Outer membrane of gram-negative bacteria. XVIII. Electron microscopic studies on porin insertion sites and growth of cell surface of Salmonella typhimurium

Abstract

S. typhimurium contains 3 major proteins or porins (34K, 35K and 36K) in the outer membrane. A mutant strain producing only the 35K porin was first grown in media containing high concentrations of NaCl to repress the porin synthesis and then was shifted into a medium without NaCl. The newly made porin molecules were then labeled with the ferritin-coupled antibody at various times after the shift, and the samples were examined by whole-mount, freeze-etching and thin-section electron microscopy. These experiments showed that newly inserted porins appeared as discrete patches uniformly distributed over the surface of the cell and that the sites of adhesion between the inner and outer membrane were most probably the pathway by which the newly made porin molecules appeared on cell surface. The 34K and 36K porins were also inserted in the same manner, since the appearance of new porins at discrete sites all over the cell surface was also observed when cells with wild-type porin phenotype were treated with unlabeled antibody to block existing antigenic sites, subsequently regrown, and labeled with the ferritin-coupled antibody. Since porins comprise a major portion of the densely packed, relatively immobile, protein framework of the outer membrane, these results showed that the outer membrane grows predominantly by diffuse intercalation rather than by the zonal growth mechanism.