Outer membrane as a diffusion barrier in Salmonella typhimurium. Penetration of oligo- and polysaccharides into isolated outer membrane vesicles and cells with degraded peptidoglycan layer

Abstract

In Escherichia coli and Salmonella typhimurium, the cell wall that contains both the outer membrane layer and the peptidoglycan layer acts as a barrier of the molecular sieve type for the penetration of uncharged saccharides (G. Decad, T. Nakae, and H. Nikaido (1974) Fed. Proc. 33, 1240). Here we examined which of the layers of the cell wall limited the size of the penetrating molecules, by studying the penetration of saccharides into (a) cells whose peptidoglycan layer had been destroyed by lysozyme treatment or growth in the presence of penicillin and (b) isolated outer membrane vesicles. We found that peptidoglycan-defective cells were similar to intact, plasmolyzed cells in that they allowed a partial penetration of stachyose (molecular weight 666), but essentially excluded saccharides with molecular weights higher than 900 to 1000. We also found that the isolated outer membrane acted as a penetration barrier for saccharides. These observations led us to conclude that the outer membrane, rather than peptidoglycan, sets the size limit for the penetration of uncharged, hydrophilic molecules through the E. coli or S. typhimurium cell wall. The isolated outer membrane, however, had an exclusion limit much higher than that found in intact cells. This "leakiness" could be decreased either by the use of mutants producing extremely deficient lipopolysaccharide, or by trypsin treatment of the isolated membrane followed by heating and slow cooling in the presence of Mg2+. We feel that these observations are consistent with the hypothesis that the resealing of the ruptured outer membrane during the isolation procedure is often incomplete, and that cracks and holes thus generated are responsible for the "leakiness" of the isolated membrane vesicles.