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Journal of Bacteriology, October 2008, p. 6458-6466, Vol. 190, No. 19
0021-9193/08/$08.00+0     doi:10.1128/JB.00780-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of the Dicarboxylate Uptake System DccT in Corynebacterium glutamicum

Jung-Won Youn,^1,# Elena Jolkver,^2,# Reinhard Krämer,² Kay Marin,^2* and Volker F. Wendisch^1*

Institute of Molecular Microbiology and Biotechnology, Westfalian Wilhelms University Muenster, Muenster, Germany,¹ Institute of Biochemistry, Cologne University, Cologne, Germany²

Received 3 June 2008/ Accepted 21 July 2008

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Many bacteria can utilize C₄-carboxylates as carbon and energy sources. However, Corynebacterium glutamicum ATCC 13032 is not able to use tricarboxylic acid cycle intermediates such as succinate, fumarate, and L-malate as sole carbon sources. Upon prolonged incubation, spontaneous mutants which had gained the ability to grow on succinate, fumarate, and L-malate could be isolated. DNA microarray analysis showed higher mRNA levels of cg0277, which subsequently was named dccT, in the mutants than in the wild type, and transcriptional fusion analysis revealed that a point mutation in the promoter region of dccT was responsible for increased expression. The overexpression of dccT was sufficient to enable the C. glutamicum wild type to grow on succinate, fumarate, and L-malate as the sole carbon sources. Biochemical analyses revealed that DccT, which is a member of the divalent anion/Na⁺ symporter family, catalyzes the effective uptake of dicarboxylates like succinate, fumarate, L-malate, and likely also oxaloacetate in a sodium-dependent manner.

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Corynebacterium glutamicum is a predominantly aerobic nonsporulating and biotin-auxotrophic gram-positive bacterium which is used for the industrial production of amino acids, mainly of L-lysine (>750,000 tons/year) (61) and glutamate (>1,500,000 tons/year) (50). C. glutamicum is well studied not only with respect to amino acid biosynthesis but also regarding carbon metabolism and its regulation (2, 6, 57). C. glutamicum grows aerobically on a variety of carbohydrates and organic acids as the sole sources of carbon and energy, e.g., on sugars like glucose, fructose, and sucrose and on organic acids like gluconate, acetate, propionate, pyruvate, and L-lactate, but also on ethanol, glutamate, vanillate, 4-hydroxybenzoate, and protocatechuate (1, 7-9, 19, 31, 35, 59). In general, growth on substrate mixtures is characterized by coutilization, e.g., of glucose with acetate (59), and even a nongrowth substrate like serine can be utilized simultaneously with glucose (35). Rarely, preferential utilization of glucose before glutamate or ethanol, e.g., has been observed (1, 28).

Citrate is the only tricarboxylic acid (TCA) cycle intermediate described as supporting the growth of C. glutamicum (42). C. glutamicum can grow on citrate as the sole carbon source and coutilizes citrate and glucose (42). In a combined DNA microarray and proteome analysis, it was revealed that the expression of genes for two citrate uptake systems, CitM and TctABC, was induced when citrate was present in the medium (42). Although succinate uptake by C. glutamicum has been observed in biochemical assays, growth on succinate has not been reported (11, 41). Other bacteria, e.g., Bacillus subtilis, are able to grow not only with citrate but also with the dicarboxylic TCA cycle intermediates succinate, fumarate, and L-malate (55). The transporter YdbH in B. subtilis is responsible for the uptake of fumarate and succinate (3) but not for that of L-malate, which is taken up via MaeN (52) or the malate/lactate antiporter YqkI (56). In Escherichia coli, fumarate, succinate, and L-malate are taken up by DctA under aerobic conditions (10) and by DcuA, DcuB, DcuC, or DcuD under anaerobic conditions (25). The Dcu antiporters are responsible for the uptake of malate and fumarate and the efflux of succinate, which can also be excreted in antiport with citrate by CitT under anaerobic conditions (26, 43). Deletion of dctA, dcuA, dcuB, dcuC, dcuD, and citT leads to a deficiency in growth on succinate and fumarate (25).

In this study, we identify and characterize the divalent anion/Na⁺ symporter (DASS) family protein DccT as a sodium-dependent dicarboxylate uptake system specific for C₄-dicarboxylic acid intermediates of the TCA cycle in C. glutamicum.

