CAREER: Form and Function of Bacterial Amyloid Fibers

在几乎所有的生态系统中，细菌与表面联系的倾向远远超过了在悬浮状态下自由生活的倾向。细菌分泌蛋白质、多糖和其他成分，组装成蛛网状的基质，包围细胞，促进被称为生物膜的保护性群落的形成。这种保护性的物理基质使细菌能够在一系列令人惊讶的环境中定居，甚至茁壮成长，从黄石公园恶劣的酸性微生物垫到船体和工业输油管道。改进的生物膜模型对于理解它们如何发挥作用至关重要。在生物学、化学和材料研究中，测量化学、物理和机械性能参数是理解复杂组件如何发挥作用的关键。在本项目中，实验旨在通过高分辨率技术实现突破性发现，将生物膜描述符从“胶水”和“黏液”等模糊术语转变为基于化学成分和分子结构的科学定量描述。细菌对这些大分子结构的自组装也激励我们使用或构建这种机械上强大的分子框架来实现新功能。在这个项目中，PI将广泛传播科学研究和教育宣传活动。她将指导本科生和研究生，包括许多来自STEM中代表性不足的群体；设计课程改革，在生物化学课程中引入更多的定量概念；领导全面的外展活动，吸引学生、教师和公众参与。主要的具体活动包括：(i)在每年夏天加入PI实验室的高中老师的帮助下，为高中科学课程开发生化生物膜实验模块；（ii）在本科实验课程中整合基于探索的实验模块，以鼓励真正的参与和动手的“a-ha”时刻，增强学习；（iii）协助辅导即将入学的第一代和代表性不足的本科生，为大学的STEM课程做准备；（iv）共享定制的主动控制反馈系统的工程协议，以稳定长采集时间内的核磁共振脉冲功率水平，提高核磁共振实验的稳定性，并在光谱仪监测中具有普遍价值。总体而言，该项目致力于教育和科学推广，将研究与教学、指导和K-12推广活动相结合，以激励和授权他人影响和改善社会。在这个项目中，将研究大肠杆菌产生的淀粉样蛋白纤维，即curli。Curli介导细菌粘附并促进生物膜的形成。该项目将为天然卷曲纤维的原子结构和功能提供前所未有的细节，并将通过整合独特的固态核磁共振（NMR）策略来测量原子水平距离，以及超分辨率显微镜来检查卷曲纤维和生物膜中其他成分的更大尺度空间排列，绘制它们与淀粉样蛋白染料及其同源生物膜多糖伙伴的相互作用。该项目由分子和细胞生物科学部的分子生物物理学和细胞动力学以及功能集群共同资助。

The propensity for bacteria to associate with surfaces in nearly all ecosystems far exceeds the tendency to persist in suspension, living freely in a planktonic state. Bacteria secrete proteins, polysaccharides, and other components to assemble a spider-web like matrix that surrounds cells to promote the formation of protective communities termed biofilms. The protective physical matrix enables bacteria to colonize and even thrive in an astonishing range of environments, in niches ranging from the harsh acidic microbial mats in Yellowstone to ship hulls and industrial oil pipelines. Improved biofilm models are crucial to understanding how they function. Across biology, chemistry, and materials research, measuring parameters of chemical, physical, and mechanical properties is key to understanding how complex assemblies function. In this project, experiments are designed to achieve breakthrough discoveries needed to transform biofilm descriptors from vague terms like "glue" and "slime" to scientific and quantitative descriptions based on chemical composition and molecular architecture by using high resolution techniques. The self-assembly of these macromolecular architectures by bacteria also inspire us to use or build such mechanically robust molecular frameworks for new functions.In this project, the PI will engage in broad dissemination of scientific research and educational outreach activities. She will mentor undergraduate and graduate students, including many from groups under-represented in STEM; design coursework changes to introduce more quantitative concepts into biochemistry courses; and lead comprehensive outreach activities, engaging students, teachers, and the general public. Major specific activities include: (i) developing a biochemical biofilm laboratory module for the high-school science curriculum with assistance from a high-school teacher who joins the PI's laboratory each summer, (ii) integrating exploratory-based lab modules in undergraduate laboratory courses to encourage true engagement and the hands-on "a-ha" moments that enhance learning; (iii) assisting in the mentoring of incoming first-generation and under-represented undergraduate students in preparation for university coursework in STEM; and (iv) sharing engineering protocols for a custom-built active-control feedback system to stabilize NMR pulse power levels over long acquisition times, enhancing the stability of NMR experiments and also of general value in spectrometer monitoring. Overall, the commitment to educational and scientific outreach in this project integrates research with teaching, mentoring, and K-12 outreach activities to inspire and empower others to impact and improve the society.In this project, amyloid fibers produced by E. coli, known as curli, will be studied. Curli mediate bacterial adhesion and contribute to biofilm formation. This project will provide unprecedented detail into the atomic structure and function of native curli fibers and will map their interactions with amyloid dyes and their cognate biofilm polysaccharide partners by integrating unique solid-state nuclear magnetic resonance (NMR) strategies to measure atomic-level distances together with super-resolution microscopy to examine the larger-scale spatial arrangement of curli and other components in the biofilm. This project is jointly funded by Molecular Biophysics and Cellular Dynamics and Function Clusters in the Division of Molecular and Cellular Biosciences.