Outer membranes of gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PAO1 and use in reconstitution and definition of the permeability barrier

Abstract

A method for separating the outer and inner membranes of P. aeruginosa PAO1 in the absence of added EDTA was devised. The method yields 2 outer membrane fractions which show the same protein pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but differ substantially in their relative contents of phospholipids. One of these outer membrane fractions and the inner membrane fraction are less than 4% cross-contaminated, as judged by the content of typical inner and outer membrane markers. The outer membrane contains 4 major protein bands with apparent MW of 37,000, 35,000, 21,000 and 17,000. Vesicles reconstituted from lipopolysaccharide and phospholipids were impermeable to all saccharides included in the vesicles during vesicle formation. When the vesicles contained outer membrane proteins, they fully retained only those saccharides of greater than 9000 MW, suggesting that the exclusion limit of the outer membrane of P. aeruginosa for saccharides is substantially larger than the figure (500-600 daltons) obtained for certain enteric bacteria. The advantages and potential disadvantages of having an outer membrane with a higher exclusion limit for hydrophilic substances are discussed.