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Bacteria, media, and growth conditions. The strains and plasmids used are listed in Table 1. Standard Luria-Bertani (LB) medium was used for E. coli (48) and brain heart infusion medium (Difco) and LB were used as a complex medium for C. glutamicum. CgXII was used as the minimal medium for C. glutamicum (12), and 100 mM glucose, 100 mM fumarate, 100 mM succinate, 100 mM L-malate, 100 mM acetate, and 100 mM L-lactate were added as carbon sources. When appropriate, kanamycin (25 mg liter^–1) and isopropyl-β-D-thiogalactopyranoside (IPTG; 1 mM) were added to the medium. E. coli was grown at 37°C and C. glutamicum at 30°C in 50 ml of medium in 500-ml baffled shake flasks and at a 120-rpm agitation. For anaerobic cultivations, the media were flushed with nitrogen for 30 min in anaerobic test tubes to displace the oxygen, and cysteine-HCl (0.5 g liter^–1) and reazurin (1 mg liter^–1) were added to control and remove the presence of residual oxygen. The cultivation mixtures contained 30 mM sodium nitrate as the terminal electron acceptor (36) and were incubated for 20 h.

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TABLE 1. Bacteria and plasmids used in this study

Construction of plasmids and strains. All plasmids were constructed in E. coli DH5 from PCR-generated fragments (KOD Hot Start DNA polymerase; Novagen) by using C. glutamicum ATCC 13032 genomic DNA prepared as described previously (15) as a template. E. coli was treated and transformed by standard methods (48). The plasmids were introduced into C. glutamicum by electroporation (12). All transformants were analyzed by plasmid analysis and/or PCR with appropriate primers. The absence of mutations in the cloned genes was verified by sequence analysis.

Homologous overexpression of dccT. The construction of pVWEx1-dccT is based on the expression vector pVWEx1 (40). dccT was amplified from genomic DNA of C. glutamicum by use of the primer Ex-dccT-fw (5'-CGGATCC GAAAGGAGGCCCTTCAGATGAGCACACCTGACATTAA-3'; the nucleotide corresponding to nucleotide [nt] 239935 of BX927147 is underlined, the BamHI restriction site is shown in boldface, and nucleotides in italics correspond to an artificial ribosome binding site) and the primer Ex-dccT-bw (5'-GCGAGCTCTTAAAGCATGATGCCAAAGAG-3'; the nucleotide corresponding to nt 241518 of BX927147 is underlined and the SacI restriction site is shown in boldface). The fragment dccT was cloned via BamHI and via a blunted SacI site into a BamHI- and Acc65I-blunted site vector, pVWEx1. The vector constructed, pVWEx1-dccT, allows the IPTG-inducible expression of dccT in C. glutamicum.

Construction of a reporter gene fusion of dccT with the promoterless cat gene. In order to monitor the activity of the dccT promoter from the C. glutamicum wild type (WT) and SSM (for succinate spontaneous mutant) during growth on differential carbon sources, transcriptional fusions with the promoterless cat gene were used, based on the corynebacterial promoter-probe vector pET2 (54). The dccT promoter region (–227 to +27) was amplified by PCR from genomic DNA of C. glutamicum WT and SSM by using primers pET-dccT-fw (5'-CGGGATCCCTCTGTCGCGGTTAATCATC 3'; the nt corresponding to nt 239610 of BX927147 is underlined, and the BamHI restriction site is given in bold) and pET-dccT-bw (5'-CGGAGCTCCCGGTCATCAATGACCATGT 3'; the nt corresponding to nt 239864 of BX927147 is underlined, and the SacI restriction site is given in bold). Promoter activities of C. glutamicum WT carrying either pET2-dccT-WT or pET2-dccT-SSM were measured by determining the chloramphenicol acetyltransferase activity.

Preparation of crude extracts and chloramphenicol acetyltransferase assay. Crude extracts were obtained from 50-ml exponentially growing cultures. The cells were harvested by centrifugation (10 min, 3,200 x g, and 4°C) and washed in 40 ml 0.08 M Tris-HCl (pH 7.0) buffer. The pellets were resuspended in 1 ml of the same buffer and mechanically disrupted twice by 20 s of bead beating with 250 mg of 0.1-mm zirconia-silica beads (Roth, Karlsruhe, Germany) by use of a Silamat S5 (Vivadent, Ellwangen, Germany). After centrifugation (1 h, 14,000 x g, 4°C) of the suspension, the supernatant was used for measuring the chloramphenicol acetyltransferase activity as described previously (49).

