Cellular homeostasis pathways in bacteria

细菌的细胞稳态途径

• 批准号: 10205911
• 负责人: CAROL Anne GROSS
• 金额: $ 100.39万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-07 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10205911
• 关键词: Address; Affect; Animal Model; Area; Bacteria; Bacterial Genome; Base Pairing; CRISPR interference; Coupled; Coupling; Data; Databases; Environment; Essential Genes; Evolution; Genes; Genetic Transcription; Goals; Grant; Growth; Homeostasis; Measurement; Measures; Metagenomics; Mutation; Organism; Output; Pathway interactions; Phylogenetic Analysis; Planets; Process; Proliferating; Property; Recording of previous events; Regulator Genes; Research; Research Personnel; Ribosomal Proteins; Ribosomes; Stress; Technology; Transcription Process; Translations; Vision; cell envelope; environmental change; fighting; fitness; gene function; genetic analysis; genome-wide; knock-down; mRNA Transcript Degradation; new technology; novel; prevent; success; tool

The overarching goal of my research is to uncover the fundamental principles that drive bacterial success, enabling them to colonize and proliferate in every corner of this planet. To accomplish this goal, I have developed a unified bacterial-centric research vision cutting across classically defined subfields. I meld my long history of unraveling the intricacies of bacterial control mechanisms with an ability to develop and implement novel global technologies to open up understudied areas and computational approaches to extend findings beyond model organisms. The current grant explores three important and related areas. First, fueled by two novel CRISPRi strategies that we developed, we continue our quest to identify cellular construction principles by exploring three understudied sets of genes: cell envelope genes, essential genes, and genes that accelerate growth transitions. We tackle the redundancy of function that has prevented genetic analysis of the envelope with double CRISPRi, a technology that allows simultaneous knockdown of two genes via adjacently encoded sgRNAs. We unravel the tradeoffs underlying the expression of essential genes with mismatched CRISPRi, which uses single mismatches in the base pairing region of sgRNAs to predictably titrate their efficacy. By measuring the fitness impact of graded knockdown, we determine the expression-fitness relationships of essential genes and how they are affected by environmental and genetic changes. Finally, we identify essential and non-essential genes that accelerate growth transitions. Second, we continue our studies of the general principles controlling translational output both by exploring the extent to which ribosomes themselves influence the upstream process of transcription (transcription/translation coupling) and the downstream process of mRNA degradation, and by determining whether alternative ribosomal proteins produced under stress conditions result in new translational properties. These studies are enabled by new technologies we developed for genome-wide measurement of ribosome spacing and mRNA degradation. Third, we have begun an exciting new study of gene regulatory networks throughout the bacterial kingdom. This effort is fueled by our new statistically rigorous, phylogenetic foot-printing approach, which we have validated to have a low false positive rate coupled with high recall and precision. We plan to leverage the vast existing database of bacterial genomes to examine evolution of gene regulatory networks across bacteria. Our studies also address an overwhelming current challenge: to develop experimental and computational approaches that enable researchers to comprehensively explore the regulatory wiring and functional diversity of bacteria that thrive in a wide variety of rapidly changing environments. Such approaches can synergize with and exploit metagenomic data to empower mechanistic interrogation of gene function in understudied organisms.

我研究的首要目标是揭示推动细菌成功的基本原理， 使他们能够在这个星球的每个角落殖民和扩散。为了实现这个目标，我有 制定了一个以细菌为中心的统一研究愿景，跨越了经典定义的子领域。我融合了我的长 揭示细菌控制机制复杂性的历史，并具有开发和实施的能力 开拓待研究领域的新颖的全球技术和扩展研究结果的计算方法 超越模式生物。目前的赠款探索了三个重要且相关的领域。 首先，在我们开发的两种新颖的 CRISPRi 策略的推动下，我们继续寻求确定 通过探索三组正在研究的基因来了解细胞构建原理：细胞包膜基因、必需基因 基因，以及加速生长转变的基因。我们解决了阻止功能冗余的问题 使用双 CRISPRi 对包膜进行遗传分析，该技术可以同时敲除 通过相邻编码的 sgRNA 来连接两个基因。我们揭示了基本表达背后的权衡 CRISPRi 错配的基因，它利用 sgRNA 碱基配对区域中的单个错配来 可预测地滴定其功效。通过测量分级击倒对健康的影响，我们确定 必需基因的表达-适应性关系以及它们如何受到环境和遗传的影响 变化。最后，我们确定了加速生长转变的必需和非必需基因。 其次，我们继续研究控制翻译输出的一般原则 探索核糖体本身对转录上游过程的影响程度 （转录/翻译耦合）和 mRNA 降解的下游过程，并通过确定 在应激条件下产生的替代核糖体蛋白是否会产生新的翻译特性。 这些研究是通过我们为核糖体全基因组测量而开发的新技术来实现的 间距和 mRNA 降解。 第三，我们已经开始了一项令人兴奋的新研究，研究整个细菌的基因调控网络。 王国。这项努力是由我们新的严格的统计、系统发育足迹方法推动的，我们 经验证具有低误报率以及高召回率和精确率。我们计划利用 现有的大量细菌基因组数据库来检查细菌基因调控网络的进化。 我们的研究还解决了当前面临的巨大挑战：开发实验和 计算方法使研究人员能够全面探索监管线路和 在各种快速变化的环境中茁壮成长的细菌的功能多样性。这样的做法 可以协同并利用宏基因组数据来对基因功能进行机械询问 未充分研究的生物体。