Synthesis of lipid classes from [14C]acetate after blocking phospholipid synthesis at the PlsY step. (A) Strain PDJ28 (ΔgpsA) was grown to an OD600 of 0.5, the culture was harvested, washed and split into media either with or without the glycerol supplement. The cells were then labeled with [14C]acetate for 30 min, the lipids were extracted and the total amount of label incorporated into cellular lipids was determined. The extracted lipids were analyzed by thin-layer chromatography on Silica Gel G layers developed with chloroform:methanol:acetic acid (98/2/1, v/v/v). The distribution of radioactivity was determined using a Bioscan Imaging detector for the cultures containing the glycerol supplement (B) and the glycerol-deprived cultures (C). The composition of the free fatty acids that accumulated in the glycerol starved cultures was determined by preparative thin-layer chromatography to isolate the fatty acids, followed by the preparation of methyl esters and quantitative analysis by gas–liquid chromatography as described in Methods. The weight percent of the two major fatty acids detected is shown in the figure. All other fatty acids were present at less than 1% of the total.