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

使用冷冻透射电镜阐明原核生物及其生物膜的结构

• 批准号: 6718-2006
• 负责人: Beveridge, Terrance
• 金额: $ 4.97万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2006
• 资助国家: 加拿大
• 起止时间: 2006-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=337056
• 关键词: Use; cryoTEM; elucidation; structure; prokaryotes

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.

这项研究将继续我对原核表面和细菌生物膜的结构和物理特性的基础研究。我们在低温透射电子显微镜 (cryoTEM) 和原子力显微镜 (AFM) 的开发方面取得了巨大进展，并打算利用这两个系统来推进微生物表面的基础知识。每种形式的显微镜都可以观察其自然水合状态的高级结构。为此，该研究旨在利用各种菌株和突变体阐明革兰氏阴性和革兰氏阳性细胞壁的分子结构，以选择性地控制构成每个细胞壁的成分网络。这样，通过高分辨率成像，可以确定每个成分的贡献和位置。对S层细菌的类似研究将为大分子（蛋白质-蛋白质、蛋白质-脂质和蛋白质多糖）相互作用的研究提供独特的模型系统。此外，首次通过冷冻置换和冷冻切片以高分辨率研究水合生物膜。还将在异化铁还原细菌希瓦氏菌上进行氧化铁粘附实验，以进一步了解微生物的铁呼吸作用。直到此时，在自然水合状态下准确可视化这些独特的重要微生物结构仍然是不可能的。这些观察结果将有助于理解诸如病原体如何与组织相互作用、生物膜如何对抗菌药物具有如此强的抵抗力以及微生物如何与地质圈相互作用等不同的主题。圭尔夫实验室拥有专业知识和专用设备，现已成为全球独一无二的微生物设施。