Investigating the contextual essentiality of the Escherichia coli efflux system

外排泵是一种跨膜蛋白质，能主动地将分子跨膜运输到细胞外环境。细菌外排泵因参与抗生素耐药而受到广泛关注。尽管存在这种联系，但越来越多的证据表明，这些蛋白质在细菌生理学中也发挥着重要作用。在新陈代谢过程中，生物体会产生废物代谢物，如果它们达到细胞内的高水平，就可能是有毒的。据推测，外排泵负责清除有毒的废物代谢物。此外，也有证据表明，外排泵参与了pH动态平衡。以大肠杆菌作为模式生物，该研究计划旨在全面了解外排泵在不同环境和遗传环境中的生理作用，特别是它们与新陈代谢和细胞动态平衡的联系。这项建议的目标1是描述我们观察到的两个外排泵之间的条件合成-病态相互作用。当大肠杆菌在营养受限的培养基中生长时，两个主要促进剂外排泵的缺失会导致严重的生长缺陷。我们假设，这些外排泵的丧失会导致有毒代谢物的积累和/或细胞无法调节细胞质的pH。为了研究这一假设，我们将整合转录-代谢分析，并评估该菌株的细胞内pH。通过研究这种条件合成-病态相互作用的基础，我们将深入了解外排泵在细菌生理学中的重要性。目的2是首次在系统水平上研究不同环境和遗传条件下大肠杆菌外排泵的重要性。通过描述各种各样的生长条件，结合广泛的遗传操作，我们将确立大肠杆菌外排家族的背景重要性。我们将使用目标1中描述的转录/代谢组学方法，结合pH耐受性和表型分析的变化评估，来表征任何条件必需的基因和遗传交互作用。这些研究将提供外排代谢废物底物、外排丧失引起的细菌应激反应以及系统水平上对参与细胞动态平衡的外排泵的了解。尽管大肠杆菌是一种具有良好特性的有机体，但关于外排泵的生理作用仍然存在一些基本问题。这项研究旨在解决这些问题，并将提供第一个系统水平的调查，背景的重要性的大肠杆菌外排家族。这些信息将有助于我们更广泛地了解细菌生理学，并将为这些蛋白质的起源提供重要的见解，这可能适用于其他细菌物种和生物体。

Efflux pumps are membrane-spanning proteins that actively transport molecules across membranes to the extracellular environment. Bacterial efflux pumps have received significant attention due to their involvement in antibiotic resistance. Despite this association, a growing body of evidence suggests that these proteins also play important roles in bacterial physiology. During metabolism, organisms produce waste metabolites that can be toxic if they reach high intracellular levels. It has been hypothesized that efflux pumps are responsible for the removal of toxic waste metabolites. Furthermore, there is also evidence that efflux pumps are involved in pH homeostasis. Using Escherichia coli as a model organism, the proposed research program is designed to provide a holistic understanding of the physiological roles of efflux-pumps in different environmental and genetic contexts, with a particular focus on their connection to metabolism and cellular homeostasis. Aim 1 of this proposal is the characterization of a conditional synthetic-sick interaction that we have observed between two efflux pumps. Deletion of two major facilitator efflux pumps causes severe growth defects when E. coli is grown in a nutrient-limited medium. We hypothesize that the loss of these efflux pumps leads to the accumulation of toxic metabolites and/or an inability of the cell to regulate cytoplasmic pH. To investigate this hypothesis we will integrate transcriptomic-metabolomic analyses and we will assess the intracellular pH of the strain. By investigating the basis of this conditional synthetic-sick interaction, we will gain insight into the essentiality of efflux pumps in bacterial physiology. Aim 2 is the first systems-level study of E. coli efflux pump essentiality in different environmental and genetic conditions. By profiling a wide variety of growth conditions in combination with extensive genetic manipulation, we will establish the contextual essentiality of the E. coli efflux family. We will characterize any conditionally essential genes and genetic interactions using the transcriptomic/metabolomic approach described in Aim 1, combined with an assessment of changes in pH tolerance and phenotypic analysis. Such studies will provide a repertoire of efflux metabolic waste substrates, bacterial stress responses induced by the loss of efflux, and a systems-level understanding of the efflux pumps involved in cellular homeostasis. Despite E. coli being a well-characterised organism, fundamental questions remain regarding the physiological roles of efflux pumps. This study is designed to address these questions and will provide the first systems-level investigation into the contextual essentiality of the E. coli efflux family. Such information will contribute to our broader understanding of bacterial physiology and will offer important insight into the origins of these proteins, which could be applicable to other bacterial species and organisms.