INTRODUCTION

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Escherichia coli can utilize C₄-dicarboxylates for energy generation under both aerobic and anaerobic conditions (8). Under aerobic conditions, the uptake of C₄-dicarboxylates (fumarate, malate, and succinate) and L-aspartate is mediated by a secondary transporter and/or a binding protein-dependent system, designated Dct (19, 22). Three mutations (cbt at 16.2 min, dctA at 79.2 min, and dctB at 16.1 min) have been reported to result in the inactivation of components involved in aerobic C₄-dicarboxylate transport (23). The dctA gene has been sequenced, and the role of its product (DctA) in the utilization of C₄-dicarboxylates (and the cyclic monocarboxylate orotate) is supported by complementation studies of Salmonella typhimurium dctA or outA mutants (2, 37). The dctB and cbt genes are predicted to encode inner membrane and periplasmic binding proteins, respectively (21), but the genes have yet to be identified in the E. coli genome sequence.

Uptake, exchange, and efflux of C₄-dicarboxylic acids under anaerobic conditions are mediated by the Dcu systems (K_m for fumarate uptake, 50 to 400 µM), which are genetically distinct from the aerobic Dct system (8, 9, 45). Transport studies suggest that the Dcu systems are expressed exclusively under anaerobic conditions, activated by the anaerobic activator protein FNR, and repressed in the presence of nitrate. Three independent Dcu systems have been identified, DcuA, DcuB, and DcuC (36, 45). DcuA and DcuB are homologous proteins (36% identical), whereas DcuC is only 22 to 24% identical to DcuA and DcuB. The DcuA protein is encoded by a gene (dcuA, at 94.0 min) located 117 bp downstream of the anaerobically expressed aspartase gene aspA (see Fig. 1). DcuB is encoded by dcuB (at 93.5 min), which is 77 bp upstream of the gene (fumB) encoding the anaerobic fumarase, FumB (see Fig. 1). DcuC is encoded by the dcuC gene at 14.1 min.

Growth tests and transport studies using dcuA, dcuB, and dcuC single, double, and triple mutants have shown that DcuA, DcuB, and DcuC each mediate exchange as well as uptake (36, 45). The triple mutant was almost completely devoid of Dcu activity and was unable to use fumarate for anaerobic respiratory growth, but growth could be supported by fermentation. The single mutants exhibited no phenotype, but the dcuA dcuB double mutant was markedly defective in both C₄-dicarboxylate transport and fumarate respiratory growth, suggesting that DcuA and DcuB have analogous and mutually complementary transport functions in the anaerobic uptake of C₄-dicarboxylates (36, 45). The affinities of DcuA and DcuB for C₄-dicarboxylates are similar, except for the lower affinity of DcuA for malate (45).

Expression of the aspA gene is repressed by glucose (~10-fold) under aerobic conditions and enhanced anaerobically (~10-fold) in a manner which is partially (~2-fold) FNR dependent (16, 44). Expression of fumB is also induced anaerobically (1.5- or 5-fold) in a manner which is dependent on both FNR and ArcA (41, 44). The anaerobic expression of fumB is reduced by ~10- or 2-fold in an fnr background, and unlike aspA expression, fumB expression is not subject to glucose-mediated repression (41, 44). The anaerobic induction of aspA and fumB is appropriate since both aspartase and fumarase B are thought to function in the generation of fumarate for utilization as an anaerobic electron acceptor. Predicted FNR and cyclic AMP receptor protein (CRP) sites at suitable distances from putative promoter sequences are located immediately upstream of aspA and dcuB (36, 44). However, a previous analysis of the aspA-dcuA and dcuB-fumB intergenic regions showed no potential CRP sites and the putative FNR sites were poorly placed, suggesting that the dcuB-fumB and aspA-dcuA gene pairs are cotranscribed (36).