Preparation of total RNA for 5' RACE-PCR and DNA microarray analysis. Total RNA was isolated from exponentially growing cells by using the RNeasy system (Qiagen, Hilden, Germany) with on-column DNase I treatment prepared as described previously (34). The quantity and quality of purified RNA were analyzed by UV spectrometry, and RNA was stored at –20°C until use. Total RNA (2 to 5 µg) was used to perform 5' rapid amplification of cDNA ends-PCR (5' RACE-PCR). After amplification of the cDNA with random primers (Invitrogen, Karlsruhe, Germany) by reverse transcription-PCR using Superscript II (Invitrogen, Karlsruhe, Germany), the fragment was treated with terminal deoxynucleotidyltransferase and dATP or dCTP before use as a template for PCR. The nucleotide sequences of the primers for the 5' RACE-PCR and nested PCR were as follows: for primer RACE-dccT-1, 5'-ACAGCTAGCACGACCGCTAGA-3' (the nucleotide corresponding to nt 240395 of BX927147 is underlined); for primer RACE-dccT-2, 5'-GGCAATGATG CCGGTTGCTTG-3' (the nucleotide corresponding to nt 240039 of BX927147 is underlined); for primer OligoT, 5'-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTTT-3; for primer OligoG, 5'-GACCACGCGTATCGATGTCGACGGGGGGGGGGGGGGGGGG-3'; and for primer Antioligo, 5'-GACCACGCGTATCGATGTCGAC-3'. The PCR fragment was sequenced without cloning at the Klinikum Münster, Labor für molekulare Diagnostik (Muenster, Germany).

DNA microarray analysis. DNA microarrays based on PCR products of C. glutamicum genes were used for global gene expression analysis (58). The methods for the synthesis of fluorescently labeled cDNA from total RNA microarray hybridization, washing, and gene expression analysis were carried out as described previously (34, 42, 58).

Transport assays. Cells were grown to mid-exponential phase in minimal medium MM1 (29) supplied with glucose as the sole carbon source and 1 mM IPTG for the induction of pVWEx1-dccT expression if appropriate. Subsequently, cells were washed three times with 2-(N-morpholino)ethanesulfonic acid (MES)-Tris buffer (50 mM MES, 50 mM Tris, pH 8.0, 10 mM NaCl, 10 mM KCl) and incubated on ice until the measurement. Before the transport assay, cells were incubated for 3 min at 30°C with 10 mM glucose at an optical density at 600 nm of 2 in an assay volume of 1 ml for energization. As tracers, ¹⁴C-labeled succinate, fumarate, and L-malate, with specific activities of 2, 0.13, and 2.04 GBq/mmol, respectively (MP-Biochemicals, Illkirch, France), were used with the indicated concentrations of the corresponding sodium salts of the substrates in 200 µl. Finally, 100-, 29-, and 67-Bq radioactivity levels were applied in the assay for succinate, fumarate, and malate, respectively. Samples of 200 µl were taken each 5 s for 1 to 2 min in the case of succinate and each 30 s in the case of malate and fumarate in order to determine initial uptake rates and to avoid saturation of transport. Cells were collected on GF55 glass fiber filters (Schleicher and Schuell, Dassel, Germany) and washed twice with 2.5 ml of 0.1 M LiCl, 22°C. After the resuspension of cells in scintillation fluid (Rotiszinth, Roth, Germany), the radioactivity of the sample was counted in a scintillation counter (Beckman, Krefeld, Germany). In order to analyze the transport mechanism or the substrate specificity, uptake measurements were performed in the presence of saturating substrate concentrations (100 µM succinate) and different sodium concentrations as well as putative substrates in 100-fold excesses. All assays were performed at least in triplicate and standard deviations are indicated.

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Isolation of the spontaneous mutants FSM and SSM of C. glutamicum able to grow on fumarate or succinate as the sole carbon source. On minimal medium containing either fumarate or succinate as the sole carbon source, C. glutamicum WT did not show significant growth for at least 20 h regardless of whether the dicarboxylates were present or absent from the preculture medium (Table 2 and Fig. 1). However, after prolonged incubation (24 h) on minimal medium with fumarate, substrate consumption and biomass formation were detected. When cells of such a culture were cultivated intermittently on LB complex medium and subsequently transferred to minimal medium with fumarate as the sole carbon source, rapid growth occurred immediately without a lag phase, indicating that a mutant had been selected during the first culture on fumarate-containing minimal medium. The selected mutant strain was named C. glutamicum FSM (for fumarate spontaneous mutant). Similarly, a spontaneous mutant was isolated on succinate, which was named C. glutamicum SSM. To determine the frequencies of occurrence of succinate- and fumarate-utilizing mutants, defined numbers of cells were plated on CGXII minimal medium agar plates containing 20 mM succinate or fumarate. Small colonies appeared after 7 days at 30°C on succinate and fumarate plates with frequencies of 2.7 x 10^–7 and 2.5 x 10^–7, respectively. Both of the spontaneous mutants analyzed further, FSM and SSM, grew without lag phases on minimal medium containing either fumarate or succinate (Table 2) when inoculated from complex medium. Additionally, both mutants were able to grow on L-malate. However, the observed growth rate on L-malate was lower, while the biomass yield was comparable to that seen for growth on fumarate or succinate (Table 2). In contrast to what was seen for C. glutamicum WT, the altered capacity for the transport or metabolic conversion of fumarate, succinate, and L-malate in FSM and SSM sustained growth and might be due to altered expression of a particular gene(s).

