (NADH dehydrogenase I) (reviewed in references 22, 23, 24, 25). The repression under iron limitation is effected by the small regulatory RNA RyhB, the synthesis of which is derepressed under iron limitation by the iron uptake regulator Fur (22, 26). Details on citric acid cycle function are given in the EcoSal Plus article by Cronan and Laporte (27).

Growth on Noncommon C₄-Dicarboxylates (D-Malate and Tartrate)

D-Malate and L-tartrate support aerobic growth of E. coli (4, 28, 29). Growth on D- and meso-tartrate has been observed only for some strains (29), whereas others do not grow on the substrates (28). For aerobic growth on C₄-dicarboxylates like D-malate or the tartrate stereoisomers, additional enzymes are required to convert the substrates to common C₄-dicarboxylates or other central metabolites (Fig. 2). The source and occurrence of D-malate is not clear, but a specific pathway for D-malate oxidation has been described. Uptake of D-malate is achieved by DctA (28), and the cytoplasmic D-malate is oxidized to pyruvate by the (decarboxylating) D-malate dehydrogenase DmlA (4). The enzyme is presumably identical to the inducible “D-malic enzyme” described previously (30) catalyzing the same reaction. The induction of DmlA is mediated by LysR-type transcriptional regulator DmlR in the presence of D-malate or tartrate stereoisomers (28, 30). The reaction of DmlA resembles that of isocitrate dehydrogenase, and both enzymes are members of the same dehydrogenase family. Details of aerobic tartrate metabolism are not known. Competition experiments indicate that L- and D-tartrate are substrates of DctA (3). It has been suggested that aerobic tartrate metabolism does not involve tartrate dehydration (31). Alternative routes for tartrate oxidation could be via glycerate using tartrate dehydrogenase and oxaloglycolate reductase, or tartrate decarboxylase (32, 33, 34).

ANAEROBIC UTILIZATION OF C₄-DICARBOXYLATES BY FUMARATE RESPIRATION

The basic 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 reduced to succinate. In most bacteria, growth by fumarate respiration requires a supply of additional substrates like H₂, glycerol, or glucose. The latter two substrates provide glycerol-3-P or NADH as the direct electron donors for fumarate respiration. Energy conservation by fumarate respiration is also the basis for anaerobic growth of E. coli by substrates that are metabolized to fumarate before being subjected to fumarate respiration. This applies to growth on L-malate and L-aspartate, and on the noncommon C₄-dicarboxylates (L-tartrate, D-tartrate, D-malate), as well as on citrate. The corresponding enzymes and proteins for anaerobic C₄-dicarboxylate metabolism are summarized in Table 1.

Succinate cannot be oxidized by E. coli under anaerobic conditions (35, 36, 37), and succinate produced in fumarate respiration has to be excreted. Succinate dehydrogenase that is active under aerobic conditions uses ubiquinone as an electron acceptor (38). The ubiquinol produced cannot be reoxidized by anaerobic fumarate, nitrite, dimethyl sulfoxide, or trimethylamine–N-oxide respiration (39, 40, 41, 42), since the corresponding terminal reductases accept only menaquinol (MKH₂) or demethylmenaquinol (DMKH₂) as the electron donor. Of the anaerobic terminal reductases, only nitrate reductase is able to use ubiquinol (as well as menaquinol) as an electron donor. E. coli is therefore able to slowly oxidize succinate and some other C-sources in the absence of glucose when nitrate is present (36, 43, 44, 45). In addition to the incompatibility of ubiquinol as an electron donor, the citric acid cycle is repressed and incomplete under anaerobic conditions and excess glucose (23, 24, 25, 26).

Fumarate Respiration Requires Fumarate Reductase and C₄-Dicarboxylate Transporters DcuAB

Fumarate is found only in low concentrations in the gut, but fumarate respiration is the basis for the utilization of various related substrates (Fig. 3). Thus, L-malate, L-aspartate, and L-tartrate or citrate are converted to fumarate and represent important physiological substrates for fumarate respiration. Fumarate reductase, which catalyzes the reverse reaction of succinate dehydrogenase, was previously shown to differ genetically from succinate dehydrogenase (46). E. coli requires an electron donor for anaerobic growth on fumarate and related compounds (idealized fermentation balances: H₂ + fumarate → succinate, or glucose + 2 fumarate → 2 acetate + 2 succinate + 2 CO₂). Fumarate is taken up by the C₄-dicarboxylate/succinate (or fumarate/succinate) antiporter DcuB, which catalyzes substrate/product antiport (47, 48, 49). Fumarate is reduced by fumarate reductase to succinate, which is excreted. Fumarate respiration is coupled to the generation of a proton gradient over the membrane, which drives the phosphorylation of ADP by ATP synthase or other proton potential (Δp)-dependent reactions. Synthesis of fumarate reductase and DcuB is stimulated under anaerobic conditions in the presence of C₄-dicarboxylates (49, 50, 51, 52, 53, 54, 55). The homologous DcuA is constitutively synthesized and is able to replace DcuB in dcuB mutants. In dcuA dcuB double mutants, a third anaerobic C₄-dicarboxylate carrier (DcuC) can substitute DcuB and DcuA in part (56, 57). Antiport activity and anaerobic growth by fumarate respiration are lost in the dcuA dcuB dcuC triple mutant, demonstrating that no further carrier for fumarate-succinate antiport is available.

Figure 3.

Anaerobic catabolic reactions of common C₄-dicarboxylates (fumarate, l-malate, aspartate) and relation to fumarate respiration (A) and use of aspartate for anabolic reactions (B). (A) The scheme shows the pathways for the uptake of the C₄-dicarboxylates, conversion to fumarate, and formation of succinate by fumarate reduction. From the fumarate reductase system, only fumarate reductase is given; details of fumarate respiration are shown in Fig. 4. DcuA is shown to function in L-Asp/Succ antiport where L-Asp utilization is linked to fumarate respiration (right part). (B) The scheme shows the role of DcuA in the constitutive uptake of L-Asp (or other C₄-dicarboxylates) for anabolism (lower part), or in a hypothetical L-Asp/Fum shuttle (upper part) where L-Asp is supposed to serve as a source for ammonia only. AspA, aspartase; ET, electron transport; DcuA, constitutive C₄-dicarboxylate carrier; DcuB, (anaerobic) C₄-dicarboxylate/succinate antiporter; Frd, fumarate reductase; FumB, (anaerobic) fumarase B; MKH₂, menaquinol. See text for details.

E. coli and Salmonella require additional electron donors, such as H₂ or glycerol, for growth by fumarate respiration. In contrast, Proteus (Providencia) rettgeri is able to grow anaerobically with fumarate as the sole energy substrate (7 fumarate → 6 succinate + 4 CO₂) (58). Part of the fumarate is oxidized to CO₂ by 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 then reduced to succinate by fumarate respiration (5 fumarate + 10 [H] → 5 succinate), whereby the excess 10 [H] from fumarate oxidation are reoxidized. Fumarate disproportionation by this reaction sequence requires a citric acid cycle with sufficient activity under anaerobic conditions, which is in contrast to the situation in E. coli and other enteric bacteria.

The Dehydrogenases of the Fumarate Respiratory Chain

In fumarate respiration various dehydrogenases are linked functionally by menaquinone to FrdABCD, in particular, the respiratory NADH, glycerol-3-P, and formate dehydrogenases and hydrogenase. E. coli encodes for most of the dehydrogenase isoenzymes, which catalyze the same redox reaction but differ in their energetic properties (reviewed in references 27, 59, and 60). In fumarate respiration, the energy-conserving variant of dehydrogenase is generally used, since fumarate reductase is non-energy conserving by itself (see below); thus, the energy-conserving NADH dehydrogenase I (encoded by the nuoA-N genes) is used and highly expressed under conditions of fumarate (Fig. 4) or dimethyl sulfoxide respiration (61, 62, 63). The noncoupling NADH dehydrogenase II (encoded by the ndh gene), on the other hand, is important during aerobic growth (37).

Figure 4.

Fumarate respiration with NADH-quinone oxidoreductase (NuoA-N) and hydrogenase 2 (HybCOAB) as the primary dehydrogenases. The scheme gives the topology of the enzymes, including the sites for H⁺ release and consumption. For NADH-quinone reduction, a translocation of 4 H⁺/2 e⁻ is suggested. The reaction of fumarate reductase is not electrogenic (H⁺/2 e⁻ ratio of 0), whereas hydrogenase 2 is a redox-loop enzyme with a H⁺/2 e ratio of 2. More details on fumarate respiration, see Unden et al. (60) and Tomasiek et al. (91).

NADH dehydrogenases I from E. coli and Klebsiella pneumoniae are redox-driven proton pumps and couple NADH oxidation to the translocation of 4 H⁺/NADH. The membrane arm of the E. coli enzyme consists of 55 transmembrane helices from six subunits, three of which are antiporter like and supposed to pump one proton each per redox cycle (NADH oxidation) (64, 65, 66, 67, 68, 69). The enzyme is also able to drive Na⁺ translocation under some conditions, supposedly through the function of a secondary Na⁺/H⁺ antiport activity of the antiporter subunits (70, 71, 72).

In fumarate respiration using glycerol as the H donor, anaerobic glycerol-3-P dehydrogenase (GlpABC) is used instead of the noncoupling “aerobic” glycerol-3P dehydrogenase (GlpD). GlpABC has not been characterized in detail (73, 74, 75, 76; reviewed in reference 60). The midpoint potential of glycerol-3-P (E^′₀ Gly3P/dihydroxyacetone phosphate = −190 mV) is close to that of menaquinone (ΔE^′₀ = −80 mV). The low redox potential difference (110 mV) makes conservation of redox energy in a proton potential difficult considering the Δp value of approximately −140 mV over the membrane during fumarate respiration (60, 77). Of the alternative hydrogenases, hydrogenase 2, or HybCOAB (encoded by the hybCOAB genes), is used preferentially in fumarate respiration (Fig. 4) (60, 78, 79, 80, 81). Since H₂-fumarate respiration supports growth of the bacteria (82, 83), the hydrogenase has to be able to generate a proton potential for driving ADP phosphorylation. The proton potential is supposed to stem from a redox half-loop (Fig. 4).

