Genetic Analysis of Escherichia coli lac Permease Biogenesis

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Manoil, ColinMCB-9905048Abstract - Genetic Analysis of Escherichia coli lac permease biogenesisThe project has two general objectives. The first is to develop a procedure for gene discovery in bacteria which is based on identifying the subcellular locations or biochemical activities of genetically tagged proteins. Exported proteins are selectively tagged with a 63 amino acid inserted sequence which permits immunological detection and rapid purification. The tags are generated by chromosomal insertion of a newly constructed transposon. Pools of strains are screened for individuals producing tagged proteins with a desired property, such as localization to a particular membrane or a particular enzymatic activity, followed by sequence identification of corresponding tagged genes. The second general objective of the project is to analyze how a number of previously identified mutations inactivate E. coli lac permease, and how the cell responds to such mutant proteins. Specifically, the experiments aim, 1, to determine whether several different mutations altering a key region of lac permease (cytoplasmic segment C3) inactivate the protein by primary effects on membrane insertion, folding, or function, and 2, to determine whether several known stress responses, or a new stress response, are induced by mutant lac permeases. This research will use a functional genomics approach to develop strategies for identifying the biological function and intracellular localization of bacterial proteins. Additional experiments will examine how an important subclass of bacterial proteins, integral membrane proteins, are altered by mutational changes, as well as how such altered proteins affect cell physiology. Both of these approaches will contribute to an increased understanding of the integration of cellular processes required for microbial life.