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TABLE 2. Growth of C. glutamicum WT and mutants FSM and SSM on minimal medium with different carbon sources

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FIG. 1. Growth of C. glutamicum WT(pVWEx1-dccT) on CgXII minimal medium with different carbon sources in the absence (filled symbols) or presence (open symbols) of 1 mM IPTG. Glucose (circles), fumarate (diamonds), succinate (triangles), and L-malate (squares) were used as carbon sources at concentrations of 100 mM. Averages and experimental errors from at least two independent growth experiments are shown. OD600 nm, optical density at 600 nm.

Transcriptome analysis of C. glutamicum WT and mutants FSM and SSM by DNA microarray experiments. Global gene expression patterns of C. glutamicum WT, FSM, and SSM were compared by DNA microarray experiments in order to identify genes showing altered expression in the spontaneous mutants. C. glutamicum WT, FSM, and SSM were grown in LB complex medium, i.e., under presumed noninducing conditions, and total RNA was isolated during the exponential growth phase and used for DNA microarray analysis. In total, 10 genes showed mRNA levels in both mutants that were altered compared to those seen for C. glutamicum WT. The expression of three genes was increased in mutants FSM and SSM, while seven genes showed mRNA levels lower than those seen for C. glutamicum WT (Table 3). None of the genes were found to encode an enzyme involved in the metabolism of fumarate, succinate, or L-malate. However, the gene cg0277 encodes a putative transport protein which belongs to the DASS family (TC 2.A.47) (60). Functionally characterized members of the DASS family have been shown to transport inorganic sulfate, inorganic phosphate, and dicarboxylic amino acids along with di- and tricarboxylic organic acids. Consequently, we hypothesized that mutants FSM and SSM had gained the ability to grow on dicarboxylates due to high expression levels of cg0277, which encodes an uptake system for such compounds. We named the gene dccT, for dicarboxylic acid corynebacterial transporter.

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TABLE 3. Gene expression differences in C. glutamicum mutants FSM and SSM as compared to C. glutamicum WT during growth on LB complex medium

Overexpression of the dccT gene in C. glutamicum permits growth on fumarate, succinate, or L-malate as the sole carbon source. In order to determine whether plasmid-borne expression of dccT is sufficient to allow the growth of C. glutamicum WT on fumarate, succinate, or L-malate as the sole carbon source, the plasmid pVWEx1-dccT, mediating the IPTG-inducible expression of dccT, was constructed and transferred into C. glutamicum WT. The resulting strain, C. glutamicum WT(pVWEx1-dccT), was grown in the presence or absence of 1 mM IPTG on CgXII minimal medium containing 100 mM of either glucose, fumarate, succinate, or L-malate. On glucose, C. glutamicum WT(pVWEx1-dccT) grew like C. glutamicum WT in the presence or absence of IPTG (Fig. 1; also data not shown). In contrast, significant growth of C. glutamicum WT(pVWEx1-dccT) on fumarate, succinate, or L-malate occurred only when the plasmid-borne expression of dccT was induced by 1 mM IPTG (Fig. 1). The overexpression of dccT sustained growth on succinate and fumarate, with high growth rates compared to that on L-malate (0.44 h^–1and 0.43 h^–1, respectively, compared to 0.33 h^–1). Thus, high expression levels of dccT enabled C. glutamicum WT to utilize fumarate, succinate, and L-malate as the sole carbon and energy sources for growth.