Overall, many, but not all, of the dehydrogenases of fumarate respiration conserve redox energy in a proton gradient (59, 60). However, not all of the respiratory dehydrogenases of anaerobic respiration have been studied in detail.

Fumarate Respiration and Energetics

In fumarate respiration (Fig. 4), the dehydrogenases are linked to fumarate reductase by menaquinone (MK) or by demethylmenaquinone (DMK). Reduced menaquinone (MKH₂, E^′ MK/MKH = −80 mV) and reduced demethylmenaquinone (DMKH₂, E^′ DMK/DMKH = +36 mV) serve as the electron donors for fumarate reductase, whereas ubiquinol (QH₂, E^′ Q/QH = +110 mV) is not suitable for fumarate reduction (E^′₀ fumarate/succinate = +30 mV) (23, 52). MKH₂ is the preferred electron donor for this electron acceptor and the major quinone synthesized during growth by fumarate respiration (41, 42, 84).

The reaction of fumarate reductase (MKH₂ + fumarate → MK + succinate) is not electrogenic since consumption of protons at the fumarate site and release of protons from menaquinol occur at the same side of the membrane in the cytoplasm (Fig. 4). Fumarate reduction serves as an electron sink for the dehydrogenase reactions, many of which couple the redox reaction to proton translocation. For NADH → fumarate respiration, the H⁺/e⁻ ratio can be as high as 2, which allows an ATP/fumarate ratio as high as 0.66 (assuming a H⁺/ATP ratio of 3).

A high-resolution three-dimensional structure is available for the fumarate reductase found in E. coli (85, 86, 87, 88). The active site for menaquinol oxidation is located in the membrane (subunits FrdCD) and the active site for fumarate reduction is in the cytoplasm (89, 90). 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. Details of fumarate reductase function and fumarate respiration are given in the EcoSal Plus article by Tomasiek et al. (91).

The free energy of fumarate respiration is sufficient for generation of a proton potential and for ADP phosphorylation (H₂ + fumarate → succinate; (ΔG^′₀ = −87 kJ/mol). E. coli is able to grow by fumarate respiration with H₂ as the electron donor (E^′₀ H₂/2H + = −420 mV), 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 is formed per fumarate (82), which is in accordance with the growth and ATP yields for fumarate respiration by other bacteria (92). In fumarate respiration, a proton potential (Δp ≈ −140 mV) similar to the potential for nitrate respiration has been found, which is slightly lower than that of aerobic respiration (Δp ≈ −160 mV) (77).

L-Malate or L-Aspartate as the Substrates for Fumarate Respiration

L-Malate and L-aspartate concentrations in the intestine are believed to exceed those for fumarate and may represent the most important physiological substrates for fumarate respiration. Use of these substrates requires a minor addition to fumarate respiration (Fig. 3). DcuB and DcuA catalyze the uptake of L-malate in exchange for succinate with rates comparable to fumarate/succinate antiport. The significance of L-malate for fumarate respiration is implied by the colocalization of fumB encoding fumarase B (FumB) with dcuB, and the observation that L-malate is the preferred substrate of the sensor DcuS (93). In the cytoplasm, L-malate is dehydrated to fumarate by FumB. E. coli possesses three fumarases: FumA, FumB, and FumC (94, 95, 96, 97). FumB is produced during fumarate respiration under anaerobic conditions (14, 15, 94). Under microaerobic conditions, FumA might contribute to L-malate dehydration.

The conversion of L-aspartate to fumarate is catalyzed by L-aspartase AspA (encoded by the aspA gene) (98, 99). Only one aspartase enzyme (AspA) is found that shows high sequence identity with FumC (99). aspA is located adjacent to dcuA (49, 51), and both genes are expressed constitutively under aerobic and anaerobic conditions (51). The colocalization of aspA and dcuA and their concurring expression pattern may indicate their involvement in the same metabolic pathway. This could be the supply of L-aspartate for anaerobic fumarate respiration (Fig. 3) or for aerobic catabolism and degradation (Fig. 2), involving aspartate uptake and deamination. Alternatively, aspartate may be used as a source for assimilation (Fig. 3B), and it is even feasible that aspartate functions as a source for ammonia when the fumarate produced by AspA is excreted by DcuA functioning as a supposed aspartate/fumarate shuttle (100) (Fig. 3B, upper).

In summary, there are specific pathways that allow rapid growth on L-malate or L-aspartate (together with an electron donor) by providing transporters for substrate/product antiport and hydratase/aspartase enzymes.

Noncommon C₄-Dicarboxylates (L- and D-Tartrate, D-Malate) as Substrates for Fumarate Respiration

L-Tartrate can be degraded by two different pathways involving either fumarate respiration (E. coli) or oxaloacetate decarboxylation (Salmonella and Klebsiella) as the key reactions (next section). In the E. coli pathway, L-tartrate is converted to fumarate by an L-tartrate-specific feeding reaction, whereas D-tartrate and D-malate are metabolized to fumarate by side reactions of L-malate-specific enzymes (DcuB and FumB) (Fig. 5).

Figure 5.

Anaerobic utilization of noncommon C₄-dicarboxylates (L-tartrate, D-tartrate, D-malate) and relation to fumarate respiration. DcuB, C₄-dicarboxylate/succinate antiporter; TtdT, L-tartrate/succinate antiporter; TtdAB, tartrate dehydratase; Mdh, malate dehydrogenase; FumB, (anaerobic) fumarase B; Frd, fumarate reductase; ET, electron transport; MKH₂, menaquinol. Other details as described in Fig. 3 and in the text.

L-Tartrate

E. coli is able to grow on L-tartrate and D-tartrate, provided that an additional electron donor like glycerol is present (29, 31, 101, 102). Tartrate utilization is sometimes termed “tartrate fermentation.” L-Tartrate metabolism uses fumarate respiration for energy conservation, and the tartrate is converted to fumarate by a specific feeding reaction sequence. L-Tartrate is metabolized to oxaloacetate by L-tartrate dehydratase, and then via malate to fumarate (31, 102) (Fig. 5). L-Tartrate dehydratase (TtdAB) is induced during anaerobic growth on L- and meso-tartrate. TtdAB is oxygen labile and is a tetramer of two subunits of TtdA and TtdB each, which are encoded by the ttdAB operon (31, 103). The TtdA subunit is similar in sequence and other properties to the class I fumarases (FumA and FumB) of E. coli that contain catalytic iron-sulfur centers (31). Fumarate is reduced by fumarate reductase to succinate, which is excreted.

L-Tartrate appears to be the only physiological C₄-dicarboxylate that is not transported by the Dcu carriers of E. coli and instead requires the transporter TtdT (102). TtdT catalyzes L-tartrate/succinate antiport, preferentially operating in L-tartrate uptake and succinate export (Fig. 5). TtdT is similar in sequence to the citrate/succinate antiporter CitT of E. coli, and both enzymes represent a carboxylate/C₄-dicarboxylate carrier subgroup of the DASS (divalent anion: Na⁺ symporter) family (102, 104, 105, 106). Carriers of this family catalyze a heterologous antiport and are different in function and sequence from the DcuA/B and DcuC carrier families (107).

D-Tartrate

D-Tartrate is a minor form of tartrate in nature and is metabolized by enteric and other bacteria (31, 108). In E. coli D-tartrate supports only slow growth and, like L-tartrate, requires an additional electron donor for fermentation (31). An enzymatic activity dehydrating D-tartrate but not L-tartrate was found in cell extracts of E. coli grown anaerobically on D-tartrate, but TtdAB was not induced during growth on D-tartrate. D-Tartrate is apparently metabolized by enzymes of the general anaerobic fumarate metabolism, and no D-tartrate-specific enzymes are involved (101) (Fig. 5). Thus, D-tartrate is dehydrated by fumarase FumB, and D-tartrate/succinate antiport is effected by DcuB.

D-Malate

E. coli is able to slowly convert D-malate to fumarate under anaerobic conditions, which is then used for fumarate respiration when glycerol is present as an electron donor (28). Equimolar amounts of succinate are produced from D-malate. The metabolism supports growth that depends on fumarase FumB, DcuB, and fumarate reductase FrdABCD, but not on D-malate dehydrogenase DmlA (28). Thus, D-malate is metabolized by the enzymes of the general L-malate pathway (FumB, FrdABCD, and transporter DcuB) to fumarate, which is then used for fumarate respiration (Fig. 5).

Stereochemistry of noncommon C₄-dicarboxylates

Figure 6 gives the structure and stereochemistry of the C₄-dicarboxylates containing hydroxyl substituents at C-2 and/or C-3, and for fumarate and maleate. The hydroxyl group of L-malate shows 2S, that of D-malate 2R configuration. The two hydroxyl groups of tartrate at C-2 and C-3 can be found in three isomeric forms: L-tartrate with 2R/3R configuration, D-tartrate with 2S/3S configuration, and meso-tartrate with 2R/3S configuration of the hydroxyl groups. L-Malate is metabolized in E. coli by fumarase FumB and antiporter DcuB in fumarate respiration. D-Tartrate with the same configuration at C-2 (S) is anaerobically metabolized in E. coli by the L-malate pathway using FumB and DcuB (101). In contrast, L-tartrate and D-malate with the hydroxyl groups at C-2 in R configuration require specific enzymes: TtdAB and TtdT for L-tartrate, and DctA and DmlA for D-malate, respectively (102). TtdAB and TtdT are produced under anaerobic conditions only, and DctA and DmlA are produced under aerobic conditions only. Anaerobic growth on D-malate therefore depends on the enzymes of L-malate metabolism (FumB and DcuB), which allow only poor growth of the bacteria because of stereochemical incompatibility (28).

