Single-cell elucidation of transcriptional regulatory mechanisms that govern cell surface variation of the human symbiotic bacteria Bacteroidetes

项目概要和摘要 基因表达的相位变化使细菌能够产生异质群体并组织起来 共同能够抵御各种环境扰动的社区。这种离散的开/关模式 基因表达的变化同时发生在多个位点以产生广泛的表型变异，但是如何 来自多个阶段变量基因座的表达是否协调是未知的。我们开发了一种突破性的单 细胞微流体技术可直接、同时、随时间研究多个位点的相位变化 跟踪专门的细菌亚群并学习决定其相对关系的基本原理 丰富度、发展速度和互联性。我们了解脆弱拟杆菌的这些原理， 一种重要的人类肠道共生体和相变大师。 B. fragilis 直接抑制病原体，例如 艰难梭菌迅速进化出大量可移动、相变的抗生素抗性基因。 研究脆弱拟杆菌的相变机制将增强人类微生物群的工程和合理性 设计共生体友好型抗生素，以限制抗生素耐药性的进化和随后的动员 基因。我们将单细胞微流体与基因组学和生物化学相结合，专门剖析了两部分 调节系统可实现协调的相变：启动子反转和终止控制。学习 这些控制相变基因表达的基本原则，我将主要接受基因组学方面的培训 以及我的合作导师 Robert Landick 博士和 Ophelia Venturelli 博士的单细胞微流体学。兰迪克博士的 与博士结合数十年研究原核基因调控基本机制的经验。 Venturelli 在厌氧细菌学、工程学和微流体学方面的专业知识为我提供了最佳的培训 实现我的职业目标。威斯康辛大学麦迪逊分校和美国威斯康星大学麦迪逊分校提供的最先进的设施和资源 生物化学系和细菌学系为我提供了最佳的环境 这个项目。

Project Summary and Abstract Phase variation of gene expression enables bacteria to generate heterogenous populations and organize communities that collectively can withstand diverse environmental perturbations. This discrete ON/OFF pattern of gene expression occurs at multiple loci concurrently to create extensive phenotypic variation, but how expression from multiple phase variable loci is coordinated is unknown. We developed a breakthrough single- cell microfluidics technology to study phase variation at multiple loci directly, simultaneously, and over time to track specialized bacterial sub-populations and learn fundamental principles determining their relative abundances, rates of development, and interconnectedness. We learn these principles for Bacteroides fragilis, a crucial human gut symbiote and master of phase variation. B. fragilis directly inhibits pathogens such as Clostridium difficile and rapidly evolves a vast reservoir of mobile, phase variable antibiotic resistance genes. Studying phase variation mechanisms in B. fragilis will enhance engineering of human microbiota and rational design of symbiote-friendly antibiotics to limit evolution and subsequent mobilization of antibiotic-resistance genes. We combine single-cell microfluidics with genomics and biochemistry to specifically dissect a two-part regulatory system enabling coordinated phase variation: promoter inversion and termination control. To study these fundamental principles governing phase variable gene expression, I will be trained primarily in genomics and single-cell microfluidics by my co-mentors, Dr. Robert Landick and Dr. Ophelia Venturelli. Dr. Landick’s decades of experience studying fundamental mechanisms of prokaryotic gene regulation combined with Dr. Venturelli’s expertise in anaerobic bacteriology, engineering, and microfluidics provide me optimal training to achieve my career goal. The state-of-the-art facilities and resources provided by UW-Madison and the Departments of Biochemistry and Bacteriology provide me with the optimal environment in which I will carry out this project.