The proposed research is designed to study the regulation of lipid metabolism using bacteria as a model system. Our approach to this problem involves the characterization of E. coli mutants defective in fatty acid degration (fad). We have recently shown that there are separate mechanisms for long and medium chain fatty acid transport in E. coli. The transport of long-chain fatty acids in this organism requires the functional activity of the fadL gene. In and effort to understand the role of the fadL gene product(s), we are planning to 1) characterize by restriction mapping subcloning, and complementation hybrid-plasmid bearing the fadL gene(s), 2) sequence the fadL gene, 3) identify the fadL gene(s) products, and 4) construct a fadL-lacZ gene fusion. These studies may enable us to identify the fadL gene(s) and purity its gene product(s). We have recently mapped the fadR gene and determined that it's product behaves like a repressor. We now wish to determine the molecular details of the mechanism(s) by which the fadR gene product interacts with the fad genes. To accomplish this, we plan to (1) clone the fadR gene, (2) purify and characterize the fadR gene product(s), and (3) determine in vitro if the fadR gene(s) product binds to hybrid-plasmids carrying fad structure genes. Studies to dissect the structural and regulatory organization of the fad genes will also be undertaken. Particular amphasis will be placed on characterizing a hybrid-plasmid which complements DeltafadAB(C) mutants. These mutants lack five fad enzyme activities. Therefore, our studies may enable us to identify the genes responsible for each enzyme activity.