Figure 6.

Stereoisomers of hydroxylated C₄-dicarboxylates. The figure gives stereoisomers of C₄-dicarboxylates carrying hydroxyl groups at C2, or at C2 and C3. Malate is found as L-malate (2S configuration) and D-malate (2R configuration), but only the L-isomer is of natural origin. Tartrate is represented by three stereoisomers (L-, D- and meso-tartrate). L-tartrate (2R, 3R configuration) is present in many plants, whereas D-tartrate (2S, 3S) is rare in nature and meso-tartrate not of natural origin. Fumarate and maleate are isomers of butenedioate. Maleate (cis-butenedioate) is chemically produced, whereas fumarate (trans-butenedioate) is a common intermediate of living cells.

Anaerobic Growth of E. coli on Citrate

E. coli metabolizes citrate under anaerobic conditions when an additional substrate like glycerol or glucose is provided to serve as electron donor. The products of citrate are succinate and acetate (109, 110). This metabolic reaction, also termed “citrate fermentation,” depends on fumarate respiration, but the degradation of the glycerol or glucose cosubstrates provides additional ATP by substrate level phosphorylation (109). Citrate uptake is catalyzed by the citrate/succinate antiporter CitT (104) and cleaved by citrate lyase (CL) to oxaloacetate and acetate (Fig. 7). Oxaloacetate is metabolized to fumarate by the reactions of malate dehydrogenase (Mdh) and fumarase B (FumB), as described in L-tartrate fermentation (Fig. 5), terminating in fumarate respiration. Succinate is excreted by the substrate/product antiporter CitT. The pathway requires NADH and MKH₂ for oxaloacetate and fumarate reduction, respectively, and the reducing equivalents are derived from additional oxidizable substrates like glucose or glycerol (110). Citrate utilization is therefore based on fumarate respiration (representing the energy-conserving reaction), as well as fueling or feeding reactions that convert citrate to fumarate (109). Metabolism of the cosubstrates (glycerol or glucose) is linked to substrate-level phosphorylation, which exceeds the ATP yield of fumarate respiration.

Figure 7.

Pathway, genes, and transcriptional regulation of the genes for citrate fermentation by citrate via the CitA-CitB and the DcuS-DcuB two-component systems. (A) Synthesis of the citrate fermentation specific enzymes and transporters (CitT, citrate/succinate antiporter; CL, citrate lyase)is induced by CitA-CitB and citrate (blue labeling). Synthesis of the fumarate respiration pathway (FrdABCD [or Frd], FumB, presented in green) is induced by the citrate response of DcuS-DcuR (using the side-activity of DcuS for citrate). (B) This scheme, for comparison, gives the fumarate respiratory system (FrdABCD, Frd) and fumarate/succinate antiporter (DcuB) that are induced by DcuS-DcuR in response to fumarate (or C₄-dicarboxylates). The enzymes and the carrier shown in blue are unique for citrate fermentation; the enzymes shown in green are used both in citrate fermentation and fumarate respiration. Cit, citrate; Fum, fumarate; Mal, malate; OAA, oxaloacetate; Succ, succinate. Modified from reference 209.

Succinate Production by Glucose Fermentation

The reactions of anaerobic C₄-dicarboxylate metabolism discussed in the previous sections result in succinate production at the expense of ambient C₄-dicarboxylates. E. coli is able to ferment glucose by mixed acid fermentation with acetate, ethanol, and formate as the products (reaction 1), or by a mixed acid fermentation combined with succinate formation (reaction 2) (Fig. 8).

Figure 8.

Succinate production from endogenous fumarate (glucose fermentation). The scheme shows the major intermediates for the formation and excretion of succinate from PEP, and of formate, acetate, and ethanol formation (mixed acid fermentation) during glucose fermentation. Residual activities of the repressed and interrupted tricarboxylic acid (TCA) cycle are shaded with broken lines. AcCoA, acetyl-CoA; DcuC, succinate export carrier; FumB, (anaerobic) fumarase; FumC, (aerobic) fumarase; Frd, fumarate reductase; Mdh, NADH-dependent cytosolic malate dehydrogenase; OAA, oxaloacetate; PTS, PEP-dependent phosphotransferase uptake for glucose; PFL, pyruvate formate lyase; Ppc, PEP carboxylase; Pyr, pyruvate; Sdh, succinate dehydrogenase SdhABCD.

$$1\text{Glucose}\to 1\text{acetate}+1\text{ethanol}+2\text{formate}+3{\text{H}}^{+}$$ (1)

(ΔG^′₀ = −218 kJ/mol glucose)

$$1\text{Glucose}+1{\text{HCO}}_{\text{3}}^{-}\to 1\text{acetate}+1\text{succinate}+1\text{formate}+3{\text{H}}^{\text{+}}$$ (2)

(ΔG^′₀ = −260 kJ/mol glucose)

Fermentation generally occurs via a mixture of both reactions, resulting in up to 0.2 mol of succinate per mol of glucose. For succinate production, PEP from the glycolytic pathway is carboxylated by PEP-carboxylase, yielding oxaloacetate, which is then converted by the reductive branch of the citric acid cycle, including malate dehydrogenase (Mdh), fumarase (FumB), and fumarate reductase (FrdABCD) (Fig. 8). The pathway can be regarded as a feeding reaction for fumarate production from glucose (see Fig. 2), but in wild-type E. coli only a small portion of the glucose is directed into this pathway. The pathway and its engineering has gained significant interest for biotechnological production of succinate in recent years (111). The succinate is not further metabolized because of the repression of the citric acid cycle under anaerobic conditions and a lack of capacity for ubiquinol reoxidation. Succinate is excreted by DcuC, a succinate exporter (5, 56, 57), in an electrogenic mode, presumably by symport of three H⁺ with succinate^2–. DcuC is produced under anaerobic conditions in the presence of glucose. When dcuC is deleted, DcuB and DcuA, as well as other unknown carriers, take over the function of DcuC (5, 56). In addition, diffusion of Hsuc^1– (other than succinate^2–) may contribute to some extent to the export of succinate (5, 112).

Succinate Production in Engineered E. coli

E. coli has been modified by genetic and metabolic engineering to produce up to 1.6 mol succinate per mol glucose (113, 114, 115) compared with 0.2 mol succinate production per mol glucose by the wild type, where succinate is a minor product (Fig. 8). Several approaches were important to improve succinate production (Fig. 9): blocking pathways for alternative fermentation products, adjusting redox balancing, strengthening the C3 → C4 branch, activating alternative succinate production pathways, and optimizing transport activities.

Figure 9.

Succinate production pathways by engineered succinate production strains of E. coli. The scheme summarizes reactions engineered in E. coli to improve succinate production. Dotted arrows, either nonfunctional or decreased steps; Bold solid arrows, the primary route for carbon flow; Δ, gene deletion; +, overproduced or transformed genes; red letters, target genes; Ac-CoA, acetyl-CoA; Ace, acetate; Cit, citrate; DcuB, fumarate-succinate antiporter; DcuC, succinate export carrier; EMP, Embden-Meyerhof-Parnas pathway; EtOH, ethanol; For, formate; Fum, fumarate; GalP, galatose permease; G6P, glucose 6-phosphate; Isoc, isocitrate; Lac, lactate; Mal, malate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PTS, PEP-dependent phosphotransferase uptake for glucose; Pyr, pyruvate; Succ, succinate;. Genes: aceA, isocitrate lyase; aceB, malate synthase; ackA, acetate kinase; adhE, alcohol dehydrogenase; fumB, anaerobic fumarase; frdABCD, fumarate reductase; glk, glucokinase; iclR, isocitrate lyase regulator; ldh, lactate dehydrogenase; mdh, NADH-dependent malate dehydrogenase; pflB, pyruvate formate lyase; pk, pyruvate kinase; ppc, PEP carboxylase; pps, PEP synthethase; pta, phosphate acetyl transferase.

The first attempt to block alternative fermentation was the inactivation of lactate dehydrogenase and pyruvate-formate lyase genes (Δldh, Δpfl), which resulted in poor growth, low succinate production, and pyruvate accumulation. Growth was restored by overproduction of a NAD⁺-dependent malic enzyme that carboxylates pyruvate and reoxidizes NADH (114, 116, 117, 118, 119, 120). Alternatively, replacement of the glucose PTS system by a glucose transporter allowed anaerobic growth on glucose with enhanced succinate production, but without lactate production (120, 121).

The C3 → C4 pathway was strengthened by heterologous overexpression of PEP carboxykinase (PCK) from Actinobacillus succinogenes (122), PEP carboxylase (PPC) from Sorghum vulgare (123), or pyruvate carboxylase (PYC) from Lactobacillus lactis (123). These approaches increased the succinate production up to 1 mol/mol glucose (114).

Use of the glyoxylate shunt as an alternative succinate-producing pathway with low NADH need improved succinate production further (114, 120, 124). The glyoxylate shunt was activated by deletion of the repressor IclR. Overall, a strain with deletions for ldhA, adhE, ack, pta, and iclR and containing pyruvate carboxylase (PYC) from L. lactis produced 1.6 mol succinate per mol glucose (115).