DccT is a sodium-dependent transporter for dicarboxylic intermediates of the TCA cycle. With the aim of demonstrating the direct participation of the DccT protein in the dicarboxylate uptake of C. glutamicum, the uptake of succinate, fumarate, and malate was determined using radioactively labeled substrates. For C. glutamicum WT, very low transport activities were found, in agreement with the impaired utilization of these substrates (Fig. 2). In contrast, significant transport activities were observed for mutant strains FSM and SSM for all three substrates. In order to characterize the transport activities present in the mutant strains, kinetic parameters were obtained after subtraction of the WT values and fitting of the curves by nonlinear regression according to the Hill equation (Fig. 2). Thus, apparent concentrations supporting half-maximal transport rates (K_0.5) of 30 ± 4 µM for succinate in the SSM strain and 79 ± 7 µM for fumarate in the FSM strain were derived. Values for the maximum rate of transport (V_max) of DccT in C. glutamicum SSM and FSM were comparable and reached 35 ± 2 nmol min^–1 mg (dry weight)^–1 for succinate and 30 ± 2 nmol min^–1 mg (dry weight)^–1 for fumarate. In each case, a Hill coefficient of greater than 1.5 was calculated, suggesting cooperative binding of the substrate (2.2 ± 0.7 for succinate and 2.4 ± 0.5 for fumarate).

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FIG. 2. Uptake of succinate (A), fumarate (B), and malate (C) by C. glutamicum WT (squares), WT(pVWEx1-dccT) (triangles), SSM (circles in panel A), and FSM (circles in panel B) as measured with 14C-labeled substrates. Uptake rates were determined as a function of substrate concentration and data fitted according to the Hill equation. The presented data are averages of at least three measurements. DW, dry weight.

The kinetic parameters obtained for C. glutamicum WT(pVWEx1-dccT) were comparable to those obtained for mutant strains SSM and FSM (Fig. 2A and B). In addition, the kinetic parameters for the uptake of malate were determined for C. glutamicum WT(pVWEx1-dccT) (K₀.₅ of 361 ± 87 µM, V_max of 33 ± 4 nmol min^–1 mg (dry weight)^–1, and a Hill coefficient of 1.3 ± 0.2). Thus, dccT encodes a dicarboxylate transport system with comparable capacities for the import of succinate, fumarate, and L-malate, whereby the affinity for succinate is about 2-fold higher than that for fumarate and about 12-fold higher than that for L-malate. Furthermore, the comparable kinetic parameters determined for dicarboxylate uptake in C. glutamicum WT(pVWEx1-dccT) and in mutant strains SSM and FSM indicated that the high expression levels of dccT are a sufficient explanation for the observed ability of C. glutamicum SSM and FSM to utilize dicarboxylic acids.

In order to address the driving force of the transport, the dependency of DccT succinate uptake activity on Na⁺ ions was assayed (Fig. 3). A clear dependency of succinate transport on the presence of Na⁺ was observed, since succinate uptake rates were diminished 10-fold in the presence of residual amounts of Na⁺ (50 µM). The apparent K_m value for sodium was derived as 1.6 mM (Fig. 3).

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FIG. 3. Sodium dependence and inhibition of succinate uptake. (A) Succinate uptake by C. glutamicum SSM was measured in terms of dependence on the external NaCl concentration and measurements were performed at Vmax in the presence of 800 µM succinate. (B) Inhibition of succinate uptake upon the addition of several organic acids as measured for the strain C. glutamicum WT(pVWEx1-dccT) with 100 µM succinate. Competing substrates were added in 100-fold excesses (10 mM). The control value of 38 nmol (min mg dry weight –1) was set as 100%. The presented data are the average of at least three measurements.

To test whether the DccT uptake system also facilitates the transport of other TCA cycle intermediates or structurally related compounds, uptake assays for succinate were performed in the presence of 100-fold excesses of unlabeled potential substrates (Fig. 3). This experiment revealed the expected inhibition of succinate transport by L-malate and fumarate but no inhibition by the tricarboxylic intermediates of the TCA cycle citrate and isocitrate. Furthermore, neither 2-oxoglutarate and glyoxylate, nor the monocarboxylic acids pyruvate and L-lactate, nor the structurally related amino acids L-glutamate and L-aspartate inhibited succinate transport. In contrast, oxaloacetate, which also is a dicarboxylic acid with four carbon atoms (C₄-dicarboxylate), strongly inhibited succinate uptake by DccT (Fig. 3). Thus, the substrate spectrum of DccT is rather narrow and restricted to dicarboxylic C₄ intermediates of the TCA cycle.

