The effects of inhibition of Escherichia coli
phospholipid synthesis on the accumulation of intermediates of the
fatty acid synthetic pathway have been previously investigated with conflicting results. We report construction of an E. coli strain that allows valid [14C]
acetate labeling of
fatty acids under these conditions. In this strain,
acetate is a specific precursor of
fatty acid synthesis and the intracellular
acetate pools are not altered by blockage of
phospholipid synthesis. By use of this strain, we show that significant pools of
fatty acid synthetic intermediates and
free fatty acids accumulate during inhibition of
phospholipid synthesis and that the rate of synthesis of these intermediates is 10 to 20% of the rate at which
fatty acids are synthesized during normal growth.
Free fatty acids of abnormal chain length (e.g., cis-13-eicosenoic acid) were found to accumulate in
glycerol-starved cultures. Analysis of extracts of [35S]
methionine-labeled cells showed that
glycerol starvation resulted in the accumulation of several long-chain acyl-
acyl carrier protein (ACP) species, with the major species being ACP acylated with cis-13-eicosenoic
acid. Upon the restoration of
phospholipid biosynthesis, the abnormally long-chain acyl-ACPs decreased, consistent with transfer of the acyl groups to
phospholipid. The introduction of multicopy plasmids that greatly overproduced either E. coli
thioesterase I or E. coli
thioesterase II fully relieved the inhibition of
fatty acid synthesis seen upon
glycerol starvation, whereas overexpression of ACP had no effect.
Thioesterase I overproduction also resulted in disappearance of the long-chain acyl-ACP species. The release of inhibition by thiosterase overproduction, together with the correlation between the inhibition of
fatty acid synthesis and the presence of abnormally long-chain acyl-ACPs, suggests with that these acyl-ACP species may act as feedback inhibitors of a key
fatty acid synthetic
enzyme(s).