For optimizing sugar uptake, the PEP-consuming PTS glucose system was replaced by glucose import with galactose permease (GalP) from E. coli or glucose facilitator (GlF) from Zymomonas mobilis (125, 126). Glucose was then phosphorylated with glucokinase (Glk). Recently, improved expression of succinate exporter DcuC and fumarate/succinate antiporter DcuB led to a significant increase in succinate production in an E. coli succinate producer (127). E. coli succinate producers can be further engineered to utilize cost-beneficial feedstocks such as sucrose, fructose, xylose, glycerol, or other industrial waste (114, 128, 129).

USE OF C₄-DICARBOXYLATES FOR CELL SYNTHESIS (ASSIMILATION)

E. coli is able to grow anaerobically with H₂ + fumarate or H₂ + L-malate as the sole substrates in mineral medium (82, 83), which demonstrates, as discussed above, that fumarate respiration is an energy-conserving process. In addition, during growth on C₄-dicarboxylates E. coli has to use the substrate for the synthesis of all cell constituents. Approximately 7% of the L-malate was converted into cell mass during anaerobic growth on H₂ + L-malate (83). In contrast to information on C₄-dicarboxylate catabolism, limited information on C₄-dicarboxylate anabolism is available. Based on the gene and enzyme equipment of E. coli, pathways for the synthesis of oxaloacetate, PEP, pyruvate, acetate, and oxoglutarate from fumarate or L-malate can be predicted (130), representing building blocks for the synthesis of major cell constituents, such as sugars, carbohydrates, amino acids, hydrocarbons, and nucleotides. Wolinella succinogenes, a member of the Epsilonproteobacteria that is able to grow on H₂ + fumarate, produces cell constituents from fumarate and H₂ by anabolic reactions (131), as suggested above for E. coli.

ANAEROBIC C₄-DICARBOXYLATE METABOLISM WITHOUT FUMARATE RESPIRATION

Tartrate Fermentation Involving Na⁺-Dependent Oxaloacetate Decarboxylase

Salmonella enterica serovar Typhimurium strain LT2 and K. pneumoniae are able to grow by tartrate fermentation by using L- or D-tartrate as the sole carbon and energy source (132, 133, 134). In these bacteria, growth on tartrate is Na⁺ dependent and induces the synthesis of a Na⁺-activated oxaloacetate decarboxylase, which is located in the membrane (see reference 135). Oxaloacetate decarboxylase couples decarboxylation to the export of sodium ions and the generation of a sodium motive force (136). Pyruvate formed by oxaloacetate decarboxylation enables fermentative metabolism and ADP phosphorylation by acetate production without the need for fumarate respiration and without an external electron donor as in E. coli.

Conversion of Succinate to Propionate

A new pathway for the conversion of succinate into propionate (succinate → propionate + CO₂) was found in E. coli (137) after identifying genes for paralogues of the crotonase enoyl-CoA hydratase. The gene for one of the paralogues (YgfG) is part of a four-gene operon (ygf or scp operon). The YgfG protein has methylmalonyl-CoA decarboxylase activity. The other genes encode propionyl-CoA-succinate CoA transferase (YgfH), methylmalonyl-CoA mutase (Sbm), and a putative protein kinase (YgfD/ArgK). YgfD/ArgK does not function as a succinate or propionate CoA ligase, leading to the possibility that YgfD/ArgK represents a protein kinase that regulates the activities of other enzymes in E. coli that utilize succinyl-CoA, propionyl-CoA, propionate, or succinate. The metabolic context of the cycle and the conditions for the synthesis of the enzymes or operation of the pathway are not known. On principle, the enzyme reactions can be linked to a metabolic cycle for the decarboxylation of succinate to propionate (137) or for providing CoA-activated precursors for biosynthetic pathways (138).

Recombinant Mesaconate Production from Glutamate or Glucose

Recently, an engineered glutamate mutase pathway was established in E. coli for the production of mesaconate (methylfumarate) from glutamate (139). Glutamate mutase and 3-methylaspartate ammonia lyase were recombinantly expressed in E. coli. By improving the availability of the adenosylcobalamin cofactor for glutamate mutase and the activity of the glutamate mutase-reactivatase, significant levels of mesaconate were produced either from glutamate or glucose. In addition, class I fumarases FumA and FumB are capable of hydrating mesaconate to citramalate. A significant number of E. coli strains, most of which are pathogenic, possess an additional class I fumarase (FumD) (140). The fumD gene clusters with genes encoding glutamate mutase and methylaspartate ammonia lyase, which are involved in converting glutamate to mesaconate. FumD is a mesaconase/fumarase with a preference for mesaconate over fumarate. As a consequence, the bacteria have the genetic potential to convert glutamate to citramalate. The use of citramalate in E. coli is unclear, however.

C₄-DICARBOXYLATE TRANSPORTERS

E. coli contains transporters for the uptake, antiport, and excretion of C₄-dicarboxylates to meet all metabolic requirements. The C₄-dicarboxylate carriers can be grouped in 6 families (DAACS, SulP, AceTr, DcuAB, DcuC, DASS) that contain the DctA, DauA, SatP, DcuA/B, DcuC, and TtdT transporters (Fig. 10, Table 1). In addition, a putative transporter of the tripartite ATP-independent periplasmic transporter (TRAP) family (141, 142) with sequence similarity to C₄-dicarboxylate transporters of this class has been identified, but the function of this transporter in E. coli is not clear (10).

Figure 10.

Phylogenetic tree of C₄-dicarboxylate carriers of E. coli (DctA, DauA, DcuAB, DcuC, TtdT, CitT, and TRAP, but without SatP transporters) as derived from protein sequences. The distances represent the differences in identical amino acid residues of the carriers or the corresponding gene products. The amino acid sequences were aligned with Clustal Omega program (http://www.ebi.ac.uk/Tools/msa/clustalo/) and the tree was reconstructed by the neighbor-joining method MEGA 6.0 (213). Abbreviations of strains: As, Actinobacillus succinogenes; Bs, Bacillus subtilis; Cg, Corynebacterium glutamicum; Ec, E. coli; Ms, Mannheimia succinoproducens; Pa, Pseudomonas aeruginosa; Pf, Pseudomonas fluorescens; Ph, Pyrococcus horikoshii; Rl, Rhizobium leguminosarum; Rm, R. meliloti; Se, Salmonella enterica; Tk, Thermococus kodacarensis; Vc, Vibrio cholerae; Ws, Wolinella succinogens.

C₄-Dicarboxylate Transporters for Aerobic Growth

The main transporter at neutral pH: DctA

DctA transporters for C₄-dicarboxylates are found in many Gram-negative and Gram-positive aerobic, but not anaerobic, bacteria (Fig. 10) (10, 107). DctA has a broad substrate specificity and accepts succinate and L-malate as substrates, in addition to fumarate, D-malate, L-aspartate, L-tartrate, and orotate, an aromatic monocarboxylate (2, 3, 4). dctA mutants show poor growth on C₄-dicarboxylates, indicating that DctA is the main C₄-dicarboxylate carrier in aerobic growth at neutral pH (10).

DctA is a member of the dicarboxylate amino acid:cation symporter (DAACS) family of transporters, which catalyze the uptake of C₄-dicarboxylates or amino acids in symport with H⁺ or Na⁺. The DAACS family comprises the DctA transporters and the glutamate/aspartate transporters Glt, including the aspartate transporter GltP of E. coli. Topology analysis and homology modeling show that DctA is composed of 8 transmembrane helices (143) and two helical hairpins (HP1 and HP2) that are located before and behind TM7 (144). TM7 and TM8 together with HP1 and HP2 bind the substrate and cotransported ions. The close homology of DctA to the Glt_Ph and Glt_Tk aspartate transporters from Pyrococcus horikoshii and Thermococcus kodacarensis (145, 146, 147) suggests very similar structure and transport modes for DctA. The Glt proteins are trimeric, with the trimerization domains mediating the subunit contacts and the transport domains with the sites for aspartate and Na⁺ binding. According to the model, the transport domains move across the membrane like an elevator and shuttle the aspartate and the Na⁺ ions from one side of the membrane to the other. A very similar transport mode is feasible for DctA, but protons drive the symport instead of Na⁺.

DctA functions preferentially at neutral pH when the dicarboxylates are present in dianionic form (Fig. 11). The K_m values for the C₄-dicarboxylates are ∼10 to 30 μM, and the V_max is ∼25 to 50 μmol/min/g (dry weight). The transport is electrogenic and depends on the presence of the proton potential. About three H⁺ ions enter the cell with each C₄-dicarboxylate (143, 148) (reaction 3).

Figure 11.

Carriers and transport modes for the exchange, uptake, and efflux of C₄-dicarboxylates in E. coli under aerobic (A) and anaerobic growth conditions (B). (C) shows the transport modes for exchange, uptake, and efflux of C₄-dicarboxylates that can be effected by the Dcu carriers (DcuA, DcuB, or DcuC). Uptake and efflux are electrogenic by the symport of 3 H⁺ with the C₄-DC, whereas antiport is electroneutral (48). In aerobic growth (A), DctA is the major carrier for uptake at pH 7. DauA and potentially SatP replace DctA function at pH 5 and pH 6. The transporters catalyze electrogenic transport as presented in the figure. In anaerobic growth (B), during growth by fumarate respiration, DcuB is the major carrier and catalyzes an electroneutral fumarate/succinate antiport. DcuB can be supported or replaced by DcuA. During glucose fermentation, succinate efflux is effected by DcuC, which can be supported by DcuB, DcuA, and other unknown efflux carriers. During anaerobic growth on tartrate, tartrate-succinate antiport is catalyzed by carrier TtdT. The function of the dcuD gene product (DcuD) is unknown. References and details in the text. Modified from reference 107.

$${\text{Succinate}}_{\text{out}}^{\text{2}-}+3{\text{H}}_{\text{out}}^{\text{+}}\to {\text{succinate}}_{\text{in}}^{\text{2}-}+3{\text{H}}_{\text{in}}^{\text{+}}$$ (3)

DctA is a bifunctional protein and forms a complex with the sensor kinase DcuS. In the DctA/DcuS complex, DctA functions as a coregulator of the DcuS sensor kinase (144, 149, 150, 151).

