Fatty acid and biotin metabolism in Escherichia coli

The enzyme beta-ketoacyl-ACP synthase II (KAS II) encoded by the fabF gene of Escherichia coli is a component of the fatty acid biosynthetic machinery that performs a key reaction in modulating membrane fluidity. While this enzyme is dispensable for growth of E. coli under the conditions tested, its overexpression from an heterologous promoter on a plasmid is lethal to the cell.;In this study I have shown that the overexpression of the enzyme leads to cell death due to an inhibition of fatty acid synthesis. The immediate effect of overexpressing FabF is the accumulation of malonyl-CoA to about 40% of the total cellular CoA pool from a steady state level of 0.5%. This increase is due to a deleterious interaction between the FabF and FabD proteins resulting in the unavailability of FabD for the FabB and FabH proteins to perform some essential reaction.;In trying to identify other genes regulated by the regulatory proteins FadR (fatty acid metabolism) and BirA (biotin protein ligase and bio operon repressor) I developed a technique to isolate all the binding sites on the chromosome for any DNA binding protein. This technique, a combination of a gel shift assay and PCR, is applicable to any DNA binding protein and is sensitive at the chromosomal level. This technique was tested on the FadR protein which has multiple binding sites on the chromosome and the BirA protein with a single defined binding site on the E. coli chromosome.;Biotin which is essential for the synthesis of fatty acids in E. coli is thought to be derived from a modified fatty acid pathway. In experiments used to verify this hypothesis I discovered a new intermediate in biotin synthesis. This intermediate `X', was purified from the spent growth medium of strain sup151 (isolated as a bypass suppressor of a bioH mutation). Genetic data suggests that this intermediate is made by the action of the bioH and bioC gene products and its production is repressed by high levels of biotin in the growth medium via the BirA protein.