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

项目摘要和摘要 基因表达的阶段变化使细菌能够产生异质种群并组织起来 集体能够承受各种环境干扰的社区。这种离散的开/关模式 的基因表达同时发生在多个基因座上，以创造广泛的表型变异，但如何 多个阶段变量的表达是协调的是未知的。我们开发了一款突破性的单曲- 细胞微流控技术，直接、同时和随时间研究多个位点的相变化，以 跟踪专门的细菌亚群，并学习确定其相对关系的基本原则 丰度、发展速度和相互关联性。我们学习了脆弱类杆菌的这些原理， 一种至关重要的人类肠道共生体，也是阶段变化的大师。脆弱杆菌可直接抑制病原体，如 艰难梭菌迅速进化出一个巨大的可移动的、相变的抗生素耐药性基因储存库。 研究脆弱芽孢杆菌的相变机制将促进人类微生物群的工程化和合理 设计共生体友好的抗生素以限制进化和随后的抗生素耐药性动员 基因。我们将单细胞微流控技术与基因组学和生物化学相结合，专门剖析了由两部分组成的 能够协调阶段变化的调控系统：启动子倒置和终止控制。学习 这些控制阶段变量基因表达的基本原理，我将主要在基因组学方面进行培训 以及我的合作导师罗伯特·兰迪克博士和奥菲莉亚·文图雷利博士的单细胞微流控技术。兰迪克医生的 几十年来研究原核生物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培训 实现我的职业目标。由威斯康星大学麦迪逊分校和 生物化学系和细菌系为我提供了最佳的环境，我将在其中进行 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.