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: 10464643
• 负责人: Johnson Jargese Saba
• 金额: $ 3.35万
• 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/10464643
• 关键词: Anaerobic Bacteria; 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; Communities; Complex; Core Facility; Cryoelectron Microscopy; Crystallization; DNA-Directed RNA Polymerase; Data; Defense Mechanisms; Development; Engineering; Environment; Evolution; Family; 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; 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.

项目概要和摘要 基因表达的时相变化使细菌能够产生异质群体并组织 能够共同承受各种环境扰动的社区。这种离散的开/关模式 的基因表达发生在多个位点同时创造广泛的表型变异，但如何 来自多个相位可变基因座的表达是否协调尚不清楚。我们开发了一个突破性的单曲- 细胞微流体技术，直接、同时和随时间研究多个位点的相位变化， 跟踪专门的细菌亚群，并了解决定其相对 丰度、发展速度和相互联系。我们从脆弱拟杆菌身上学到了这些原理， 一种重要的人类肠道共生体和相位变化大师B。fragilis直接抑制病原体， 艰难梭菌和迅速演变的一个巨大的水库移动的，时相可变抗生素耐药基因。 研究了B的相变机制。fragilis将加强人类微生物群的工程， 设计共生体友好的抗生素，以限制进化和随后的抗药性动员 基因.我们将联合收割机单细胞微流体技术与基因组学和生物化学相结合， 调控系统，使协调相位变化：启动子倒位和终止控制。研究 这些基本原则管理时相可变基因表达，我将主要在基因组学培训 和单细胞微流体技术。我的共同导师，罗伯特·兰迪克博士和奥菲利亚·文图雷利博士。兰迪克医生 几十年的经验，研究原核基因调控的基本机制结合博士。 Venturelli在厌氧细菌学，工程和微流体方面的专业知识为我提供了最佳培训， 实现我的职业目标。国家的最先进的设施和资源由威斯康星大学麦迪逊分校和 生物化学和细菌学系为我提供了最佳的环境，我将在其中进行 这个项目