[2] Utilization of labeled Escherichia coli as phospholipase substrate

Escherichia coli marked with 14C-labeled fatty acid have proved to be convenient for the assay of deacylating phospholipases. They have been used in two ways: as intact bacteria and after autoclaving. As is the case for phospholipids in the membranes of most unperturbed cells, the phospholipids of intact E. coli are refractory to the action of both endogenous and added deacylating phospholipases. This helps in studying the ability of various agents to activate selectively or indiscriminately phospholipolysis by a broad range of secretory and cellular phospholipases. Autoclaving of E. coli tagged with 14C-labeled fatty acid inactivates all bacterial phospholipases and exposes the phospholipids in the E. coli membranes to all deacylating phospholipases. The use of autoclaved labeled E. coli as substrate has now been widely adopted, the advantages of which are the ease of preparation and the high degree of sensitivity of the assay. The retention of the typical rod shape and hence of the gross envelope structure of the E. coli after autoclaving suggests that the presentation of the bacterial phospholipids is more similar to that of phospholipids in natural membranes than is the case for phospholipids in most of the other commonly used assays. The labeling of E. coli with 14C-labeled fatty acid during several generations produces uniform labeling of the E. coli phospholipids so that the distribution of the label fairly reflects the bacterial phospholipid composition. As phospholipid and fatty acid composition of lipids extracted from autoclaved and untreated E. coli are the same, the exposure of the bacteria to the autoclave conditions does not appreciably change the bacterial phospholipids.