The media was extracted and analyzed, and no extracellular labeled fatty

acids were detected. The accumulation of fatty acid was not a linear function of time, but rather became progressively slower. These data indicated that fatty acid and phospholipid synthesis were coupled at the PlsY step, however, the continued synthesis of free fatty acids showed that there was a biochemical pathway to bypass the regulatory steps and accumulate an intermediate that is usually not detected. The fatty acids could come from the hydrolysis of acyl-ACP, but this seems unlikely in light of the observation that fatty acids did not accumulate in a strain depleted of PlsX [23] where acyl-ACP, but not acyl-PO4, would be formed. Thus, it #check details randurls[1|1|,|CHEM1|]# was likely that long-chain fatty acids accumulated due to the hydrolysis Sepantronium cell line of the unstable acyl-PO4 formed from acyl-ACP by PlsX when the PlsY step was blocked by glycerol removal. Figure 5 Time course for the incorporation of [ 14 C]acetate into the lipids of strain PDJ28. Strain PDJ28 was grown to an OD600 of 0.5, the cells were harvested, washed and resuspended in media without glycerol. [14C]acetate was added to the culture 30 min after the cells were resuspended in the new growth medium, samples were removed at the indicted times, the lipids were extracted, and the distribution of label between the phospholipid

and fatty acid pools were determined by thin-layer chromatography. Intracellular intermediate pools following glycerol deprivation The decrease in the overall rate of fatty acid synthesis suggested a feedback regulation mechanism that may be similar to that in E. coli where acyl-ACPs are key negative regulators of FASII [4]. We examined the intracellular concentrations of acyl-ACP in strain PDJ28 (ΔgpsA) as a function of time following glycerol withdrawal. Interestingly, much we consistently observed that there was more acyl-ACP in strain PDJ28 supplemented with glycerol compared to its wild-type counterpart suggesting that PlsY activity was somewhat compromised by GpsA inactivation even in the presence of the

media supplement (Figure 6A). Within 30 min of glycerol removal, the acyl-ACP pool reached 50% of the total ACP and remained constant for the remainder of the time course. The gel electrophoresis system separates acyl-ACP based the nature of the acyl chain, and the fact that the acyl-ACP in the glycerol-starved cultures migrated faster than the 17:0-ACP standard indicated that these acyl-ACP chains were longer than 17 carbons. This conclusion was consistent with the finding that 19:0 and 21:0 fatty acids accumulated in the glycerol-deprived cells (Figure 4C), and these fatty acids would be derived from the acyl-ACP end-products of de novo fatty acid synthesis. These data showed that acyl-ACP did accumulate in the absence of PlsY function, but that not all the ACP was converted to acyl-ACP.