This paper describes studies on the transcriptional regulation and organization of the dcuA and dcuB genes that provide further information concerning the roles of DcuA and DcuB. The results show that the dcuB gene is expressed exclusively under anaerobic conditions in a manner that is FNR dependent and that it is repressed by NarL in the presence of nitrate and is subject to CRP-mediated catabolite repression. Furthermore, dcuB is strongly induced by C₄-dicarboxylates presumably mediated by an uncharacterized C₄-dicarboxylate-responsive gene regulator. In contrast, the dcuA gene shows little variation in expression under the growth conditions investigated. These findings suggest that DcuB functions primarily as a C₄-dicarboxylate transporter during fumarate respiration, whereas DcuA has a general role in anaerobic C₄-dicarboxylate transport.

	MATERIALS AND METHODS

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Strains and plasmids. All E. coli strains, plasmids, and phages used in this study are listed in Table 1. A plasmid containing the aspA-dcuA region was obtained by subcloning the 6.2-kb SphI-SalI fragment of pGS73 (13) into the corresponding sites of pBR322 to generate pGS745 (Fig. 1A). A dcuA'-lacZYA transcriptional fusion was produced by subjecting pGS745 to digestion with NarI, treatment with DNA polymerase I (Klenow fragment) to generate blunt-ended fragments, and digestion with EcoRI and then subcloning the resulting 1-kb EcoRI-NarI dcuA'-containing fragment into EcoRI- and SmaI-digested pRS415 (35) to generate pGS929 (Fig. 1A).

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Restriction maps and transcriptional organization of the aspA-dcuA (A) and dcuB-fumB (B) regions. The inserts of plasmids pGS745, pGS748, and pGS929 (A) and of plasmids pGS744, pGS749, and pGS931 (B) are shown. Restriction site abbreviations: B, BamHI; Bc, BclI; E, EcoRI; H, HindIII; N, NarI; P, PstI; S, SalI; Sp, SphI. Restriction sites in parentheses were introduced during PCR amplification. Asterisks indicate restriction sites that have been inactivated by digestion with the corresponding restriction enzyme followed by filling in with the Klenow fragment of DNA polymerase I. Open arrows indicate the positions and polarities of the aspA, dcuA, dcuB, and fumB structural genes. Closed arrows indicate primary transcripts and putative processed transcripts (sizes in kilonucleotides) as detected by Northern hybridization experiments. The relative abundance of each transcript is indicated by arrow thickness. The small downward arrow indicates a potential endoribonucleolytic processing site. Hatched bars represent DNA fragments used as hybridization probes, and strongly predicted stem-loop structures are indicated.

Growth media and conditions. Cultures were grown at 37°C, either aerobically in shaking 250-ml conical flasks or anaerobically in 10-ml bijou bottles (for -galactosidase measurements) or in 300-ml medical flats (for the preparation of total RNA). Bacteria were usually grown aerobically in L broth for DNA manipulation and in L broth supplemented with glycerol and fumarate for the extraction of total RNA. For measurements of -galactosidase activity, strains were grown in M9 minimal salts medium (Sigma), unless otherwise stated, with glucose (0.4%) or glycerol (0.4%) as a carbon source, and supplements of 0.5% Casamino Acids, 1 mM MgSO₄, 0.1 mM CaCl₂, and 0.5 mg of vitamin B₁ per ml. When used, fumarate, nitrate, and trimethylamine N-oxide (TMAO) were present at 50 mM.

-Galactosidase measurements. Samples with an optical density at 650 nm (OD₆₅₀) of 0.5 were withdrawn from cultures grown in duplicate at regular intervals during the growth cycle. The samples were cooled on ice, and the bacteria were pelleted by centrifugation, resuspended in ice-cold saline, and resedimented at 4°C. After complete removal of the supernatant by aspiration, the bacteria were frozen at 20°C and used within 3 days.

