The long-term goal of the research is to understand the biochemical and genetic mechanisms that regulate membrane lipid biosynthesis in Escherichia coli. Fatty acid and phospholipid synthesis is a vital facet of bacterial physiology that is tightly regulated at several levels. The enzymes have been identified, their genes have been located and cloned, and their placement within the biosynthetic pathway determined. The three major challenges in the field are: a) to precisely define the physiological role for each pathway enzyme and isozyme, and to understand how their activities are regulated; b) to determine how the overall pathway activity is regulated and coordinated with major branches of intermediary metabolism; and c) to determine how expression of individual genes are modulated to transcribe the correct proportion of pathway enzymes. Dr. Rock and his colleagues have developed several important leads in each of these areas that form the cornerstones of the proposal. First, they have identified two key enzymes in the fatty acid biosynthetic pathway that are modulated by acyl-acyl carrier protein (ACP). Second, they found the glycerol-phosphate acyltransferase (plsB) to be regulated by the intracellular concentration of guanosine-3`-diphosphate-5`-diphosphate, and is envisioned as a sensor that coordinates fatty acid and phospholipid production with macromolecular biosynthesis. Third, they have verified that the ratio of the fabA and fabB gene products is a key element governing the proportion of unsaturated fatty acids produced by the pathway and have preliminary data that the expression of these two genes is coordinately regulated. The research plan will build on these advances and is organized around three specific aims: 1) To verify the proposed role of acyl-ACP and its target enzymes in the regulation of fatty acid biosynthesis; 2) To substantiate the role of the glycerol-phosphate acyltransferase system in the regulation of fatty acid and phospholipid biosynthesis; 3) To define biochemical and transcriptional mechanisms that regulate unsaturated fatty acid biosynthesis. The investigation of lipid synthesis in E. coli is a paradigm for the study of lipid metabolism in more advanced organisms, and recent results show that the fatty acid synthetic pathway is an important target for antimicrobial chemotherapy.
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