| The work described in this dissertation is composed of two parts. The first describes the multiple regulatory factors required for the transcriptional control of eda, which encodes the Escherichia coli Entner-Doudoroff aldolase. The second part of this dissertation comes from the characterization of KdgR regulation of eda, and describes the negative regulation of genes required for the metabolism of 2-keto-3-deoxygluconate (KDG) by the DNA binding protein KdgR.;eda is thought to be transcribed from multiple promoters and induced by growth on sugar acids, phosphate starvation, SOS recovery and entry into stationary phase. Here, it is shown that Eda synthesis is elevated in cells growing on gluconate, glucuronate or methyl-beta-D-glucuronide, and in cells subject to phosphate starvation, carbon starvation, and during the transition to stationary phase. Transcription of eda initiates from each of three promoters, designated here as P1, P2 and P4, under different growth conditions. P1 is responsible for eda expression on gluconate and is under control of GntR. P2 is responsible for eda expression on glucuronate and galacturonate and is under direct control of KdgR. P4 is active under conditions of phosphate starvation and is directly controlled by PhoB. In addition, Eda synthesis is controlled by CsrA. The complex regulation of eda is discussed with respect to its several physiological roles.;Metabolism of KDG in E. coli requires the products of three genes, kdgT, kdgK and eda. Enzyme assays have shown that regulation of these genes is probably under control of a regulatory protein, KdgR, and that the true inducer of these genes is most likely KDG. However, control of KDG metabolism genes in E. coli has not been characterized at a molecular level. Now, it is shown that kdgT, kdgK, and eda are regulated in response to growth on the hexuronate glucuronate, and that this regulation is mediated at the transcriptional level by KdgR. KdgR binds specific KdgR binding sites upstream of kdgK, kdgT and eda in vitro, and this binding is abolished in the presence of KDG. Results of gel mobility shift assays allowed the identification of a consensus sequence for KdgR binding in E. coli. Finally, the mechanism of regulation of KDG metabolism genes by KdgR in E. coli is compared to the well-characterized mechanism of regulation of pectinolysis genes by KdgR in Erwinia chrysanthemi. |