Northern hybridization and primer extension analysis. Total RNA present in MC4100 was extracted by using a method based upon that described by Aiba et al. (1). Cultures (500 ml) were harvested by centrifugation and resuspended in 3 to 5 ml of a solution containing 20 mM sodium acetate (pH 5.5), 0.5% SDS, and 1 mM EDTA at 4°C. An equal volume of acid-phenol (phenol equilibrated with 20 mM sodium acetate [pH 5.5]), was added, and the mixture was incubated for 5 min at 65°C. After centrifugation, the aqueous layer was removed, extracted twice more with acid-phenol, and then extracted once with chloroform and once with a mixture of phenol-CHCl₃-isoamyl alcohol (25:24:1; equilibrated with 0.1 M Tris-Cl [pH 8.0]). RNA in the aqueous layer was precipitated with 3 volumes of ethanol and dissolved in ~1 ml of water. The RNA content was determined spectroscopically by assuming that an OD₂₆₀ of 1 corresponds to ~40 µg of RNA per ml.

	RESULTS

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Effects of growth conditions on the expression of single-copy dcuA- and dcuB-lacZ transcriptional fusions. The expression of the dcuA and dcuB genes was investigated by using derivatives of MC4100 (lacZ) containing single copies of the corresponding lacZ transcriptional gene fusion (see Materials and Methods). The fusions were transferred from plasmids to RS45 via homologous recombination for use in constructing the monolysogens JRG3834 (MC4100 [RS45::dcuA'-lacZYA]) and JRG3835 (MC4100 [RS45::dcuB'-lacZYA]). In order to include the entire dcuA and dcuB operator-promoter regions, 1.1- and 1.4-kb segments of DNA located immediately upstream of the respective structural genes were fused to the lacZ reporter gene. Both fusions were active (see below), indicating that dcuA and dcuB each possess independent promoters and that dcuA transcription is not dependent on the upstream aspA gene.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Expression of dcuA-lacZ and dcuB-lacZ transcriptional fusions during aerobic (A) and anaerobic (B) growth at 37°C in M9 minimal salts medium containing 0.4% glycerol, 50 mM fumarate, and 0.05% Casamino Acids. The strains used were JRG3834 (dcuA-lacZ) ( and ) and JRG3835 (dcuB-lacZ) (, ). Growth (open symbols) and -galactosidase activities (closed symbols) are shown.

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Expression of the dcuA- and dcuB-lacZ fusions during aerobic (open bars) and anaerobic (closed bars) growth at 37°C in M9 minimal salts medium containing 0.05% Casamino Acids plus either 0.4% glycerol and 50 mM TMAO (A), 0.4% glycerol with 50 mM nitrate (B), or 0.4% glucose (C). The plus and minus signs indicate the presence and absence of 50 mM fumarate, respectively. -Galactosidase activities were assayed in samples of mid-logarithmic to late logarithmic cultures of JRG3834 (dcuA-lacZ) and JRG3835 (dcuB-lacZ).

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Effects of carboxylates on expression of the dcuB-lacZ fusion during anaerobic growth in M9 salts medium containing 0.4% glycerol and 50 mM TMAO but lacking Casamino Acids (except where indicated). The alternative carboxylates (50 mM) or 0.5% Casamino Acids (CA) (A) and the fumarate concentrations (B) are indicated on the x axis. Other details are as described for Fig. 3.

Effect of global regulators on dcuA- and dcuB-lacZ expression. The glucose, oxygen, and nitrate repression of dcuB expression suggests that dcuB could be subject to regulation by the global transcriptional regulatory proteins FNR, CRP, ArcA, NarP, and NarL. This was tested by measuring dcuB-lacZ (and dcuA-lacZ) expression in the appropriate regulatory mutants (Fig. 5). The anaerobic expression of dcuB in minimal medium containing glycerol plus TMAO and fumarate was 15-fold lower in a fnr strain, JRG3839, than in the parental strain, JRG3835 (Fig. 5Aii). However, even in the absence of FNR, the anaerobic expression level was still 19-fold higher (0.094 µmol/min/mg) than the aerobic level (0.005 µmol/min/mg). These observations suggest that the anaerobic activation of dcuB transcription is mediated by FNR-dependent and FNR-independent mechanisms. Aerobic dcuB expression was only twofold lower in the fnr strain (0.005 µmol/min/mg) than in the parental strain (0.009 µmol/min/mg), indicating that the FNR activation of dcuB expression is mainly an anaerobic process (Fig. 5A). Both the aerobic and anaerobic expression levels of dcuB were only ca. twofold lower in the arcA mutant (JRG3841), indicating that ArcA plays no more than a minor role in regulating dcuB expression in response to oxygen (Fig. 5B) and that ArcA is not responsible for the FNR-independent mechanism of anaerobic activation of dcuB transcription. The activation of dcuB expression was lowered ca. twofold by TMAO (from 2.5 to 1.4 µmol/min/mg Fig. 5Aii and Bii), although dcuA expression was unaffected by TMAO (Fig. 5Ai and Bi), suggesting that TMAO represses dcuB expression.