Transporters under acidic conditions: DauA and SatP

Mutants deficient of dctA show poor growth on C₄-dicarboxylates, but at more acidic pH (pH 6) the dctA mutant regains growth on succinate, but not on fumarate (5). At acidic pH around pH 5, the secondary transporter DauA is the sole aerobic succinate transporter (6), and deletion studies show that DauA is essential for growth on C₄-dicarboxylates under acidic conditions. DauA catalyzes an electrogenic uptake of succinate (Hsuc¹⁻_ex + 2 H⁺ → Hsuc¹⁻_in + 2 H⁺) (Fig. 11). The common C₄-dicarboxylates, with the exception of L-malate, are substrates of DauA. Although most dicarboxylates are present in monocarboxylate form at pH 5, DauA is able to transport the dicarboxylate forms of succinate and fumarate as well. The K_m for the uptake of succinate at pH 5 is higher than at pH 7, where essentially DctA catalyzes transport, by a factor of 22. At pH 5, the V_m of transport decreases by a factor of 3 compared with that at neutral pH. DauA is a member of the Solute carrier 26 and sulfate transporter superfamily of secondary anion transporters (SulP) that are conserved from bacteria to humans (152). Transporters of this family have various functions, are widespread from bacteria to humans, and are characterized by a C-terminal cytoplasmic sulfate transporter and anti-sigma factor antagonist (STAS) domain (153). The crystal structure of a DauA homologous fumarate transporter from Deinococcus geothermalis reveals a 7 + 7 TM topology for each monomer within the homodimeric transporter, with a new type of core and gate domain (154). The core domain contains an anion binding site that is alternately accessible through a “rocking movement” to either side of the membrane (154, 155).

The SatP monocarboxylate transporter is able to catalyze the uptake of acetate and of succinate in the monocarboxylate form (Hsuc^–) in symport with two H⁺ (Fig. 2 and 11) (156). SatP is a member of the acetate uptake transporter family (AceTr) (Table 1). It has rather low affinity for succinate (K_m of 1.18 mM for Hsuc^– at pH 6.0) and is supposed to function at pH 6, between the pH optima of DauA (pH 5) and DctA (pH 7). Growth experiments of mutant strains are required to evaluate the physiological significance and role of SatP in succinate or C₄-dicarboxylate uptake at pH 6 or other pH values.

In addition to the general C₄-dicarboxylate transporters DctA and DauA, E. coli contains the glutamate-aspartate carrier GltP (157, 158, 159), a secondary (H⁺-dependent) carrier that appears to be of minor significance for aspartate catabolism, but its physiological relation to DcuA in aspartate transport has not been characterized. The relative contribution of DctA, DcuA, or GltP for the uptake of L-aspartate into E. coli is not known.

C₄-Dicarboxylate Transporters for Anaerobic Growth

The DcuA/DcuB carriers

The DcuA and DcuB carriers form an independent family (DcuAB or Dcu family) (49, 152) of secondary C₄-dicarboxylate transporters (Fig. 10) that is found in anaerobic and facultatively anaerobic bacteria capable of fumarate respiration (107). DcuA and DcuB carriers are capable of C₄-dicarboxylate exchange, uptake, and efflux (Fig. 11) but have, as described below, preferred transport modes according to their major physiological function. In antiport mode, transport is electroneutral, whereas uptake or export is an electrogenic symport of 3 H⁺ with the dianionic C₄-dicarboxylates (48). Like DctA under aerobic conditions (150), DcuB is a bifunctional protein that functions under anaerobic conditions as a coregulator of the sensor kinase DcuS (151, 160).

DcuB

DcuB is the most important fumarate/succinate antiporter of E. coli and is ∼2.3 times as active as DcuA (57). The DcuB transporter is required for growth by fumarate respiration and catalyzes fumarate/succinate antiport. DcuB also catalyzes uptake and efflux, which represent partial reactions of the antiport (48, 49, 57, 107), and is produced during fumarate respiration (50, 51, 55). The fumB gene, encoding anaerobic fumarase B, is located downstream of dcuB and cotranscribed with it (49, 51). DcuB has a broad substrate specificity for C₄-dicarboxylates (48). Its expression under conditions of fumarate respiration and its capacity for C₄-dicarboxylate/succinate antiport show that DcuB is the major transporter for fumarate respiration and related processes (Fig. 3, 5, 11). The colocalization and cotranscription with fumB indicate furthermore that the major physiological substrate of DcuB may be L-malate. L-Malate and aspartate are supposed to represent the most important sources or precursors for fumarate in the intestine or rumen, where fumarate is scarce.

The protein sequence suggests 12 transmembrane helices for DcuB, and this has been confirmed by topology studies (161). N- and C-terminal ends of the protein have periplasmic locations. TM11 and TM12 are separated by a large periplasmic loop with a postulated α-helical region. DcuB, like DctA, is bifunctional and has, in addition to its function as transporter, a role as a coregulator of the C₄-dicarboxylate sensor DcuS (151, 160). The TM11/TM12 cytoplasmic loop region and TM11 comprise residues which are important for coregulation of DcuS (151).

The kinetic parameters for C₄-dicarboxylate/succinate antiport were determined in cells grown under conditions of fumarate respiration and represent essentially the parameters for DcuB (48). The K_m values for fumarate and succinate are approximately 100 μM for the exchange or uptake reactions. DcuB catalyzes an electroneutral antiport (reaction 4), which exceeds uptake or efflux activities by factors of approximately 3. At neutral pH, the substrates are transported as divalent anions.

$${\text{Succinate}}_{\text{in}}^{2-}+{\text{fumarate}}_{\text{out}}^{2-}\leftrightarrow {\text{succinate}}_{\text{out}}^{2-}+{\text{fumarate}}_{\text{in}}^{2-}$$ (4)

Net uptake of C₄-dicarboxylates is observed when no internal counter substrate is present (48, 57). 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.

$${\text{Fumarate}}_{\text{out}}^{2-}+3{\text{H}}_{\text{out}}^{+}\to {\text{fumarate}}_{\text{in}}^{2-}+3{\text{H}}_{\text{in}}^{+}$$ (5)

DcuB is 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 (48, 107).

$${\text{Succinate}}_{\text{in}}^{2-}+3{\text{H}}_{\text{in}}^{+}\to {\text{succinate}}_{\text{out}}^{2-}+3{\text{H}}_{\text{out}}^{+}$$ (6)

DcuA

The dcuA gene is preceded by aspA encoding aspartase AspA (49, 51), and dcuA/aspA cotranscripts have been identified in addition to monocistronic dcuA and aspA transcripts (51). The dcuA and aspA genes are expressed constitutively under aerobic and anaerobic conditions (51) but are apparently not induced by C₄-dicarboxylates. DcuA is not able to replace DctA with comparable rates in C₄-dicarboxylate uptake and growth. The physiological role of DcuA is not clear (51), but the colocalization and coexpression of aspA and dcuA suggests that DcuA and AspA form a pathway for the uptake of aspartate and production of fumarate for aerobic (Fig. 2) or anaerobic catabolism (Fig. 3). Despite constitutive expression, DcuA apparently does not significantly support aerobic growth on C₄-dicarboxylates at pH 7 in the absence of DctA (5), whereas DcuA is able to maintain fumarate/succinate antiport when present without DcuB and DcuC (56, 57, 107). Thus DcuA is able to catalyze C₄-dicarboxylate antiport, uptake, and efflux, and to replace DcuB and DcuC in these functions. Alternatively, it has been hypothesized that DcuA and AspA might supply aspartate to aerobically and anaerobically grown bacteria (49, 51) as an N or C source (Fig. 2 and 3). It was further suggested that uptake of aspartate, followed by deamination with AspA and excretion of the fumarate, provides a pathway for supply of ammonia from aspartate (100), with DcuA functioning as an aspartate/fumarate shuttle. Interestingly, proteome analysis indicates that DcuA levels are high when the growth medium contains high levels of amino acids (162), supporting the latter suggestions. Experimentally, 10 transmembrane helices were determined for DcuA (163). 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.

DcuC and DcuD

DcuC is able to catalyze the exchange, uptake, and efflux of C₄-dicarboxylates. Although DcuC is produced only under anaerobic conditions, the synthesis is not repressed by glucose and not stimulated by C₄-dicarboxylates, in contrast to that of DcuB (56, 57, 107). The physiological role of DcuC therefore appears to be succinate efflux during fermentative growth. Succinate efflux by anaerobically grown E. coli is electrogenic (Fig. 8 and 11), as described in reaction 6. It is assumed that DcuC is the major transporter for this reaction and that succinate efflux during anaerobic growth contributes to Δp generation.

The DcuC carriers form a distinct family separate from the DcuAB transporters (57, 107). Carriers of the DcuC family are found in bacteria capable of hexose fermentation and succinate production. Bacteria that are limited to fumarate respiration without hexose fermentation contain DcuAB-type transporters, which supports the view that DcuC is the succinate exporter of hexose fermentation. DcuD from E. coli is a member of the DcuC family with unknown function (5, 107, 164). The dcuD gene encodes an intact protein and heterologous expression of DcuD does not support growth on common C₄-dicarboxylates, although a role for growth on or excretion of noncommon C₄-dicarboxylates has not been tested.

