Biochemical Characterization of the C4-Dicarboxylate Transporter DctA from Bacillus subtilis

Bacterial secondary transporters of the DctA family mediate ion-coupled uptake of C₄-dicarboxylates. Here, we have expressed the DctA homologue from Bacillus subtilis in the Gram-positive bacterium Lactococcus lactis. Transport of dicarboxylates in vitro in isolated membrane vesicles was assayed. We determined the substrate specificity, the type of cotransported ions, the electrogenic nature of transport, and the pH and temperature dependence patterns. DctA was found to catalyze proton-coupled symport of the four C₄-dicarboxylates from the Krebs cycle (succinate, fumurate, malate, and oxaloacetate) but not of other mono- and dicarboxylates. Because (i) succinate-proton symport was electrogenic (stimulated by an internal negative membrane potential) and (ii) the divalent anionic form of succinate was recognized by DctA, at least three protons must be cotransported with succinate. The results were interpreted in the light of the crystal structure of the homologous aspartate transporter Glt_Ph from Pyrococcus horikoshii.The DctA family is one of several diverse families of secondary transporters that catalyze the uptake of C₄-dicarboxylates from the Krebs cycle in bacteria (16, 27). In Escherichia coli, DctA mediates the uptake of succinate, fumurate, and malate under aerobic conditions; genomic disruption of dctA in E. coli prevents growth with malate or fumarate as the sole carbon source, and the mutant grows poorly on succinate (5). Similarly, a dctA knockout mutant of Bacillus subtilis cannot grow with succinate or fumarate as the sole carbon source (1). DctA plays a major role in the symbiotic relationship between nitrogen-fixing rhizobia (43) and root nodule-forming plants (30, 37, 38). Transport assays with Sinorhizobium meliloti cells showed previously that in addition to succinate, malate, and fumarate, orotate is transported and that a range of other substrates such as succinamic acid and succinamide may be transported, because they inhibit the transport of orotate (42). In Corynebacterium glutamicum, malate transport by DctA is inhibited by α-ketoglutarate, oxaloacetate, and glyoxylate, indicating that these compounds may be substrates also (41).DctA transporters belong to a large family of secondary transporters (the DAACS dicarboxylate/amino acid:cation symporter] family), which also comprises well-characterized glutamate/aspartate transporters and neutral amino acid transporters (32, 33). While DctA-type dicarboxylate transporters are found only in bacteria, glutamate/aspartate transporters of the DAACS family are found both in prokaryotes (e.g., GltT in Bacillus stearothermophilus, GltP in E. coli, and Glt_Ph in Pyrococcus horikoshii 2, 7, 34]) and in higher eukarya, where they play a pivotal role in the reuptake of the excitatory neurotransmitter glutamate from the synaptic cleft (4). Neutral amino acid (alanine, serine, and threonine) transporters are found in mammals (see, e.g., references 36 and 44) as well as bacteria (17).Secondary transporters of the DAACS family use (electro)chemical gradients of cations across the membrane to drive transport. The type of cotransported ions varies among family members: eukaryotic glutamate transporters couple the transport of glutamate to the symport of one proton and three sodium ions and the antiport of one potassium ion (24, 45). Bacterial and archaeal glutamate transporters utilize either sodium ions or protons for symport (2) and are independent of potassium ions (28, 31). The bacterial and mammalian neutral amino acid transporters are sodium ion coupled. Glutamate/aspartate transporters and bacterial serine/threonine transporters (SstTs) are electrogenic, but mammalian neutral amino acid transporters are obligate electroneutral amino acid antiporters (44).Insight into the structure-function relationships of the DAACS family members has greatly increased since crystal structures of the P. horikoshii aspartate transporter Glt_Ph have been determined (2, 29, 40). The protein consists of eight membrane-spanning helices and two reentrant regions (helical hairpins HP1 and HP2) (40). The C-terminal part of the protein (helices 7 and 8 and HP1 and HP2) is most strongly conserved with respect to other family members and binds the substrate and cotransported ions, with HP1 and HP2 functioning as lids that allow alternating access to the substrate- and ion-binding sites from either side of the membrane (3, 29). Glt_Ph forms a homotrimeric complex in which each protomer functions independently of the other subunits (11, 12, 18, 19, 23). The fold and oligomeric state are likely to be conserved throughout the family.Whereas the transport mechanisms of bacterial glutamate and neutral amino acid transporters of the DAACS family have been studied extensively in vitro, the C₄-dicarboxylate transporters of the DAACS family (DctA proteins) have been studied using whole cells only. To fully characterize these transporters, in vitro activity assays using either membrane vesicles or proteoliposomes containing purified protein are necessary. In such assays, the internal and external buffer compositions can be controlled, thus allowing manipulation of the chemical ion gradients and the electrical potential across the membrane. Here, we present the first biochemical characterization of a DctA family member in membrane vesicles. We have studied the DctA homologue from B. subtilis, which is annotated as DctP (1) but which we propose to rename DctA to reflect the homology to other DctA proteins. B. subtilis DctA (DctA_Bs) has 30 to 32% sequence identity to the aspartate transporter Glt_Ph and human excitatory amino acid transporter (EAAT) family members, over 40% sequence identity to the characterized bacterial glutamate transporters from E. coli and B. stearothermophilus, and 41 and 56% identity to DctA homologues from C. glutamicum and E. coli, respectively. We determined the substrate specificity of DctA_Bs, the type of cotransported ions, the electrogenic nature of transport, and the pH and temperature dependence patterns.