View larger version (20K): [in this window] [in a new window]   FIG. 5.   Effect of fnr (A), arcA (B), crp (C), and narL (D) on dcuA and/or dcuB expression. Growth was performed under both aerobic (open bars) and anaerobic (closed bars) conditions in M9 minimal medium containing either 0.4% glycerol, 50 mM TMAO, 50 mM fumarate, and 0.05% Casamino Acids (A and D); 0.4% glycerol and 50 mM fumarate (B and Ci); and 0.4% glucose with (+) or without () 50 mM fumarate (Cii). The strains used were JRG3834 (Ai and Bi), JRG3835 (Aii, Bii, and D), JRG3837 and JRG3846 (C), JRG3838 (Ai), JRG3839 (Aii), JRG3840 (Bi), JRG3841 (Bii), and JRG3842 (D). Other details are as described for Fig. 3. wt, wild type.

Northern blot analysis of the dcuA, dcuB, aspA, and fumB transcripts. To determine whether the lacZ fusion analyses reflect transcript abundance and to examine the transcriptional organization of the aspA-dcuA and dcuB-fumB genes, Northern hybridization experiments were performed on the dcuA, dcuB, aspA, and fumB gene transcripts. Total RNA was extracted from MC4100 grown aerobically and anaerobically to mid-logarithmic phase in L broth containing glycerol plus fumarate. RNA was hybridized with labeled dcuA, dcuB, aspA, and fumB gene fragments (Fig. 1 and 6).

View larger version (76K): [in this window] [in a new window]   FIG. 6.   Northern hybridization analysis of the dcuA (A), aspA (B), dcuB (C), and fumB (D) transcripts. Total RNA was extracted from MC4100 grown aerobically and anaerobically in L broth containing 0.4% glycerol and 50 mM fumarate and, after electrophoresis and capillary transfer, was hybridized with labeled dcuA, aspA, dcuB, and fumB gene fragments (see Materials and Methods). The positions of RNA molecular weight standards (in kilonucleotides) are indicated. The arrows mark hybridizing transcripts (sizes in kilonucleotides) and a minor band (2.5 kilonucleotides) possibly corresponding to nonspecifically hybridizing 23s rRNA.

Determination of the transcriptional start sites for the dcuA, dcuB, and aspA genes. The transcriptional start sites of the dcuA, dcuB, and aspA genes were determined by primer extension analysis (Fig. 7). Four primer extension products were obtained for the dcuA transcript; they correspond to transcriptional start sites at C-2058, T-2059, T-2060, and T-2061 (Fig. 7A and 8A). These sites are approximately 30 bp upstream of the +1 site predicted by Six et al. (36). The relative abundances of the four cDNA species were 1:3:6:1, respectively, indicating that of the four alternative transcriptional start sites, T-2060 is preferred. The latter is 70 bp upstream of the dcuA initiation codon, and suitably positioned and well predicted 10 and 35 sequences are centered at bp 2047.5 and bp 2025.5, respectively. No potential FNR or CRP binding site sequences were detected directly upstream of the dcuA promoter, which is consistent with the expression studies.

View larger version (33K): [in this window] [in a new window]   FIG. 7.   Determination of the dcuA (A), dcuB (B), and aspA (C) transcriptional start sites by reverse transcriptase-mediated primer extension. Lanes 1 indicate primer extension products. Sequencing ladders (lanes A, C, G, and T) were generated by using the primers used for the reverse transcriptase reaction. The sequencing ladders and primer extension products shown in panels A, B, and C were generated by using, respectively, primers P1dcuA, P2dcuB, and P2aspA (see Materials and Methods). Similar results were obtained with primers P2dcuA, P1dcuB, and P1aspA (data not shown). The sequence, its complement, and the transcriptional start sites (indicated by asterisks) are shown. Horizontal arrows indicate primer extension products.

