Escherichia coli membrane vesicles with elevated phosphatidic acid levels. A detergent-free system for in vitro phospholipid synthesis.

A new method for studying phospholipid biosynthesis in Escherichia coli is described. The method makes use of the previously reported observation that E. coli cytidine auxotrophs accumulate phosphatidic acid when starved of cytidine (Ganong, B. and Raetz, C.R.H. (1982) J. Biol. Chem. 257, 389-394). We now show that phosphatidic acid that accumulates in these cells is competent for further biosynthetic use in vivo, if cytidine is re-supplied to the cells. Furthermore, phosphatidic acid-rich membranes prepared from such cells can be used for in situ assays of the later steps of phospholipid biosynthesis. Since this system does not require detergent, our in situ assays more accurately reflect the conditions of an intact membrane. We have used this system to probe the regulation of the branch-point of the biosynthetic pathway for phospholipid polar headgroups. Phosphatidic acid-rich membranes prepared from cells that overproduce either phosphatidylserine synthase or phosphatidylglycerolphosphate synthase do not have increased rates of lipid synthesis in our in situ assays. This correlates with synthetic rates measured in vivo and, thus, our in situ assays accurately reflect conditions in a growing cell's membrane.