Use of cryoTEM for the elucidation of the structure of prokaryotes and their biofilms

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This research will continue my fundamental studies into the structure and physical properties of prokaryotic surfaces and bacterial biofilms. We have made great progress in the development of cryo-transmission electron microscopy (cryoTEM) and atomic force microscopy (AFM) and intend to use both systems to advance the fundamental knowledge of microbial surfaces. Each form of microscopy allows the visualization of high-order structure in its natural hydrated state. To this end, the research is designed to elucidate the molecular architecture of Gram-negative and Gram-positive cell walls using a variety of strains and mutants in an effort to selectively control the network of constituents that make up each cell wall. In this way, through high-resolution imaging, the contribution and position of each constituent can be ascertained. Similar studies on S-layered bacteria will provide a unique model system for the study of macromolecular (protein-protein, protein-lipid, and protein polysaccharide) interaction. In addition, for the first time, hydrated biofilms will be studied at high resolution via freeze-substitution and cryo-sections. Fe-oxide adhesion experiments will also be done on the dissimilatory iron-reducing bacterium, Shewanella, in an effort to further understand microbial iron-respiration. Until this time, accurate visualization of these unique important microbial structures in a natural hydrated state has been impossible. These observations will aid in the understanding of such disparate subjects as how pathogens interact with tissue, how biofilms are so resilient to antimicrobials, and how microorganisms interact with the geosphere. This Guelph laboratory with its expertise and specialized equipment is now a unique microbiological facility worldwide.