NSF Award
9206674
The proposed research focuses on the functional and structural organization of envelope membranes of established strains of Escherichia coli and of new constructs. Biochemical, immunological and molecular-biological techniques will be used in combination with new developments in cryofixation procedures to optimize high resolution topography of the membranes of Gram-negative bacterial cells. Emphasis is placed on a biochemical, structural and genetic dissection of translocation processes involving membrane proteins and polysaccharides as they are transported from the inner membrane (IM) to their target site at the outer envelope membrane (OM). Two models of translocation pathways will be tested: after synthesis and passage through the IM, a molecule may travel either via IM/OM connections (the membrane adhesions sites), or via the periplasmic space. The proposed experiments are aimed at the study of topographic relationships of the envelope components in undisrupted cells, and at a molecular stabilization of the cell envelope. The work will be subdivided into two aspects: 1, the topology of export pathways of outer membrane polysaccharides and proteins; and 2, stabilization of the membrane organization using cryofixation and UV-flash crosslinking, to determine the in vivo structure of adhesion sites and periplasm. It is hoped that development of the UV-flash and cryo preparation methodologies will permit future investigations into the spatial distribution of components of the protein export machinery (e.g., localization of Sec A, Sec E, and export sites of precursor proteins in wild type and mutant cells). %%% Gram-negative bacteria are bounded by envelopes consisting of an inner membrane (equivalent to the plasma membrane of the bacterial cell) and an outer membrane (a true biological membrane, with proteins associated with a lipid bilayer), and a layer of wall (peptidoglycan) sandwiched between the two membranes. The envelope forms a barrier against macromolecules, drugs, and viruses, and provides mechanical stability toward turgor pressure and physical injury. The envelope membranes also have to provide for the specific transmembrane transfer of large molecules (both coming in and going out). It also must be dynamic in structure, to allow for insertion of new membrane constituents during cell growth. The goal of this work is to elucidate the structural and functional organization of the Gram-negative bacterial surface. It is expected that some of the methodological advances will have broader applications in cell and membrane biology. This research will contribute significantly to the understanding of the dynamic organization of the bacterial envelope membranes, their export machinery, and their structural and functional mosaic.
