Characterization of the Dicarboxylate Transporter DctA in Corynebacterium glutamicum

Transporters of the dicarboxylate amino acid-cation symporter family often mediate uptake of C₄-dicarboxylates, such as succinate or l-malate, in bacteria. A member of this family, dicarboxylate transporter A (DctA) from Corynebacterium glutamicum, was characterized to catalyze uptake of the C₄-dicarboxylates succinate, fumarate, and l-malate, which was inhibited by oxaloacetate, 2-oxoglutarate, and glyoxylate. DctA activity was not affected by sodium availability but was dependent on the electrochemical proton potential. Efficient growth of C. glutamicum in minimal medium with succinate, fumarate, or l-malate as the sole carbon source required high dctA expression levels due either to a promoter-up mutation identified in a spontaneous mutant or to ectopic overexpression. Mutant analysis indicated that DctA and DccT, a C₄-dicarboxylate divalent anion/sodium symporter-type transporter, are the only transporters for succinate, fumarate, and l-malate in C. glutamicum.In bacteria, the uptake of dicarboxylates, such as the tricarboxylic acid (TCA) cycle intermediates succinate, fumarate, and l-malate, is mediated by transporters of different protein families. Whereas Dcu-type transporters facilitate dicarboxylate uptake under anaerobic conditions, the most common aerobic dicarboxylate transporters are members of the dicarboxylate amino acid-cation symporter (DAACS), divalent anion sodium symporter (DASS), tripartite ATP-independent periplasmic (TRAP), and CitMHS transporter families. DAACS transporters are responsible for C₄-dicarboxylate uptake under aerobic conditions in various bacteria, e.g., DctA from Escherichia coli, Bacillus subtilis, or Rhizobium leguminosarum, and are involved in different physiological functions (2, 4, 27, 41). The first described member of the TRAP family is the C₄-dicarboxylate transporter DctPQM from Rhodobacter capsulatus, which facilitates substrate uptake by the use of an extracytoplasmic solute receptor (8). An example of the DASS family, members of which occur in bacteria, as well in eukaryotes, is the well-characterized transporter SdcS from Staphylococcus aureus (13). Members of the CitHMS family import citrate in symport with the cation Mg²⁺ or Ca²⁺. Whereas E. coli possesses one DctA and four different Dcu carriers, no Dcu transporter-encoding genes were found in Corynebacterium glutamicum (16, 19), which is used for the industrial production of amino acids, such as glutamate (33) or l-lysine (39), and is capable of succinate and l-lactate production under oxygen deprivation conditions. A dctA gene was annotated (19); however, C. glutamicum is not able to utilize succinate, malate, or fumarate as a sole carbon source. The uptake systems CitH and TctCBA have been characterized recently as citrate uptake systems (3, 26). Interestingly, we and others have shown that C. glutamicum possesses a DASS family transporter (DccT) for uptake of the C₄-dicarboxylates succinate, fumarate, and l-malate (36, 40). Spontaneous mutants showing fast growth in succinate or fumarate minimal medium were isolated and shown to possess promoter-up mutations in the dccT gene (40). In l-malate minimal medium, these spontaneous mutants showed relatively slow growth, and the affinity of DccT for succinate and fumarate was found to be 5- and 12-fold higher than for l-malate, respectively (40). These findings prompted us to search for other uptake systems for l-malate in C. glutamicum. Here, we describe the identification and characterization of the DAACS family protein DctA from C. glutamicum as a proton motive force-driven uptake system for C₄-dicarboxylate intermediates of the TCA cycle. Additionally, we compare both uptake systems, DccT and DctA, from C. glutamicum.