A point mutation in the promoter region of dccT is responsible for the high dccT expression level in C. glutamicum SSM. The increased expression levels of dccT in C. glutamicum SSM and FSM compared to what was seen for C. glutamicum WT implicated either a cis mutation in the promoter region of the dccT gene or a trans mutation affecting a regulatory gene. To test for mutations in the dccT promoter region, first the transcription start point of dccT was determined using 5' RACE-PCR. Transcription initiated at a G 98 nt upstream of the start codon of dccT (Fig. 4). A –10 hexamer, TAATAT, similar to the –10 core hexamer consensus sequence (TAtAAT; less conserved nucleotides are indicated in lowercase) could be identified, while the –35 sequence (CTACCA) was less well conserved, as is typical for C. glutamicum promoters (38). When the upstream region of dccT from –227 to +27 was amplified by PCR using genomic DNA from mutants FSM and SSM, sequence analysis revealed C-to-T transition mutations in the dccT promoter regions of both mutants 15 bp upstream of the transcription start point (Fig. 4).

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FIG. 4. DNA sequence of the promoter region of cg0277 (dccT) from C. glutamicum WT. The transcriptional start site (TS) is underlined and given in boldface, the putative –10 and –35 hexamers are underlined, and the start codon of dccT is highlighted in bold. The CT transitions identified in the mutant SSM and FSM sequences are shown below the sequence of C. glutamicum WT and marked by an arrow. The first and last nucleotides of the sequence shown are given in italics and correspond to the indicated positions of the genome sequence BX927147.

To test whether the identified cis mutation in the dccT promoter region affects gene transcription, the dccT promoter sequences of C. glutamicum WT and SSM were cloned into the promoter probe vector pET2 (54). The resulting plasmids, pET2-dccT-WT and pET2-dccT-SSM, were introduced into C. glutamicum WT, and the expression of both dccT'-'cat fusions was analyzed (Fig. 5). Expression of the cat fusion with the WT dccT promoter region was low in C. glutamicum WT(pET2-dccT-WT) under the conditions tested, while 20- to 40-fold-higher expression levels were observed for the cat fusion with the mutant dccT promoter region in C. glutamicum WT(pET2-dccT-SSM) (Fig. 5). In comparison, the expression ratios obtained by DNA microarray analysis were low, which might be due to low signal levels and/or cross-hybridization. Thus, the identified point mutation in the dccT promoter region is responsible for high dccT expression levels.

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FIG. 5. Comparative expression analysis of dccT-cat reporter gene fusions of the upstream dccT regions from C. glutamicum WT and SSM. Expression of the dccT-cat reporter gene fusions was determined for C. glutamicum WT(pET2-dccT-WT) (empty columns) and WT(pET2-dccT-SSM) (filled columns) by measuring chloramphenicol acetyltransferase activity after cultivation on CgXII minimal medium containing 100 mM glucose, 100 mM L-lactate, or 100 mM acetate or on LB complex medium without or with 100 mM fumarate, 100 mM L-malate, or 100 mM succinate. The values represent means and standard deviations from at least three independent cultivations.

The expression of the WT dccT-cat fusion in C. glutamicum WT(pET2-dccT-WT) was low (0.02 to 0.04 µmol mg^–1 min^–1) on minimal medium with glucose, L-lactate, or acetate as well as on LB complex medium with or without the addition of C₄-dicarboxylates (Fig. 5). Under anaerobic conditions, the expression of the WT dccT-cat fusion was also low (0.04 µmol mg^–1 min^–1 on LB and 0.03 µmol mg^–1 min^–1 on glucose minimal medium [data not shown]). Thus, the expression of dccT appears to be constitutive and rather low under all conditions tested.

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Occurrence of DccT and other transport systems for dicarboxylic acids. Seven families of transport systems for dicarboxylic acids in bacteria have been defined on the basis of their sequence similarity, occurrence, and function (44). The Dcu and DcuC families are present in gram-negative bacteria only and were extensively studied for E. coli (26). The transporters act as fumarate:aspartate, fumarate:malate, or fumarate:succinate antiporters and play a role in the anaerobic utilization of aspartate and fumarate (17, 51, 53). In gram-positive bacteria like Bacillus subtilis, members of the CitMHS and 2-HCT families are known to transport citrate and D-isocitrate in complex with Mg²⁺ or Ca²⁺ and citrate and/or malate, respectively (4, 5). Transporters of the TRAP family are three-component systems for secondary uptake of malate, fumarate, and succinate like the DctPQM system of Rhodobacter capsulatus (18). Another class of transporters for di- and tricarboxylates as well as amino acids or inorganic sulfate is represented by the DASS family, of which the SdcS transporter of Staphylococcus aureus has recently been characterized (23). Furthermore, the DAACS family of (putative) dicarboxylate transporters comprises transport systems for the uptake of TCA cycle intermediates and amino acids in symport with Na⁺ or H⁺. Prominent examples are the functionally characterized DctA of Rhizobium leguminosarum and Glt of Pyrococcus horikoshii, for which the three-dimensional structure has been solved (63, 64).

