Characterization of the Dicarboxylate Transporter DctA in Corynebacterium glutamicum

Abstract

Transporters of the dicarboxylate amino acid-cation symporter family often mediate uptake of C 4 -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 C 4 -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 C 4 -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 C 4 -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 C 4 -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 Mg 2ϩ or Ca 2ϩ . 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 C 4 -dicarboxylates succinate, fumarate, and L-malate (36, 40). Spontaneous mutants showing fast growth in succinate or fumarate mi...

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

“…2 indicate that DctA Bs couples succinate transport to protons and not to sodium ions. This observation corresponds with results obtained for DctA of C. glutamicum in a whole-cell experimental setup (41). To gain more insight into both the optimal pH for transport by DctA and the protonation state of the transported dicarboxylate, we measured the pH dependence of succinate transport into membrane vesicles.…”

“…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 ␣-ketoglutarate, oxaloacetate, and glyoxylate, indicating that these compounds may be substrates also (41).…”

Mechanism of Citrate Metabolism by an Oxaloacetate Decarboxylase-Deficient Mutant of Lactococcus lactis IL1403

“…Transporters for oxaloacetate are rarely found in bacteria. The dicarboxylate transporters DccT of Corynebacterium glutamicum (31) and SdcL of Bacillus licheniformis (29) as well as the DctA transporters of C. glutamicum (32) and Bacillus subtilis (10) were shown to be competitively inhibited by oxaloacetate. In the present study, the physiological response of the oxaloacetatedeficient mutant to the potentially toxic accumulation of oxaloacetate in the cytoplasm is demonstrated.…”

Transcription of malP is subject to phosphotransferase system-dependent regulation in Corynebacterium glutamicum

“…Data represent mean values and SDs from three independent cultivations with three technical replicates. Uhde et al, 2013;Youn et al, 2008Youn et al, , 2009. No mutations were observed in sequence analyses of the malP promoter region, as well as of the malP gene itself, in DNA preparations from C. glutamicum HSM; thus, no evidence for cis-acting mutations was found.…”