Isolation and chemical composition of the cytoplasmic membrane of a gram-negative bacterium

Abstract

With procedures developed previously in this laboratory, the various layers of the cell wall of a gram-negative bacterium, a marine pseudomonad (ATCC 19855), were removed completely giving rise to true protoplasts. Membranes were isolated from the protoplasts formed. After treatment with ribonuclease, deoxyribonuclease, and washing, the membranes isolated were shown by electron microscopy and chemical analysis to be essentially free from both wall material and cytoplasmic constituents. The membranes gave rise to a single compact band in a sucrose density gradient. All of the lipid and protein were found to be associated in the membrane band. Analysis showed the membranes to contain 30.5% lipid (78% of which was phospholipid), 62.8% protein, and 2% carbohydrate. The predominant phospholipid present was phosphatidylethanolamine with a lesser amount of diphosphatidylglycerol and traces of unidentified compounds.