MULTICOPY SUPPRESSION OF AN ILV MUTATION IN E COLI K 12

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Enteropathogenic strains of Escherichia coli are increasingly important agents of many diarrheal diseases. Because it is considered a prototype organism for enteric bacteria, continuing the search for understanding of its genetic make up and cellular functions is crucial. In this proposal, the PI seeks to unravel the expression of an acetohydroxy acid synthase (AHAS) gene as a consequence of cloning it into normal copy plasmid. The "new" AHAD activity expressed is refractory to normal inhibition by valine when cloned into a high copy plasmid, pBR322. High copy phenomena have been reported in several bacterial systems, but not in the AHAS system of K-12. Specific goals involve: 1) physicochemically characterizing the resulting enzymatic activity from the cloned fragment, 2) determining the effect of other ilv genes on expression of this activity, 3) determining the number of copies of the newly expressed gene on the chromosome, 4) mapping the genes involved and 5) determining and analyzing the DNA sequence of the gene. Understanding mechanisms of gene activation under certain environmental conditions certainly facilitate understanding the recent pathogenicity of some organisms once thought to be non-pathogenic. Students involved in this project will gain an invaluable experience in conducting state-of-the-art research and in collecting, analyzing and presenting scientific data at research conferences and national meetings. Technologies used may be applied to any number of areas in the bimolecular sciences which may interest the student in planning science careers.