THE EFFECT OF SN-GLYCEROL 3-PHOSPHOROTHIOATE ON GROWTH, METABOLISM AND MEMBRANE STRUCTURE AND FUNCTION IN ESCHERICHIA COLI (PHOSPHOLIPIDS)

Abstract

The role of the phospholipid polar headgroup in membrane function was investigated by replacing the phosphate moiety of the polar headgroup with its phosphorothioate analog as a result of the selective resistance of phosphorothioate-containing compounds to phosphatase hydrolysis. Phosphorothioate-containing phospholipids were metabolically introduced into membranes of E. coli by culturing these cells in the presence of the glycerol-phosphate analog, sn-glycerol 3-phosphorothioate. These cells possess an active transport system for sn-glycerol 3-phosphate, the precursor of all phospholipids in E. coli.;Glycerol-phosphorothioate was found to be bacteriocidal to strains of E. coli containing a functional sn-glycerol 3-phosphate transport system (glpT). Transport studies demonstrated that glycerol-phosphorothioate is taken up via the specific active transport system for glycerol-phosphate with a K(,m) (11 (mu)M) in close agreement to the found for glycerol-phosphate (9 (mu)M).;The rate of macromolecular synthesis was severely inhibited at bacteriocidal concentrations of glycerol-phosphrothioate (5 (mu)M). The rates of protein and RNA syntheses were more sensitive than the rates of DNA and cell wall syntheses. The uptake of precursors into E. coli was significantly affected in the presence of sub-bacteriocidal concentrations of glycerol-phosphorothioate (0.5 (mu)M), suggesting that the affect on macromolecular synthesis may be due to inhibition of uptake rather than biosynthesis. Phospholipid polar headgroup composition was significantly altered in the presence of glycerol-phosphorothioate (5 (mu)M), i.e., a decrease in the level of phosphatidylglycerol and a concurrent fourfold increase in cardiolipin content.;Doubly-labeled glycerol-phosphorothioate was found to be incorporated into phospholipid, and the major phosphorothioate-containing phospholipid formed was identified as thiophosphatidic acid. Chromatagraphic analysis of the metabolites of the labeled compound has suggested that they are incorporated intact into membrane-derived oligosaccharides as 1-phosphoglycerol-3-phosphorothioate from the phosphorothioate analog of phosphatidylglycerol phosphate.;Analysis of E. coli strain 8 under electron microscopy revealed that these cells became osmotically unstable as seen by cell lysis and loss of membrane structure.;These studies suggest that the bacteriocidal effects of glycerol-phosphorothioate may be due to alterations of the cell envelope as a result of changes in phospholipid composition and the accumulation of the negatively-charged phosphorothioate-containing membrane-derived oligosaccharides located in the periplasmic space.