In C. glutamicum, no gene encoding DcuAB-, DcuC-, or 2-HCT-type transporters is known, whereas one TRAP (cg2568-70)-, one CitMHS (cg0088)-, three DAACS (cg2810, cg2870, cg3356)-, and three DASS (cg0277, cg2072, cg2243)-type transporter-encoding genes have been annotated (27). In spite of the presence of several putative dicarboxylate transporter genes in the C. glutamicum genome, C. glutamicum WT was not able to efficiently utilize dicarboxylic intermediates of the TCA cycle for growth. The transport of citrate, the only TCA cycle intermediate supporting the growth of C. glutamicum WT as the sole carbon source, and that of isocitrate appear to be mediated by the CitMHS system but seem to require very high Mg²⁺ concentrations (42). Here, we report the involvement of DccT in dicarboxylate import under aerobic conditions. DccT is a DASS-type transporter showing 42% amino acid identity with the recently described dicarboxylate importer SdcS from S. aureus and 26% amino acid identity with the mammalian renal NaDC-1 dicarboxylate importer (37). In addition, the plant tonoplast from Arabidopsis thaliana contains a related malate transporter named AttDT (16). Like SdcS and NaDC-1, DccT from C. glutamicum is Na⁺ dependent (22, 37), which is in agreement with biochemical data obtained by Ebbighausen et al. (11). Although DccT, SdcS, and NaDC-1 share high sequence similarity and are Na⁺ dependent, their K_m values for succinate vary. The renal NaDC-1 transporter has a low affinity of 400 µM (37), whereas SdcS has the highest affinity toward succinate (K_m = 6.6 µM) and the K_m determined for DccT was intermediate (79 µM). B. subtilis, which possesses five different (putative) dicarboxylate transporters, apparently lacks a protein with significant sequence identity to DccT. Limited sequence identity was found between DccT and the only DASS family protein in B. subtilis, YflS, which was proposed to be a malate transporter but was shown to be dispensable for growth on L-malate (52). We showed that DccT from C. glutamicum mediates the uptake of succinate, fumarate, and malate and that oxaloacetate likely is an additional substrate. To our knowledge, this would be the first bacterial carrier protein identified for this particular substrate, as oxaloacetate transporters are so far known to exist only in eukaryotes (44). Unlike uptake systems of the DAACS family, amino acids are not substrates of C. glutamicum DccT.

Based on sequence comparisons, cg2870 was proposed to encode a DctA-type uptake system and to be responsible for the succinate uptake activity observed for C. glutamicum (26). In this study, no indication for the involvement of a DctA-type uptake system was found; for example, the kinetic parameters for succinate as well as fumarate uptake in C. glutamicum SSM, FSM, and WT(pVWEx1-dccT) were comparable (Fig. 2), and C. glutamicum WT is unable to grow with dicarboxylates as the sole carbon sources (Table 2). Currently, however, it cannot be ruled out that a DctA-type uptake system is active in C. glutamicum under certain conditions.

Homologs of DccT from C. glutamicum are encoded in the genomes of C. efficiens (CE0194; 79% identical amino acids), C. diphtheriae (DIP017; 68%), C. urealyticum (CU1939; 63%) and also in, e.g., Helicobacter pylori (HPSH_01105; 50%), Shewanella denitrificans (Sden_2459; 47%), and Rhodopseudomonas palustris (RPE_1280; 44%). The genomes of C. jeikeium and of all sequenced mycobacteria, with the exception of Mycobacterium gilvum (Mflv_0935; 51%), do not encode proteins sharing high (>40%) sequence similarities with DccT from C. glutamicum. However, the role(s) of DccT homologs in other bacteria has not been analyzed yet.

Expression of dccT in C. glutamicum. Regarding the activity of DccT in C. glutamicum WT, we could show that succinate, fumarate, and L-malate cannot be utilized for growth because of an insufficient transport capacity for these substrates under standard conditions (Fig. 2). Only high dccT expression levels either as a consequence of a promoter mutation, as observed for the spontaneous mutants SSM and FSM, or due to plasmid-borne dccT overexpression enabled growth on succinate, fumarate, and L-malate as the sole sources of carbon and energy. The spontaneous mutants and the dccT overexpression strain grew as fast on succinate and fumarate as on glucose, which might indicate that growth is no longer limited by the uptake rate of succinate or fumarate but rather by their subsequent catabolism. In contrast, growth on L-malate was slower than growth on glucose even when dccT was overexpressed, suggesting that L-malate uptake still limits growth.

