Biochemical Characterization of the C ₄ -Dicarboxylate Transporter DctA from Bacillus subtilis

Abstract

Bacterial secondary transporters of the DctA family mediate ion-coupled uptake of C 4 -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 C 4 -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 Sensor Kinase DctS Forms a Tripartite Sensor Unit with DctB and DctA for Sensing C4-Dicarboxylates in Bacillus subtilis

“…However, as shown here, succinate transport by DctA is independent of DctB, and DctB does not directly interact with DctA. Additionally, in earlier transport experiments (25) there were also no indications that DctA requires additional components for transport. Therefore, the requirement of DctB for the expression of dctA (13) and the direct interaction with DctS indicate that DctB functions as a cosensor for the DctSR system only.…”

“…The transport of C 4 -dicarboxylates in B. subtilis is accomplished by DctA, which mediates the proton-coupled symport of fumarate, malate, succinate, and oxaloacetate into the cell (25). To test the role of DctA as a binding partner of the DctS sensor kinase, (BACTH).…”

“…For testing the role of DctB in succinate uptake, e.g., as a binding protein for the transporter DctA, [ 14 C]succinate uptake into cells of B. subtilis that were either deficient or proficient for DctB was measured. Succinate uptake in B. subtilis is known to depend on the DctA transporter (25). Strain B. subtilis 168, which is wild type for dctA and dctB, showed efficient uptake of […”

Ketoglutarate Transport Protein KgtP Is Secreted through the Type III Secretion System and Contributes to Virulence in Xanthomonas oryzae pv. oryzae

“…The dicarboxylate transport (DctA) system mediates the uptake of C 4 -dicarboxylates in Escherichia coli under aerobic conditions (14). However, in Corynebacterium glutamicum, malate transport by DctA is inhibited by ␣-ketoglutarate, indicating that substrates may also regulate nutrient uptake (68).…”

Mechanism of Transport Modulation by an Extracellular Loop in an Archaeal Excitatory Amino Acid Transporter (EAAT) Homolog

“…Glutamate is removed by reuptake into glia and neurons by members of the EAAT 2 family, which transport one glutamate molecule into the cell coupled to three Na ϩ ions and one H ϩ accompanied by the countertransport of one K ϩ ion (2). EAATs are members of the dicarboxylate/amino acid:cation symporter family (TCDB code 2.A.23) that also includes members that transport dicarboxylates and other amino acids (3)(4)(5). Glt Ph , from the archaeon Pyrococcus horikoshii, is 30% identical to the EAATs (6) and transports aspartate (L-Asp); it is the only dicarboxylate/amino acid:cation symporter member for which there is high resolution structural information (7)(8)(9)(10).…”