Isolation And Characterization Of The Outer And Cytoplasmic Membranes Of Pseudomonas cepacia

Abstract

A method is described for the separation of the outer membrane (OM) from the cytoplasmic membrane (CM) of Pseudomonas cepacia grown in nutrient broth and in chemically defined media under different nutrient depletions. The method is particularly valuable since it is effective when applied to stationary phase cells. Enzyme activities indicated that the contamination of the OM with the CM was less than 5%. The OM protein profile of magnesium-depleted cells was much simpler than that of the iron-depleted and nutrient broth grown cells. The apparent molecular weights of the OM proteins of magnesium-depleted cells were: 40 000, 36 000, 24 500 and 14 500. Iron depletion induced the synthesis of an OM protein with apparent molecular weight of 66 000. The OM proteins with apparent molecular weights of 40 000, 36 000 and 24 500 were heat-modifiable and the 24 500 dalton protein was found also to be affected by the presence of 2-mercaptoethanol. The OM consisted of 50% protein and 20% phospholipid and the rest was probably LPS while the CM consisted of 80% phospholipid and 20% protein. The major phospholipid in both membranes was phosphatidylethanolamine with a smaller amount of phosphatidylglycerol and a trace amount of phosphatidylcholine; the OM contained more phosphatidylethanolamine than the CM.