Cellular homeostasis pathways in bacteria

我研究的首要目标是揭示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。为了实现这一目标，我 开发了一个统一的以细菌为中心的研究愿景，跨越了经典定义的子领域。我把我的长 揭开细菌控制机制的复杂性的历史， 新的全球技术，以开辟研究不足的领域和计算方法，以扩大发现 超越模式生物。目前的赠款探索三个重要和相关的领域。 首先，在我们开发的两种新型CRISPRi策略的推动下，我们继续寻求识别 通过探索三组未充分研究的基因：细胞包膜基因，必需的 基因和加速生长转变的基因。我们解决了功能的冗余， 用双CRISPRi对包膜进行遗传分析，这是一种允许同时敲除 两个基因通过相邻编码的sgRNA。我们揭示了表达本质的权衡 具有错配CRISPRi的基因，其使用sgRNA的碱基配对区域中的单个错配， 可预测地滴定它们的功效。通过测量分级击倒的适应性影响，我们确定了 必需基因的表达-适合度关系以及它们如何受环境和遗传的影响 变化最后，我们确定了加速生长过渡的必需和非必需基因。 其次，我们继续我们的研究控制翻译输出的一般原则， 探索核糖体自身对转录上游过程的影响程度 （转录/翻译偶联）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导致新的翻译性质。 这些研究是由我们开发的用于核糖体全基因组测量的新技术实现的 间隔和mRNA降解。 第三，我们已经开始了一项令人兴奋的新研究，即在整个细菌中的基因调控网络。 王国这一努力是由我们新的统计严格，系统发育足迹的方法，我们 已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 庞大的现有细菌基因组数据库，以研究跨细菌的基因调控网络的进化。 我们的研究还解决了一个压倒性的当前挑战：开发实验性和 计算方法，使研究人员能够全面探索监管布线， 细菌的功能多样性，在各种快速变化的环境中茁壮成长。这样的方法 可以协同和利用宏基因组数据，使基因功能的机械询问， 未被充分研究的生物

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.