EAGER: Collaborative Research: Integrating microtome sectioning with isotopic tracing to study biotransformation in synthetic Escherichia coli biofilms

生物膜是由细菌菌落与细胞产生的聚合物质形成的。由于生物膜对各种压力具有很高的耐受性，因此在工业（生物污染和腐蚀）和医疗保健（持续感染）环境中都会引起严重的问题。另一方面，环境友好菌的生物膜在生物修复和生物燃料生产方面具有广阔的应用前景。本项目将为研究以大肠杆菌为模式生物的营养转运和酶活性提供一种新的方法。pi在大肠杆菌代谢分析和生物膜生理学方面具有丰富的研究经验。该团队将开发和测试一种新的生物膜研究方法，通过整合几个跨学科的方法：1)培养具有控制形态的合成生物膜，以减少基因表达和酶功能的空间差异；2)利用显微切片法获得细胞代谢/营养转运状态相似的生物膜样品的薄片；3)应用稳定同位素标记方法（即13c脉冲）来追踪生物膜层内的营养物质传递以及细胞内自由代谢物沿功能途径的转化。级联代谢物的瞬时标记可以揭示底物通过生物膜层的扩散速度以及生物转化为下游细胞内代谢物的速度。它还可以确定这些对生物膜生存和生长重要的活性生物转化途径。最终，该探索性项目的成果将成为研究多种环境微生物生物膜生理学（即细胞内生物转化）的新方法。这种生物膜研究技术将揭示生物膜在环境胁迫（如抗生素条件）下生长和持续的调节机制；它将提高我们对从环境微生物到病原体的各种生物膜系统的理解。具体而言，大肠杆菌将作为本项目的模型细菌，并在以下任务中概述补充研究：1。创造具有严格控制形态的合成生物膜，以减少结构异质性。2. 优化微生物组技术，从不同层的生物膜细胞中淬灭和取样游离代谢物，可通过液相色谱-质谱分析。3. 通过进行13c脉冲实验，用显微切片和级联代谢物的液相色谱-质谱分析来验证这种新方法，以确定葡萄糖在不同层的大肠杆菌生物膜细胞中的转移和生物转化率。该项目将产生一种新的方法来切片生物膜，以检测代谢物的生物转化，并以所需的空间分辨率探测途径功能。这项工作的跨学科性质以及本文概述的新工具的开发将为在分子水平上解决与微生物生物膜相关的许多重大挑战的高级研究和创新奠定基础。它将为生物膜基质内的营养转运和细胞内酶转化提供新的见解；并有助于揭示生物膜在不同环境条件下生存的调控机制和重要途径。这些见解可以帮助研究人员设计出更有效的控制生物膜生理的方法。

1700881/1700935Tang/RenBiofilms are created by colonies of bacteria with polymeric substances produced by the cells. With a high-level of tolerance to a variety of stresses, biofilms cause serious problems in both industrial (biofouling and corrosion) and healthcare (persistent infections) settings. On the other hand, biofilms of environmentally friendly bacteria have promising applications in bioremediation and biofuel production. This project will develop a new method for investigating nutrient transport and enzyme activity of using Escherichia coli as a model organism.The PIs have extensive research experience in E. coli metabolic analysis and biofilm physiology. This team will develop and test a new biofilm study method by integrating several interdisciplinary approaches: 1) culture synthetic biofilms with controlled morphology to reduce spatial variation in gene expression and enzymatic functions; 2) use microtome sectioning to obtain thin slides of biofilm samples with cells in similar metabolic/nutrient transport status; and, 3) apply a stable isotopic labeling method (i.e., 13C-pulse) to trace nutrient mass transfer within biofilm layers as well as intracellular free metabolite conversions along functional pathways. The transient labeling in cascade metabolites can reveal the speed of substrate diffusion through biofilm layers as well as the rate of biotransformation into downstream intracellular metabolites. It can also determine these active biotransformation pathways important for biofilm survival and growth. Ultimately, the outcome of this exploratory project will be a new method for studying biofilm physiology (i.e., intracellular biotransformation) of diverse environmental microbes. This biofilm study technology will reveal regulatory mechanisms of biofilm growth and persistence under environmental stresses (e.g., antibiotic conditions); and it will improve our understandings of diverse biofilm systems from environmental microbes to pathogens. Specifically, Escherichia coli will be used as a model bacterium in this project with complementary studies outlined in the following Tasks: 1. Create synthetic biofilms with rigorously controlled morphology to reduce structural heterogeneity. 2. Optimize microtome technology to quench and sample free metabolites from different layers of biofilm cells, which can be analyzed via liquid chromatography-mass spectrometry. 3. Validate this new method by conducting 13C-pulse experiments with microtome sectioning and liquid chromatography-mass spectrometry analysis of cascade metabolites to determine glucose transfer and biotransformation rates across different layers of E. coli biofilm cells. This project will result in a new approach to section biofilms to detect metabolite biotransformation and probe the pathway function with desired spatial resolutions. The interdisciplinary nature of this work and the development of new tools outlined herein will build the foundation for advanced research and innovation to address many grand challenges associated with microbial biofilms at molecular level. It will provide new insights into nutrient transport and intracellular enzyme conversions within the biofilm matrix; and help reveal regulatory mechanisms and essential pathways for biofilm survival under diverse environmental conditions. Such insights can help researchers design more effective way to control biofilm physiologies.