Inactivation and regulation of the aerobic C-4-dicarboxylate transport (dctA) gene of Escherichia coli

The gene (dctA) encoding the aerobic C-4-dicarboxylate transporter (DctA) o f Escherichia coli was previously mapped to the 79-min region of the linkag e map. The nucleotide sequence of this region reveals two candidates for th e dct4 gene:f428 at 79.3 min and the o157a-o424-o328 (or orfQMP) operon at 79.9 min. The f428 gene encodes a homologue of the Sinorhizobium meliloti a nd Rhizobium leguminosarum H+/C-4-dicarboxylate symporter, DctA, whereas th e orfQMP operon encodes homologues of the aerobic periplasmic-binding prote in-dependent C-4-dicarboxylate transport system (DctQ, DctM, and DctP) of R hodobacter capsulatus. To determine which, if either, of these loci specify the E. coli DctA system, the chromosomal f428 and orfM genes were inactiva ted by inserting Sp(r) or Ap(r) cassettes, respectively. The resulting f428 mutant was unable to grow aerobically with fumarate or malate as the sole carbon source and grew poorly with succinate, Furthermore, fumarate uptake was abolished in the f428 mutant and succinate transport was similar to 10- fold lower than that of the wild type. The growth and fumarate transport de ficiencies of the f428 mutant were complemented by transformation with an f 428-containing plasmid. No growth defect was found for the orfM mutant. In combination, the above findings confirm that f428 corresponds to the dctA g ene and indicate that the orfQMP products play no role in C-4-dicarboxylate transport. Regulation studies with a dctA-lacZ (f428-lacZ) transcriptional fusion showed that dctA is subject to cyclic AMP receptor protein (CRP)-de pendent catabolite repression and ArcA-mediated anaerobic repression and is weakly induced by the DcuS-DcuR system in response to C-4-dicarboxylates a nd citrate. Interestingly, in a dctA mutant, expression of dctA is constitu tive,vith respect to C-4-dicarboxylate induction, suggesting that DctA regu lates its own synthesis. Northern blot analysis revealed a single, monocist ronic dctA transcript and confirmed that dctA is subject to regulation by c atabolite repression and CRP. Reverse transcriptase-mediated primer extensi on indicated a single transcriptional start site centered 81 bp downstream of a strongly predicted CRP-binding site.