The nucleotide sequence of the entire Escherichia coli edd-eda region that encodes the enzymes of the Entner-Doudoroff pathway was determined. The edd structural gene begins 236 bases downstream ofzwf. The eda structural gene begins 34 bases downstream of edd. The edd reading frame is 1,809 bases long and encodes the 602-amino-acid, 64,446-Da protein 6-phosphogluconate dehydratase. The deduced primary amino acid sequences of the E. coli and Zymomonas mobilis dehydratase enzymes are highly conserved. The eda reading frame is 642 bases long and encodes the 213-amino-acid, 22,283-Da protein 2-keto-3-deoxy-6-phosphogluconate aldolase. This enzyme had been previously purified and sequenced by others on the basis of its related enzyme activity, 2-keto-4-hydroxyglutarate aldolase. The data presented here provide proof that the two enzymes are identical. The primary amino acid sequences of the E. coli, Z. mobiis, and Pseudomonas putida aldolase enzymes are highly conserved. When E. coli is grown on gluconate, the edd and eda genes are cotranscribed.Four putative promoters within the edd-eda region were identified by transcript mapping and computer analysis. P1, located upstream of edd, appears to be the primary gluconate-responsive promoter of the edd-eda operon, responsible for induction of the Entner-Doudoroff pathway, as mediated by the gntR product. High basal expression of eda is explained by constitutive transcription from P2, P3, and/or P4 but not P1.

The eda gene that encodes 2-keto-3-deoxy-6-phosphogluconate aldolase of the Entner-Doudoroff pathway was cloned from Zymomonas mobilis by genetic complementation of an Escherichia coli mutant. The gene is present in a single copy on the Z. mobilis genome and is not tightly linked to the edd gene. Nucleotide sequence analysis of the eda region revealed that the structural gene is 627 bp long and capable of encoding a protein of 208 amino acids with a deduced molecular weight of 21,505. The eda gene is monocistronic and is transcribed from a single promoter. The transcriptional initiation site was determined and an improved consensus promoter sequence for Z. mobilis was derived. High-level expression of the eda gene can be attributed to very efficient translational initiation caused by the high quality of the ribosome-binding site and stability of the mRNA, which has a decay rate of 7.6 min. A comparison of highly expressed Z. mobilis genes indicated that the relative quality of the ribosome-binding sites of these genes might play an important role in determining the level of enzyme synthesis. This possibility is discussed with regard to the role of gene expression in co-ordinating the enzyme levels of the Entner-Doudoroff glycolytic pathway.

Expression of the Zymomonas mobilis glf (glucose facilitator protein) and glk (glucokinase) genes in Escherichia coli ZSC113 (glucose negative) provided a new functional pathway for glucose uptake and phosphorylation. Both genes were essential for the restoration of growth in glucose minimal medium and for acid production on glucose-MacConkey agar plates.

The Entner-Doudoroff pathway was shown to be induced for oxidative glucose metabolism when Escherichia coli was provided with the periplasmic glucose dehydrogenase cofactor pyrroloquinoline quinone (PQQ). Induction of the Entner-Doudoroff pathway by glucose plus PQQ was established both genetically and biochemically and was shown to occur in glucose transport mutants, as well as in wild-type E. coli. These data complete the body of evidence that proves the existence of a pathway for oxidative glucose metabolism in E. coli. PQQ-dependent oxidative glucose metabolism provides a metabolic branch point in the periplasm; the choices are either oxidation to gluconate followed by induction of the Entner-Doudoroff pathway or phosphotransferase-mediated transport. The oxidative glucose pathway might be important for survival of enteric bacteria in aerobic, low-phosphate, aquatic environments.