Application): Many important bacteria possess surface layers (S-layers) that are additional to their typical Gram-positive or -negative cell wall. These S-layers, which may be unstable, aid the organisms in surviving adverse environmental conditions. The conditions under which these layers are present and functional are not clearly understood. Aquaspirillum serpens possesses such an S-layer and therefore is being used as a model to study this method of bacterial protection. The PI has shown that the S-layer of A. serpens is made up of an array of hexagonal units. Chemically, these units are composed of a single acidic glycoprotein (SG). The synthesized SG non-covalently assembles into the S-layer on the surface of the cell wall. The assembly of the S-layer increases the survival index of the organism. Many mechanistic aspects about the formation of the S-layer are still not known. The PI is currently investigating the chemistry of the glycan moiety of the SG. Also, little is known about the physicochemical nature of the conformation-determining cell wall macromolecular template. This project seeks to extend the current studies to investigate, at the molecular level, (1) the exact nature of the cell envelope template to which the SG is attached, and (2) the role of the glycan moiety and template conformation in the binding-adhesion process. Identifying the template will be achieved by electrophoretically separating the macromolecules from the cell wall template, transferring them by Western blotting onto a suitable solid matrix, and testing the ability of native and deglycosylated SG to bind to the separated macromolecules under conditions known to enhance S-layer formation. The binding will be assessed using rabbit SG-specific antibodies and alkaline phosphatase-conjugated antirabbit antiserum.