View larger version (30K): [in this window] [in a new window]   FIG. 8.   Nucleotide sequence of the dcuA (A), dcuB (B), and aspA (C) promoter regions. Coordinates for the aspA gene are from the work of Woods et al. (43), and coordinates for the dcuA and dcuB gene are from the work of Six et al. (36). The experimentally determined +1 sites are boxed and labeled, as are the deduced 35 and 10 sites and predicted CRP and FNR sites. Putative NarL binding sites are indicated by horizontal arrows: rightward arrows indicate that the binding site is on the coding strand, while leftward arrows indicate that the binding site is on the noncoding strand. Residues matching the corresponding consensus sequence residues for the FNR and CRP sites are in boldface type. Closed circles indicate residues matching the corresponding consensus sequence residues for the 35 and 10 sites. The positions of the predicted FNR and CRP sites with respect to the +1 sites are indicated.

	DISCUSSION

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This study shows that the dcuA and dcuB genes of E. coli are differentially regulated. The dcuB gene is expressed exclusively under anaerobic conditions in a manner that is largely FNR dependent, is repressed by nitrate through a mechanism that is mostly NarL mediated, and is strongly induced by C₄-dicarboxylates anaerobically. Expression of dcuB is also repressed by glucose, is slightly repressed by TMAO, and in the absence of glucose is threefold induced by CRP. In agreement with the expression data, the dcuB promoter region contains well-predicted binding sites for FNR, NarL, and CRP (Fig. 8). ArcA has no significant role in dcuB regulation, although it appears that a factor other than FNR also contributes to anaerobic induction of dcuB. Furthermore, in the absence of NarL, dcuB is repressed fourfold by nitrate in a manner that appears to be NarP independent, and, in addition, dcuB expression is reduced by glucose in a fashion that is mostly CRP independent. Thus, factors other than FNR, NarL, and CRP also appear to contribute to the regulation of dcuB in response to oxygen, nitrate, and glucose. In contrast to the strong regulation of dcuB, expression of the dcuA gene is virtually unaffected by the environmental and regulatory factors tested. The dcuB gene is ca. fourfold more strongly expressed than dcuA during fumarate respiration (Fig. 3A, 5Bii, and 5Ci). This supports the results of transport experiments with dcu mutants showing that DcuB is the dominant Dcu carrier during fumarate respiration (45). However, the dcuA gene is more strongly expressed than dcuB under most of the other growth conditions examined, and this is concordant with the better codon usage of dcuA relative to that of dcuB (36).

The expression and transport data are consistent with each other insofar as Dcu transport activity and the combined dcuA- and dcuB-lacZ activities are activated anaerobically by FNR, repressed anaerobically in the presence of nitrate by NarL, increased by fumarate or succinate, and weakly affected by ArcA (8, 9). The factors affecting Dcu transport activity described above clearly reflect those influencing dcuB expression. This is because dcuB expression, but not dcuA expression, is strongly modulated by these factors and also because dcuB expression exceeds that of dcuA during fumarate respiration. The slight repression effect (greater than twofold) of TMAO on dcuB expression is consistent with the lack of effect of TMAO on Dcu transport activity. It is possible that the TMAO effect on dcuB expression is mediated by the TMAO-responsive two-component sensor-regulator system, TorS-TorR (18, 34), although this has not been tested.

Surprisingly, the correspondence between transport and expression activities is not maintained under all conditions examined. Previous studies showed that Dcu activity is only slightly reduced by glucose and is unaffected by cAMP or a cya mutation, suggesting that CRP does not regulate Dcu synthesis (8, 9, 45). However, clear glucose and CRP effects on dcuB expression were observed in the studies reported here, although the CRP effect was relatively weak. The reason for these discrepancies is unclear, but they could reflect posttranscriptional effects or a lack of specificity when transport activity mediated by at least three alternative systems is measured.

