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

单细胞阐明控制人类共生细菌拟杆菌细胞表面变异的转录调控机制

• 批准号: 10682388
• 负责人: Johnson Jargese Saba
• 金额: $ 3.71万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10682388
• 关键词: Antibiotic Resistance; Antibiotics; Antibodies; Bacteria; Bacteriology; Bacteriophages; Bacteroides; Bacteroides fragilis; Bacteroidetes; Bar Codes; Binding; Biochemical; Biochemistry; Biological Assay; Biological Models; Cell surface; Cells; ChIP-seq; Clostridium difficile; Collaborations; Complex; Core Facility; Cryoelectron Microscopy; DNA-Directed RNA Polymerase; Data; Defense Mechanisms; Development; Engineering; Environment; Evolution; Family; Friends; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic Transcription; Genomics; Goals; Human; Human Engineering; Immune; In Vitro; Invertase; Knowledge; Learning; Mentors; Microbial Biofilms; Microfluidics; Modeling; Molecular; Operon; Outcome; Pattern; Phase; Phenotype; Polysaccharides; Population; Population Heterogeneity; Process; Proteins; Regulon; Reproducibility; Resources; Roentgen Rays; Sampling; Site; Stress; Structure; Surface; System; Technology; Testing; Time; Training; Universities; Variant; Work; antibiotic design; antitermination; career; combinatorial; community organizations; environmental stressor; experience; experimental study; follow-up; gene expression variation; gut microbiota; human microbiota; improved; inhibitor; innovation; innovative technologies; microfluidic technology; pathogen; pathogenic bacteria; promoter; rational design; resistance gene; single cell sequencing; single cell technology; transcription factor

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.

项目摘要和摘要 基因表达的相位变化使细菌能够产生异质种群并组织 集体可以承受潜在的环境扰动的社区。这种离散的开/关模式 基因表达的同时发生在多个局部情况下，以创造广泛的表型变异，但是如何 来自多相变量基因座的表达未知。我们开发了一个突破性的单一 细胞微流体技术，可直接，轻松，随着时间的推移研究多个语言环境的相变 跟踪专业的细菌亚人群并学习决定其相对的基本原理 抽象，发展速率和相互联系。我们学习了这些针对fragilis的原理， 至关重要的人类肠道共生和相位变异的主人。 B. Fragilis直接抑制病原体，例如 艰难梭状芽胞杆菌和迅速进化了大量流动性，相变的抗生素耐药性基因。 研究脆弱芽孢杆菌中的相变机制将增强人类微生物群的工程和理性的工程 符号友好的抗生素的设计，以限制进化和随后动员抗生素抗性 基因。我们将单细胞微流体与基因组学和生物化学相结合，以专门剖析两部分 调节系统实现协调期变化：启动子反转和终止控制。学习 这些基因可变基因表达的基本原理，我将在基因组中受过培训 以及我的联合官员Robert Landick博士和Ophelia Venturelli博士的单细胞微流体。兰迪克博士 数十年来研究核基因调节基本机制结合博士的经验 Venturelli在厌氧细菌，工程和微流体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。 UW-Madison提供的最先进的设施和资源 生物化学和细菌学部门为我提供了最佳的环境 这个项目。