Cellular homeostasis pathways in bacteria

我研究的首要目标是揭示推动细菌成功的基本原理， 使它们能够在这个星球的每一个角落殖民和繁殖。为了实现这一目标，我有 制定了一个跨越经典定义的子领域的以细菌为中心的统一研究愿景。我把我的长发 具有开发和实施能力的揭开细菌控制机制复杂性的历史 开拓研究不足领域的全球新技术和推广研究成果的计算方法 超越了模型生物。目前的赠款探索了三个重要和相关的领域。 首先，在我们开发的两种新的CRISPRi策略的推动下，我们继续寻找 探索三组未被研究的基因的细胞构建原理：细胞被膜基因，必需的 基因，以及加速生长过渡的基因。我们解决了职能的冗余问题，防止了 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sgRNAs。我们解开了Essential表达背后的权衡 CRISPRi不匹配的基因，它使用sgRNAs碱基配对区域中的单个错配来 可以预见的是，滴定它们的功效。通过测量分级击倒对健康的影响，我们确定了 必需基因的表达-适合度关系及其受环境和遗传的影响 改变。最后，我们确定了加速生长过渡的必需基因和非必需基因。 其次，我们继续研究控制翻译输出的一般原则，包括 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 在胁迫条件下产生的替代核糖体蛋白是否会产生新的翻译特性。 我们为全基因组核糖体测量开发的新技术使这些研究成为可能 间距和信使核糖核酸降解。 第三，我们已经开始了一项令人兴奋的新研究，研究遍及细菌的基因调控网络。 王国。这一努力是由我们新的统计上严格的系统进化足迹方法推动的，我们 已被证实具有低的假阳性率，以及高的召回率和准确率。我们计划利用 庞大的现有细菌基因组数据库，以检查细菌间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。这样的方法 可以与元基因组数据协同并利用元基因组数据来支持对基因功能的机械性询问 未被充分研究的生物体。

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.