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

这项研究将继续我对原核生物表面和细菌生物膜的结构和物理性质的基础研究。我们在低温电子显微镜和原子力显微镜的发展方面已经取得了很大的进展，并打算使用这两个系统来促进对微生物表面的基础知识。每种形式的显微镜都可以显示处于自然水合状态的高阶结构。为此，这项研究旨在利用各种菌株和突变体来阐明革兰氏阴性和革兰氏阳性细胞壁的分子结构，以努力选择性地控制组成每个细胞壁的成分网络。这样，通过高分辨率成像，就可以确定每个成分的贡献和位置。对S层状细菌的类似研究将为大分子(蛋白质-蛋白质、蛋白质-脂肪、蛋白质多糖)相互作用的研究提供独特的模型体系。此外，将首次通过冷冻替代和冷冻切片以高分辨率研究水合生物膜。为了进一步了解微生物铁的呼吸作用，还将对异化铁还原细菌Shewanella进行氧化铁附着实验。直到现在，这些独特的重要微生物结构在自然水合状态下的准确可视化一直是不可能的。这些观察将有助于理解不同的主题，如病原体如何与组织相互作用，生物膜如何对抗菌剂具有如此大的弹性，以及微生物如何与地质圈相互作用。这个拥有专业知识和专门设备的Guelph实验室现在是世界上独一无二的微生物设施。

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.