The activity of DcuC (antiport and uptake) is distinctly lower than that of DcuA and DcuB. Thus, only DcuA and DcuB have sufficient activity to maintain normal growth by fumarate respiration, and they can fully replace DcuC in dcuC deletion strains. In recombinant strains of E. coli that are engineered for increased succinate production, both DcuC and DcuB are required for optimal succinate excretion (127). Increased expression of dcuB or dcuC allows improved succinate production, indicating that succinate efflux becomes limiting in succinate production strains.

TtdT and CitT

The anaerobic L-tartrate/succinate antiporter TtdT is similar to the citrate-succinate antiporter CitT of E. coli, and is a member of the divalent anion: Na⁺ symporter family carboxylate/C₄-dicarboxlyate antiporter (DASS) family (Fig. 11). Most of the carriers of this type are found in mitochondria and chloroplasts. The ttdT gene encoding TtdT is located downstream of the tartrate dehydratase genes ttdAB, and ttdAB and ttdT are cotranscribed (102). Deletion of ttdT abolishes anaerobic growth on L-tartrate completely, and bacteria containing TtdT catalyze L-tartrate or succinate uptake and L-tartrate/succinate antiport (Fig. 5 and 11) (102). TtdT operates preferentially in the direction of L-tartrate uptake and succinate excretion. D-Tartrate is not accepted by TtdT as a substrate but is transported by DcuB (101). The Dcu carriers, however, do not support anaerobic growth on L-tartrate or L-tartrate transport. TtdT is related in sequence and function to CitT, which catalyzes heterologous citrate/succinate antiport in citrate fermentation (104).

TRANSCRIPTIONAL REGULATION OF C₄-DICARBOXYLATE METABOLISM

The expression of the genes for C₄-dicarboxylate metabolism (dctA, dcuB, dcuC, fumB, frdABCD, ttdAB, ttdT, and dmlA) is subject to transcriptional regulation in response to electron acceptors, carbon sources, and other factors, such as the growth phase (Table 2). Regulation in response to the electron acceptors O₂ and nitrate is achieved by the fumarate nitrate reductase regulator (FNR), ArcA-ArcB, NarX-NarL, and NarP-NarQ, whereas regulation by C₄-dicarboxylates is verified by DcuS-DcuR, TtdR, and DmlR.

Table 2.

Gene expression of enzymes for aerobic and anaerobic C₄-dicarboxylate metabolism of E. coli in response to electron acceptors (O₂, nitrate, and fumarate), carbon source, and other factors^a

Gene(s)b	Protein/function	Expressionc
Aerobic C4-dicarboxylate metabolism
dctA	DctA, aerobic succinate uptake, pH 7	AN (−) (ArcA); C4-DC (+) (DcuSR); Gluc (−) (CRP)
dauA	DauA, aerobic succinate uptake, pH 5	C4-DC (+) (unknown)
dmlA	DmlA, D-malate dehydratase	D-Malate, L-, meso-tartrate (+) (DmlR, DcuSR)
fumA	FumA, Fumarase A	AN (−) (ArcA, FNR); Fe limitation (−) (RyhB)
fumC	FumC, Fumarase C	AN (−) (ArcA)
mqo	Mqo, malate dehydrogenase	AN (−) (ArcA)
satP	SatP, succinate or acetate uptake, pH 6	Unknown
sdhCDAB (sucABCD)	SdhABCD, succinate dehydrogenase	AN (−) (ArcA, FNR); C4-DC (+/−); Fe limitation (−) (Fur, RyhB)
Anaerobic C4-dicarboxylate metabolism
dcuB (fumB)	DcuB, fumarate-succinate antiporter	AN (+) (FNR); C4-DC (+) (DcuSR); nitrate (−) (NarL, NarP); Gluc (−) (CRP)
dcuC	DcuC, succinate exporter	AN (+) (ArcA, FNR); C4-DC (+/−); Gluc (+/−)
frdABCD	FrdABCD, fumarate reductase	AN (+) (FNR); C4-DC (+) (DcuSR); nitrate (−) (NarL)
fumB	FumB, fumarase B	AN (+) (FNR); C4-DC (+) (DcuSR); nitrate (–) (NarL, NarP); Gluc (–) (CRP); cotranscription with dcuB
ttdAB(ttdT)	L-Ttd, L-tartrate dehydratase	AN (+) (ArcA, FNR); L-, meso-tartrate (+) (TtdR, DcuSR), nitrate (−) (NarL, NarP)
ttdT	TtdT, tartrate-succinate antiporter	AN (+) (ArcA, FNR); L-, meso-tartrate (+) (TtdR, DcuSR), nitrate (–) (NarL, NarP); cotranscription with ttdAB
Constitutive expression
aspA	AspA, aspartase	Aerobic and anaerobic expression
dcuA	DcuA, fumarate carrier	Aerobic and anaerobic expression
mdh	Mdh, NAD-dependent malate dehydrogenase	Aerobic and anaerobic expression

^a Positive (+) or negative (–) effects are indicated, and the respective transcriptional regulators are given in parentheses.

^b Genes or operons located downstream of the respective genes that are cotranscribed in addition to separate transcription are shown in parentheses.

^c Conditions that affect gene expression: AN, anaerobic condition; C₄-DC, C₄-dicarboxyltes; Gluc, presence of glucose; effect on gene expression [(+), positive; (−), negative; (+/−), no effect]; responsible regulators shown in parentheses.

Transcriptional Regulation by Electron Acceptors

E. coli and related bacteria use electron acceptors in a specific order for respiration (25, 52, 54, 59, 87). O₂ (E^′_{0 O2/H2O} = +820 mV) is the preferred electron acceptor and represses other respiratory pathways and fermentation, whereas nitrate (E^′_{0 NO3−/NO2−} = +430 mV) represses other anaerobic respiratory pathways, including fumarate respiration (E^′_{0 fumarate/succinate} = +30 mV) and fermentation. Thus, fumarate respiration functions only in the absence of nitrate and O₂. The fumarate respiratory system of E. coli is synthesized under anaerobic and microaerobic conditions (up to 3 to 5% of air saturation) (165, 166, 167). The ΔG values, H⁺/e⁻ ratios, and ATP yields are highest in aerobic respiration, intermediate in nitrate respiration, and lowest in fumarate respiration (52, 59). In addition, growth rates during aerobic growth exceed those for growth by fumarate respiration (μ = 0.21 h⁻¹) by a factor of ∼6 (with glycerol as the electron donor) (77).

The hierarchical control is achieved by transcriptional regulators responding to electron acceptors. Transcriptional regulation by O₂ is effected by the FNR and aerobic respiratory control (ArcA) proteins. FNR is a cytoplasmic sensor-regulator containing a sensory and a helix-turn-helix DNA-binding domain. The sensory domain contains a [4Fe4S]²⁺ cluster that is converted to a [2Fe-2S]²⁺ cluster by direct reaction with O₂ (168, 169, 170, 171). ArcA is the response regulator of the ArcB-ArcA two-component system, which controls synthesis of the aerobic respiratory chain in response to O₂ availability (172). Nitrate regulation is effected by two two-component systems, NarX-NarL and NarP-NarQ, which sense the presence of external nitrate (173, 174). The C₄-dicarboxylate-specific regulation is effected by the DcuS-DcuR two-component system and two LysR-type transcriptional regulators, TtdR and DmlR. The DcuS-DcuR two-component system senses the presence of external C₄-dicarboxylate and induces the common C₄-dicarboxylate metabolism, including fumarate respiration (50, 53, 55, 107, 151). The cytoplasmic regulators TtdR and DmlR sense the presence of noncommon C₄-dicarboxylates and control the enzymes required for noncommon C₄-dicarboxylate metabolism (28, 53, 175, 176). The transcriptional regulators bind in various combinations and modes to the promoter regions of the genes involved in fumarate metabolism (Table 2).

Synthesis of DctA is maximal in the stationary phase during aerobic growth on C₄-dicarboxylates (10). The expression of dctA is subject to anaerobic repression by the ArcA-ArcB two-component system. C₄-dicarboxylates cause an approximately 2-fold induction via the DcuS-DcuR two-component system (50, 55) and glucose represses DctA approximately 30-fold by the cyclic AMP receptor protein-cyclic AMP complex (CRP-cAMP complex). CRP is also responsible for stationary-growth-phase induction (10). In addition, expression of dctA is autoregulated, and absence of DctA causes constitutive expression of dctA (10, 144, 150, 151). The transcriptional regulation of dauA and satP has not been studied in detail (6, 156), but expression of dauA is independent of DcuS. Transcription of the succinate dehydrogenase genes sdhCDAB is repressed by ArcA and FNR during anaerobic growth. C₄-dicarboxylates have no DcuS-DcuR-dependent effect on sdhCDAB expression. The genes for α-ketoglutarate dehydrogenase (sucAB genes) and of succinyl-CoA synthetase (sucCD genes) are located adjacent to the sdhCDAB genes, and their transcription is also controlled from the sdhC promoter. Expression of sdhC is also regulated by Fe ions and the ferric uptake regulator Fur. The effect is exerted by a small regulatory RNA, RyhB, the synthesis of which is repressed by Fe²⁺-bound Fur in the presence of iron (22, 177, 178). RyhB is produced under iron-limiting conditions; it binds by sequence complementarity as an antisense RNA to the promoter region of sdhCDAB and represses transcription. As a consequence, fur mutants are not able to grow on succinate. In addition, expression of fumA encoding fumarase A is controlled by RyhB (177, 178).

The genes for central anaerobic C₄-dicarboxylate metabolism (Fig. 3; Table 2), the frdABC and the dcuB fumB operons, are transcriptionally activated by FNR under anaerobic conditions and repressed by NarL in the presence of nitrate (51, 179, 180). In addition, expression of both operons is induced by C₄-dicarboxylates via the DcuS-DcuR two-component system (50, 55).

