Cardiolipin, a major phospholipid of Gram-positive bacteria that is not readily extractable

Extraction of phospholipids from stationary phase grown cells of the Gram+ bacteria, Bacillus megaterium, Bacillus subtilis, Bacillus cereus and Micrococcus lysodeikticus was found to be incomplete with various commonly used extraction procedures. Phosphatidylglycerol and phosphatidylethanolamine were readily extracted but up to 95% of the cardiolipin appeared to be retained within the cell residue. Extraction of the cardiolipin could be slightly enhanced by increasing the temperature or the acidity of the extraction solutions but complete extraction was obtained only after lysozyme treatment of intact cells or cell residues remaining after extraction. In addition complete extraction could be observed in the case of cells harvested in the early logarithmic phase. Freeze-fracture electron microscopy was carried out on the cell residue remaining after extraction of all phospholipids except cardiolipin. A fracture plane through the plasma membrane could not be observed anymore. Instead fracture planes through lipid vesicles were observed. These vesicles reside within the remnants of the cytoplasm and consist most likely of the non-extracted cardiolipin.