The expression of the phosphoglycerate transport (pgt) system of Salmonella typhimurium is induced by exogenous, but not endogenous, inducers. We have cloned the pgt system into Escherichia coli using the plasmid pBR322. The cloned DNA contains all regulatory elements needed since the cloned system is regulated identically as in S. typhimurium. Preliminary work from this laboratory indicates that the operon is negatively controlled and there exists repressor and activator which counteracts and neutralizes the action of repressor. Because of the novelty of exogenous induction of gene expression, the regulatory machinery will be genetically dissected and necessary regulatory components will be identified, isolated, purified and characterized. The proposed work involves isolation of deletion mutant clones and performing complementation test on these clones to define number of regulatory components involved; identification of the regulatory proteins using gene fusion technique (insertion of lac Z gene) developed by Beckwith; determination of nucleotide sequence of the regulatory genes; construction of clones hyperproducing the regulatory proteins so that they can be conveniently isolated in large quantity to be used for biochemical work; biochemical characterization of regulatory proteins in DNA binding, interaction between repressor and activator, effect of inducers on the DNA binding and the repressor and activator interaction to be carried out in in vitro transcription and coupled transcription-translation systems. The ultimate goal of this project is to fully understand the molecular and biochemical mechanism of the gene expression induced by exogenous inducers.
