Three-dimensional structure of an open form of the surface layer from the fish pathogen Aeromonas salmonicida

Cell-free culture supernatants of a lipopolysaccharide (LPS) O-polysaccharide-deficient, single-insertion transposon mutant of the tetragonal surface protein array (S layer)-containing fish pathogen Aeromonas salmonicida were examined by electron microscopy. Negative staining showed that the S layer was released as sheets of tetragonal material, indicating that although surface retention of assembled S layer requires the presence of wild-type LPS oligosaccharides, initial assembly of S-layer subunits into sheets does not require the presence of O-polysaccharide chains. The three-dimensional structure of the S layer was reconstructed from tilted micrographs of the released sheets. Horizontal sections through this reconstruction showed that the released sheets were composed of two identical S layers that were perfectly in register. The reconstructed layer had a lattice constant of 12.5 nm. At a resolution of 1.6 nm, the layer consisted of a major tetragon at one fourfold axis of symmetry and a minor tetragon at the second fourfold axis of symmetry. The core, composed of four of the major domains, contained a large depression and was located toward the inside of the layer. The minor tetragon provided connectivity within the layer and was located toward the outer surface of the layer. Projections through the double layer gave a type I (closed) pattern (M. Stewart, T. J. Beveridge, and T. J. Trust, J. Bacteriol. 166:120-127, 1986), yet projections through the single layer indicated that the type II (open) pattern was present. This open pattern was indistinguishable from that seen in S layer released from the surfaces of wild-type cells.