Under the conditions tested, the dicarboxylate uptake capacity of C. glutamicum WT was too low to allow growth on succinate, fumarate, or L-malate as the sole carbon source. Expression analysis of dccT also did not reveal inducing conditions, as dccT was induced neither by the presence of the dicarboxylic acid substrates of the encoded uptake system nor by anaerobiosis, conditions known to induce the expression of genes for dicarboxylate uptake systems in E. coli and B. subtilis (21, 43, 52, 62, 65). As the identified promoter mutation, a CT transversion (position –15; AATGTTAATATTC [the mutated nucleotide is underlined]), increased the similarity to the consensus sequence of the extended –10 region (TGTG[C/G]TATAATGG [33]), and as promoter activity was increased to similar extents under all conditions tested, the mutation might represent a promoter-up mutation increasing promoter recognition by RNA polymerase E^A holoenzyme and transcription initiation. In addition or alternatively, the mutation might have altered a binding site of an unknown transcriptional regulator. Indeed, the CT transition improves the symmetry of two partly overlapping imperfect palindromes (ATTCGCACTGCGAAT and GCGAATGTT AATATTCCC [mutated nucleotides underlined]). In this respect, it has to be noted that the expression of the mutant dccT promoter, but not that of the WT dccT promoter, was about twofold higher when L-lactate was added to the medium than that seen for other growth conditions (Fig. 5). However, the palindromes are not similar to known binding sites of transcriptional regulators (57) and, in particular, do not resemble the binding site of LldR, the L-lactate-dependent repressor of the L-lactate utilization operon (20). Thus, neither inducing conditions of WT dccT nor genetic control mechanisms governing dccT expression are known and, if they exist, they remain to be identified.

Physiological role of DccT. The physiological role of DccT is not fully understood, as dccT expression is too low to enable utilization of C₄-dicarboxylic acids for the growth of WT C. glutamicum cells. However, as DccT transports the TCA cycle intermediates succinate, fumarate, and L-malate into the cell, DccT-mediated uptake replenishes the TCA cycle when intermediates are withdrawn as precursors for anabolism. Usually, the C₃ carboxylating reactions catalyzed by pyruvate carboxylase (39, 41) and phosphoenolpyruvate carboxylase (14, 32) or by the glyoxylic acid cycle (45, 46) replenish the TCA cycle in C. glutamicum (13). Supplementation of the growth medium with fumarate circumvented the need for anaplerotic reactions (41). Lysine production could be improved by increased supply with lysine biosynthesis precursor oxaloacetate either by increasing anaplerosis, e.g., by overexpression of the pyruvate carboxylase gene (40); by reducing oxaloacetate decarboxylation, e.g., by deletion of the phosphoenolpyruvate carboxykinase gene (47); or by feeding fumarate (30). Thus, DccT-mediated uptake of fumarate, succinate, and L-malate—although insufficient to enable growth on these substrates—might function to enhance growth and amino acid production under conditions requiring high anaplerotic flux. In addition, as C. glutamicum secretes succinic acid into the medium at significant levels under oxygen deprivation conditions (24), the DccT-mediated uptake of succinate represents a means to reutilize this fermentation product as a cosubstrate for growth once oxygen becomes abundant again.

 
Work in our labs was supported in part by the German Ministry of Education and Research (BMBF) through grants 0313805I (GenoMik) and 9313704 (SysMAP).

 
* Corresponding author. Mailing address for Volker F. Wendisch: Institute of Molecular Microbiology and Biotechnology, Westfalian Wilhelms University Muenster, Corrensstr. 3, D-48149 Muenster, Germany. Phone: 49-251-833 9827. Fax: 49-251-833 8388. E-mail: wendisch{at}uni-muenster.de. Mailing address for Kay Marin: Institute of Biochemistry, Cologne University, Cologne, Germany. Phone: 49-221-470 6476. Fax: 49-221-470 5091. E-mail: kay.marin{at}uni-koeln.de

Published ahead of print on 25 July 2008.

^# Both authors contributed equally.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Bacteriology, October 2008, p. 6458-6466, Vol. 190, No. 19
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