A considerable amount of metabolic energy is expended in the biogenesis of membrane lipid and organisms in general exert a high degree of control over the activity of this pathway. In Escherichia coli, the bulk of the available evidence indicates that the control point is at an early step in fatty acid biosynthesis, however, the definitive experimental identification of the rate-controlling enzyme has proved elusive and remains one of the major unanswered questions about bacterial physiology. The overall goal of this research program is to elucidate the mechanisms that regulate membrane lipid production and coordinate this process with growth and protein biosynthesis. An evaluation of the available data has led to the working hypothesis that control over the fatty acid biosynthetic rate is exerted at the level of chain initiation by modulation of acetyl-CoA:acyl carrier protein transacylase activity. This enzyme is ideally positioned in the pathway to control the number of fatty acids synthesized since the transfer of an acetyl group to acyl carrier protein yields the primer molecule required for starting new rounds of elongation. A combination of physiological, biochemical and genetic experimental approaches have been designed to test this working hypothesis against the alterative candidates for the regulatory enzyme. Acyl carrier protein-specific antibodies, high-pressure liquid chromatographic systems and a conformationally sensitive gel electrophoresis method will be used to directly determine the precursor pool present in the shortest supply in vivo. These measurements will either confirm that the supply of acetyl-acyl carrier protein is the rate-limiting factor or identify which of the other candidate enzymes catalyzes the slow step in fatty acid biosynthesis. As a further test of the hypothesis, acetyl transacylase will be purified and characterized to examine the biochemical mechanism of activity regulation in vitro. Temperature-sensitive acetyl transacylase mutants and strains exhibiting abnormal regulation of fatty acid biosynthesis will also be used to evaluate the role of this enzyme and to map the location of the transacylase structural gene on the E. coli chromosome.
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