The 70-fold induction of dcuB expression by C₄-dicarboxylates (Fig. 4) is likely to be mediated by an undefined C₄-dicarboxylate-dependent regulator able to sense and respond to exogenous C₄-dicarboxylates. A good candidate for such a regulator is a putative two-component regulatory system composed of a response regulator and a membrane-associated histidine kinase sensor encoded by the yjdHG genes located just 570 bp upstream of dcuB. The corresponding proteins have strong sequence similarity to the Klebsiella CitB and CitA proteins involved in the regulation of anaerobic citrate metabolism (5, 25). The possible involvement of these genes in C₄-dicarboxylate-responsive gene regulation is being investigated. Other E. coli genes are also known to be induced, albeit weakly, by C₄-dicarboxylates. These are the frdABCD and nuo operons which are 1.5-fold induced by fumarate and ~2.5-fold induced by fumarate or succinate, respectively (4, 17). The mechanism governing this regulation is unknown.

The pattern of dcuA and dcuB expression provides important clues for the likely physiological functions of the homologous and functionally related DcuA and DcuB proteins. The profile for dcuB expression is consistent with a role for DcuB in the provision of substrate and export of product for the reaction catalyzed by Frd during fumarate respiration. Indeed, the expression profile of dcuB resembles that of the frdABCD operon. Appropriately, both are anaerobically induced by FNR and repressed by NarL in response to nitrate (14, 17), thus ensuring that oxygen and nitrate are utilized in preference to fumarate as terminal electron acceptors. The fumB gene, which is adjacent to dcuB, encodes the enzyme fumarase B, which acts as a malate dehydratase in the conversion of malate to fumarate. Fumarase B thus provides substrate for Frd during anaerobic fumarate respiration. Therefore, DcuB and fumarase B are both involved in feeding substrate to Frd and constitute consecutive steps in the anaerobic transport and metabolism of malate. The dcuB and fumB genes would therefore be expected to be expressed in a coordinated fashion, as is suggested by the Northern blotting analysis showing strong anaerobic induction and partial cotranscription (Fig. 6).

The expression of dcuA under both aerobic and anaerobic conditions is inconsistent with the previously proposed, anaerobic function for DcuA (36, 45) and suggests that DcuA has an aerobic function in addition to contributing to anaerobic C₄-dicarboxylate uptake. However, transport studies failed to demonstrate any Dcu activity, attributable to DcuA, under aerobic conditions, possibly because the Dcu systems are inactivated by oxygen (8, 9). Therefore, it is possible that the aerobic expression of dcuA produces an inactive DcuA protein. However, inactivation of Dcu activity by transient exposure to oxygen (or other oxidants) can be reversed by subsequent treatment with reducing agents (8). This offers the possibility that constitutive expression of dcuA allows E. coli to respond rapidly to transitions from aerobic to anaerobic conditions through the activation of presynthesized DcuA.

Aspartase, the product of the aspA gene located upstream of dcuA, converts L-aspartate to fumarate. Together with the constitutive aspartate aminotransferase (encoded by aspC), it is thought to provide an alternative mechanism for converting oxaloacetate to fumarate as part of the reductive branch of the noncyclic form of the citric acid cycle (6). A previous lacZ fusion analysis (44) and the Northern blot analysis reported above suggest that, like aspC and dcuA, the aspA gene is well expressed under both aerobic and anaerobic conditions, indicating that DcuA and aspartase have aerobic as well as anaerobic functions. However, it should be stressed that this is inconsistent with aspartase activity measurements that show that aspartase is anaerobically induced (16). Aspartase may also have a role, together with the anaerobically induced periplasmic asparaginase II, in the utilization of exogenous asparagine (24) and in the degradation of aspartate for use as a carbon source (29). Furthermore, aspartase is required for regenerating oxaloacetate in the aerobic and anaerobic utilization of glutamate (38). Whether the DcuA protein assists in any of these processes is uncertain, but the colocations of the aspA and dcuA genes and the related substrate specificities of their products are certainly suggestive of linked functions.