Visualization of lipid biosynthesis in isolated cytoplasmic membrane vesicles of Escherichia coli by freeze fracture electron microscopy

Abstract

The continuous addition of acyl-CoA to an incubation system in which cytoplasmic membrane vesicles of Escherichia coli are present together with substrates and cofactors for phospholipid synthesis results in a continuous de novo synthesis of anionic phospholipids. After 6 h of incubation the phospholipid to protein ratio is increased nearly fourfold. The accumulated phospholipids are cardiolipin, phosphatidic acid, and phosphatidylglycerol. Equimolar substrate mixtures of palmitoyl-CoA, palmitoleoyl-CoA, and vaccenoyl-CoA results in the synthesis of phospholipids which appear to have similar fatty acid profiles as the original constituents. Extensive accumulation of phosphatidic acid during the course of lipid biosynthesis and turnover studies carried out with membrane vesicles isolated from cells grown in the presence of [ 3H]glycerol suggest that lipid clusters with low reactivity are present in the vesicle membranes. Freeze-fracture electron microscopy of the membrane vesicles before and after accumulation of the anionic phospholipids shows a large increase in vesicle size and extensive aggregation of membrane proteins. The increase in vesicle size results from the increase in phospholipid content as well as from fusion of the membrane vesicles. The relevance of the results in relation to the aberrations in growth and division observed in Escherichia coli mutants which accumulate anionic phospholipids is discussed.