The synthesis of Dcu carriers is controlled at the transcription level. The expression of dcuB (dcuB fumB operon) is controlled by FNR and DcuS-DcuR. The repression of the dcuB fumB operon by nitrate is mediated by NarX-NarL and also NarQ-NarP (181), and catabolite repression is effected by CRP. Altogether, 150-fold induction takes place during growth by fumarate respiration in comparison with aerobic growth. In addition, synthesis of DcuS-DcuR is repressed by the NarX-NarL system in the presence of nitrate and regulated by an internal promoter responding to CRP-cAMP (179, 182). Nitrate and glucose are therefore believed to regulate expression of DcuS-DcuR-controlled genes indirectly by the control of the DcuS-DcuR levels. The anaerobic expression of dcuC is controlled by FNR and ArcA, is not repressed by nitrate, and is independent of C₄-dicarboxylates and glucose (Fig. 8; Table 2) (56, 57). Thus dcuC, in contrast to dcuB, is expressed to a high level in glucose fermentation. The genes of two Dcu carriers (dcuB and dcuC) are highly regulated, whereas the gene of DcuA (dcuA) is constitutive and shows only a slight induction (<2-fold) under anaerobic conditions (50, 51, 55, 56). Despite expression of DcuA under aerobic conditions, DcuA does not support substantial aerobic growth on succinate (5, 10).

The expression of the fumA, fumB, and fumC genes encoding the alternative fumarases differ in their response to electron acceptors, iron, carbon, superoxide, and growth rate (14, 15, 22, 183), involving regulation by ArcB-ArcA, FNR, NarX-NarL, SoxR, and RyhB. Fumarase B (fumB gene) is only produced in absence of both O₂ and nitrate with Fnr and NarX-NarL as the major regulators. Activity of fumarase A is found under aerobic and anaerobic conditions, with maximal activity under aerobic and microaerobic conditions. The fumA gene (fumarase A) is repressed under anaerobic conditions by ArcA and FNR. Fumarase A contains a catalytic FeS cluster and fumA is repressed in the absence of iron by RyhB (178). Fumarase C is produced under aerobic conditions and repressed under anaerobic conditions by ArcA. It does not require iron for function and, for this reason in particular, is utilized for aerobic growth under Fe limitation and under oxidative stress.

The ttdAB ttdT operon is expressed anaerobically (regulators FNR and ArcA) and repressed by NarL and NarP in the presence of nitrate (28, 53, 101, 175). The operon is induced by TtdR in the presence of L- and meso-tartrate. In aerobic D-malate metabolism, dmlA is induced by DmlR in the presence of D-malate, L- and meso-tartrate (28).

Regulation by C₄-Dicarboxylates and the DcuS-DcuR Two-Component System

In E. coli, the expression of the dcuB, fumB, frdABCD, and dctA genes is stimulated by the presence of fumarate, succinate, or other C₄-dicarboxylates in the medium. Other genes encoding C₄-dicarboxylate-metabolizing enzymes, such as sdhCDAB, dcuC, dcuA, or aspA, are not or are only slightly transcriptionally regulated by the addition of C₄-dicarboxylates. A regulatory system (dcuS dcuR genes) responding to the presence of external C₄-dicarboxylates is encoded upstream of dcuB (50, 55). DcuS-DcuR represents a C₄-dicarboxylate two-component system with the histidine kinase DcuS and the response regulator DcuR. DcuS is a member of the CitA/DcuS family of histidine protein kinases and shows close similarity to the citrate sensor CitA from E. coli and Klebsiella (53, 107, 184). The response regulator DcuR dimerizes upon phosphorylation at the conserved Asp56 residue. DcuR-P binds to the promoter regions of the DcuS-DcuR-regulated genes dctA, dcuB, and frdA containing a DcuR binding motif (185, 186).

DcuS contains a periplasmic sensory domain (PAS_P) that is framed by two transmembrane helices, a cytoplasmic PAS_C domain and the kinase domain with the conserved His residue (Fig. 12) (50, 53, 55, 151). 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 not effectors (187). L-Malate and fumarate are the preferred substrates. In addition to the C₄-dicarboxylates, citrate is a substrate of DcuS. The approximate K_m for the induction of the DcuS-dependent genes is in the millimolar range. The homologous citrate sensor kinase CitA, on the other hand, has high affinity and specificity for citrate and tricarboxylates with a K_D value in the low micromolar range (188, 189).

Figure 12.

Domain structure and topology of the DcuS sensor kinase (A) and compaction (B) of the periplasmic citrate/C₄dicarboxylate binding domains of CitA upon citrate binding or of DcuS upon L-malate binding, and (C) details of DctA/DcuS interaction. (A) DcuS is membrane-embedded by transmembrane helices 1 and 2 (TM1, TM2), and contains additionally the extracytoplasmic Per-Arndt-Sim domain PAS_P, a cytoplasmic PAS domain PAS_C, and a C-terminal HisKA/HATPase-type kinase. The monomers of the DcuS homodimer are presented in light and dark gray. In the dark gray monomer, the α-helical structure of TM2 and of the C-terminal helix α₆ PAS_P is indicated. + and − indicate the periplasmic and cytoplasmic sides of the membrane. (B) Structure comparison of the periplasmic PAS_P domains of DcuS with L-malate (brown; #3BY8 [93]) and CitA_Kp without citrate (gold, #2V9A). Structures were superimposed using the software Chimera (214). More details are given in (93, 151, 196, 197). (C) For the DctA/DcuS complex, only monomers of the proteins are shown. DcuS is preferentially dimeric (195), whereas DctA is presumably a trimer (215). The C-terminal cytoplasmic helix 8b of DctA plays a central role in the interaction of DctA with DcuS (144). Helix 8b interacts with the PAS_C domain of DcuS and controls by the interaction the kinase activity of DcuS (see text for details).

Signal perception occurs by the extracytoplasmic (or periplasmic) PER-Arndt-SIM (PAS_P) domain, also termed the Pho/DcuS/DctB/CitA (PDC) domain (Fig. 12). PAS_P contains the ligand binding, or sensing, site (93, 151, 187, 190, 191), whereas the transmembrane helices and PAS_C function in signal transduction from TM2 to the kinase, which is of the HisKA/HATPase type (192, 193, 194, 195). DcuS is a dimer or a higher oligomer in the membrane (53, 195).

Signal perception and sensing

In PAS_P of CitA, a close homolog of DcuS, binding of the effector results in contraction of the domain and an uplift of the C-terminal part of the β-sheet of PAS_P, away from the membrane surface (196) (Fig. 12). The corresponding region extends into TM2. The structure of DcuS-PAS_P (Fig. 12) (93) is almost identical to PAS_P from CitA_Kp, and a structural reorganization is predicted for DcuS-PAS_P upon ligand binding (53, 151).

Transmembrane signaling by DcuS by a piston-type displacement of TM2

The signaling-related movement of the TM helices of DcuS was probed by testing the water accessibility of the amino acid residues at the water-membrane interface (197). TM1 showed no significant movement upon fumarate-activation, whereas TM2 exhibited a piston-type shift to the periplasm by four residues. There is apparently only a low energy barrier for the transition between both positions of TM2, whereas a fixed “anchor” position is suggested for TM1 in agreement with the experimental data. Although the data indicate a piston-type mode of signal transfer by TM2, contribution from other modes (e.g., scissors type) of transmembrane signaling cannot be excluded to date.

Signal transfer from TM2 to the kinase domain and function of PAS_C as a regulated silencer

At the cytoplasmic side, the signal is transmitted to PAS_C and the kinase (Fig. 12). PAS_C has high plasticity (192, 194) and serves as a silencer, or inhibitor, of the kinase activity. Presence of the transporters DctA or DcuB stabilizes the dimer and silencing, but perturbation of the PAS_C/PAS_C’ homodimer upon activation by TM2 (or loss of DctA or DcuB) activates the kinase (192, 194).

The DctA/DcuS and DcuB/DcuS sensor complexes and their role for DcuS function

The transporters DctA and DcuB are obligate coregulators of DcuS under aerobic and anaerobic conditions, respectively, and their loss causes constitutive activity of DcuS (10, 149, 160, 198). DctA and DcuB interact in vivo with DcuS (144, 151, 199) by a C-terminal amphipathic helix (H8b in DctA) on the cytoplasmic side of the membrane (144).

Only binding of the effectors to DcuS, but not to the transporters, triggers sensing by the complexes (149, 151), DcuS has a defined binding site for C₄-dicarboxylates (93, 151, 187, 190), and sensing by the transporter/DcuS complexes occurs only via DcuS. Sensing is independent of the transport function of the transporters (149, 160), which instead have a structural role in converting DcuS to the sensory competent state (149, 151).

Function of DctA and DcuB as Coregulators in the Sensor Complex with DcuS: A Model for Function

Figure 13 summarizes the present model for sensing and signaling by the DctA/DcuS or DcuB/DcuS complexes. Binding of the ligand causes a contraction of PAS_P, resulting in an uplift of the C-terminal region around helix α₆ by approximately 3.8 Å. The uplift induces the displacement of TM2 toward the periplasm in a piston-type mode by four amino acid residues (or 1.1 turns of an α-helix). The displacement of TM2 is transmitted to PAS_C and the kinase. Signal transfer to the kinase apparently depends on a relief of PAS_C dimerization that abolishes kinase inhibition.

Figure 13.

Transmembrane signaling by DcuS: Control of the kinase activity by C₄-dicarboxylates and the transporter proteins DctA (or DcuB). B and C represent the functional state of DcuS in the DctA/DcuS sensor complex (C₄-dicarboxylate responsive DcuS). (A) shows DcuS without DctA (permanent active DcuS, constitutive ON). In the C₄-dicarboxylate responsive state (B, C), binding of C₄-dicarboxylates causes contraction of PAS_P with an uplift of α₆ and of TM2 (red arrows) by one helical turn in TM2. The shift of TM2 is transmitted in the cytoplasm to PAS_C, causing relieved PAS_C dimerization and relief of kinase inhibition. DctA and PAS_C collaborate in silencing (or inhibiting) the activity of the kinase domain. PAS_C is only able to silence the kinase when properly positioned by DctA. DctA is therefore a cosilencer of PAS_c, and silencing of the kinase can be abolished artificially both by deletion of PAS_C or of DctA, or physiologically by the pulling of TM2 after C₄-dicarboxylate binding at PAS_P. See text for references.

Role of DctA or DcuB as coregulators

DctA and DcuB are bifunctional and serve as transporters and as coregulators of DcuS during aerobic (DctA) or anaerobic (DcuB) growth. The transporters have no role in chemical or flux sensing but convert DcuS structurally from the permanent ON to the C₄-dicarboxylate responsive state (Fig. 13).

Polar Localization of the DcuS Sensor Complex

Bacteria reveal a complex spatial organization of some proteins within membranes or the cytoplasm. The spatial organization can be represented by an accumulation of proteins at distinct sites, or a colocalization of proteins from metabolic pathways or other cellular functions. The consequence is a spatial compartmentalization of proteins for functional reasons in bacteria (100, 200, 201, 202). DcuS exhibits dynamic, preferential polar localization in fast time lapse and fast recovery in FRAP experiments, even when it is produced at very low levels in a functionally active state (199, 203). The dynamic polar localization is therefore not caused by molecular crowding or a concentration artefact, is different from the more static polar localization described for other proteins, such as DivIVa or monocyte chemotactic proteins (204, 205, 206), and might be based on a diffusion and capture mechanism (206, 207). Factors that are commonly believed to be necessary for polar accumulation, such as cardiolipin contents, cell geometry, and high curvature at the cell poles, or guiding by the cytoskeleton protein MreB, play no direct role for DcuS localization (199). The component or mechanism responsible for the polar localization of DcuS is not known, unlike many other proteins with polar localization. DcuS intrinsic factors for polar localization, however, are the cytoplasmic PAS_C and kinase domains (199).

DctA and DcuR colocalize with DcuS at the cell poles when coexpressed at appropriate levels; hence, it can be assumed that functional DctA/DcuS/DcuR sensor units are formed (199, 203), which is in agreement with the functionally defined DctA/DcuS and DcuS/DcuR complexes. Overall, it appears that sensing by DcuS is complex not only in functional terms, but also in structural terms, because of the presence of the tripartite DctA/DcuS/DcuR complex. In this complex DcuS and DcuR can each be assumed to prevail as homodimers, whereas DctA is most likely homotrimeric in the functional state.

Cytoplasmic Regulators for Noncommon C₄-Dicarboxylates

E. coli contains cytoplasmic regulators that respond to noncommon C₄-dicarboxylates such as L-tartrate or D-malate. L-Tartrate and D-malate are perceived by the cytoplasmic regulators TtdR and DmlR, respectively (28, 175, 176). TtdR and DmlR are LysR-type transcriptional regulators consisting of an N-terminal helix-turn-helix DNA-binding domain (approximately residues 1 to 66) and a larger C-terminal effector-binding domain approximately 200 amino acids in length. The effector-binding domains of TtdR and DmlR show 52% identity in protein sequence (53). Under anaerobic conditions and in the presence of L- or meso-tartrate, TtdR stimulates the expression of the ttdAB ttdT operon, which encodes the L-tartrate/succinate antiporter TtdT and L-tartrate dehydratase TtdAB (Fig. 5), both key enzymes of L-tartrate fermentation (102). TtdR responds to either L- or meso-tartrate, but only L-tartrate is metabolized.

DmlR stimulates aerobic D-malate catabolism in the presence of D-malate (28), and then D-malate is taken up by the transporter DctA and oxidized to pyruvate and CO₂ by the D-malate dehydrogenase DmlA (Fig. 2). The induction of dmlA by DmlR responds to D-, L-, or meso-tartrate, but only D-malate supports aerobic growth in most strains of E. coli.

Neither TtdR nor DmlR respond to D-tartrate (28, 53, 175). The effectors TtdR (L-tartrate and meso-tartrate) and DmlR (D-malate or L-, meso-tartrate) carry a hydroxyl group at C-2 in the R configuration, whereas D-tartrate carries the hydroxyl group at C-2 in the S configuration like L-malate. Thus, the response to D-tartrate occurs by DcuS-DcuR (208), and D-tartrate is metabolized anaerobically by transporter DcuB and fumarase FumB (Fig. 5) (101). All reactions of anaerobic D-tartrate metabolism therefore represent side reactions of L-malate metabolism, based on a similar stereochemistry of the hydroxyl group at C-2 (Fig. 6).

Coordination of the Metabolism for Common and Noncommon C₄-Dicarboxylates

E. coli contains altogether three transcriptional regulators responding to C₄-dicarboxylates: the two-component system DcuS-DcuR, and the LysR-type regulators TtdR and DmlR (53). The common and noncommon C₄-dicarboxylate metabolic pathways are coordinated by the three regulators. DcuS-DcuR represents the general regulatory system controlling expression of the central reactions of C₄-dicarboxylate metabolism (frdABCD, fumB, dcuB, dctA genes) by extracellular substrates (Fig. 14). In accordance with this role, DcuS has broad substrate specificity, perceiving both common and noncommon C₄-dicarboxylates and inducing expression of genes encoding enzymes and transporters for the utilization of common C₄-dicarboxylates (53, 208). In contrast, cytoplasmic regulatory systems TtdR and DmlR show a narrow substrate spectrum and control the expression of the genes for the utilization of the noncommon C₄-dicarboxylates of L-tartrate fermentation (ttdAB ttdT) and D-malate utilization (dmlA), respectively. L-Tartrate fermentation also requires the enzymes of fumarate respiration (FumB and FrdABCD), and aerobic D-malate utilization also requires the transporter DctA; meanwhile, expression of the corresponding systems is accomplished by DcuS-DcuR.

Figure 14.

Coordinated regulation of common and noncommon C₄-dicarboxylates metabolism mediated by DcuS-DcuR two-component system and LysR-type regulators TtdR and DmlR. The common C₄-dicarboxylates and proteins/genes for general (central) C₄-dicarboxylate metabolism are presented in gray, proteins and genes of the noncommon C₄-dicarboxylate metabolism in orange. Dotted lines indicate that the type of regulation (direct or indirect) is not known. Arrow, induction; block, repression; common C₄-dicarboxylates, gray square; D-malate, orange triangle; L-tartrate, orange circles.

The different substrate specificities ensure that under anaerobic conditions, fumarate and L-malate induce only fumarate respiration (via DcuS-DcuR), whereas L-tartrate induces both L-tartrate-specific reactions (via TtdR) and fumarate respiration, which is also required for growth on L-tartrate (via DcuS-DcuR). In a similar strategy, presence of D-malate induces production of DctA and DmlA by the use of DcuS-DcuR and DmlR, respectively (Fig. 14).

With respect to regulation from outside (DcuS-DcuR) versus inside (TtdR, DmlR) of the bacteria, it appears that different strategies for sensing and regulation have to be applied (53). The common C₄-dicarboxylates fumarate, succinate, and L-malate are metabolites of central metabolism and permanently present within the cell. These substrates function only as inducers when supplied additionally from the exterior. On the other hand, the noncommon C₄-dicarboxylates D-malate and L-tartrate are sensed by cytoplasmic regulators, since no interference from central metabolism is expected.

Relation of C₄-Dicarboxylate Metabolism to Citrate and CitA-CitB-Dependent Regulation

Transcriptional regulation of the genes required for citrate fermentation is subject to dual control in response to citrate. Synthesis of the first, citrate-specific part comprising CitT, citrate lyase, and Mdh is induced by citrate and the two-component system CitA-CitB, which is highly specific for citrate (109, 184). Expression of the genes for the second part of the pathway (fumarate respiration; see Fig. 7) is induced by DcuS-DcuR. DcuS also responds to citrate (187, 208), and therefore expression of the fumB and frd genes is stimulated during growth on citrate by DcuS-DcuR, using the side activity of DcuS for citrate. This response ensures that citrate induces the citrate fermentation-specific reactions via CitA-CitB, and the reactions of fumarate respiration via DcuS-DcuR.

DcuR binds to the citA citB promoter region and apparently regulates citAB expression (209). This indicates that DcuS-DcuR controls CitA-CitB levels and function, and serves as an overriding regulatory system for the coordination of both metabolic systems and their interaction in citrate fermentation. In contrast, the DcuS-DcuR system appears to be independent of regulation by citrate and CitA-CitB (209).

Other Regulators Specific for C₄-Dicarboxylate Metabolism

In addition to DcuS-DcuR, TtdR, and DmlR, other regulators have been postulated to play a role in the regulation of C₄-dicarboxylate metabolism. The succinate stimulation of dctA expression is lost only in part in dcuSR mutants, indicating that further regulators are involved (50, 55). It has also been suggested that the gene regulator YhiF, which is a member of the LuxR family of helix-turn-helix transcriptional regulators, regulates succinate metabolism as well (1), although this has yet to be substantiated. Furthermore, tRNAs have been implicated in expression control of the C₄-dicarboxylate metabolism of S. enterica serovar Typhimurium (210, 211, 212). tRNAs in 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 represents a specific regulation or whether genes for succinate metabolism (and others) respond more sensitively to the loss of modification (210, 211).