RUI: Unraveling the physiological roles of multidrug efflux pumps in bacteria

该项目将确定大肠杆菌的多药外排(MDR)泵如何调节基因表达和细胞生理。外流是所有生命形式中的一种基本功能。革兰氏阴性细菌，如大肠杆菌，具有高度保守的MDR泵，在细胞生理学和细菌对内部和环境变化的适应中发挥关键作用。这些MDR泵具有一种独特的能力，可以将多种分子驱逐出细胞。这些分子包括从外部进入细胞的有毒化合物，如胆盐和抗生素；以及这些细菌在内部产生的分子，这些分子参与细菌生长、信号传递和捕获铁或其他有限营养物质等功能。在MDR泵中，大肠杆菌的AcrAB-TolC泵最为著名，因为它是该细菌中主要的MDR泵，是一种重要的模式生物、肠道共生菌、病原菌，在生物技术中应用广泛。这种泵最初被研究是因为它通过排出不同的抗生素来增强抗生素耐药性。最近的发现表明，该泵在调节基因表达和细菌生理方面也发挥着核心作用；然而，该泵如何控制这些功能仍不清楚。通过识别由AcrAB-TolC泵排出的大肠杆菌内部产生的分子，以及该泵及其调节器如何控制基因表达和生理，该项目将为科学和社会整体福祉做出多项重大贡献。更具体地说，这个项目将促进我们对细菌如何保持内部平衡和适应新环境的理解，并为如何生物工程细菌以改善生物燃料和其他工业化合物的生产提供新的见解。该项目更广泛的成果来自于它与加州州立大学北岭分校(CSUN)重新设计的微生物生理学课程的整合。CSUN主要是一个本科生和为少数群体服务的机构。因此，该项目将扩大来自不同背景的学生获得实践研究的机会，这对培养和激励下一代科学家和工程师至关重要。MDR泵调节基因表达、新陈代谢、应激反应、细菌运动和其他生理过程。然而，允许这些泵控制所有这些过程的分子机制仍然未知。特别耐人寻味的问题是，位于细胞膜中的多药耐药泵如何调节基因表达。我们的中心假设是，AcrAB-TolC泵的主要功能是外流细胞代谢物，而转录抑制因子AcrR感知不同泵底物的浓度，然后作为基因表达的全球调节因子。这一假设是基于国际和平研究所实验室和其他组织的最新发现。为了达到总体目标，我们将追求两个具体目标：1)确定AcrR直接调控的基因和控制AcrR功能的代谢物；2)确定与AcrAB-TolC结合的代谢物以及AcrB-AcrZ相互作用在调节AcrB-TolC功能中的作用。我们将通过生物信息学和电泳迁移率改变分析来鉴定AcrR直接调控的基因，以及荧光偏振结合分析来鉴定AcrAB-TolC排出的细胞代谢物，确定小蛋白AcrZ对该泵识别底物的影响，并确定哪些泵底物与AcrR结合并调节AcrR功能。最终，该项目将通过确定哪些细胞代谢物既是泵底物又是AcrR配体，揭示泵功能和基因调控之间的功能联系。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

This project will determine how a multidrug efflux (MDR) pump of the bacterium Escherichia coli, regulates gene expression and cell physiology. Efflux is an essential function in all life forms. Gram-negative bacteria such as E. coli possess MDR pumps that are highly-conserved and play a pivotal role in cell physiology and bacterial adaptation to internal and environmental changes. These MDR pumps have a unique ability to expel a wide range of molecules out of the cell. These molecules include toxic compounds entering the cells from the outside such as bile salts and antibiotics; as well as molecules internally produced by these bacteria involved in functions such as bacterial growth, signaling, and capturing iron or other limited nutrients. Among MDR pumps, the AcrAB-TolC pump of E. coli is the most well-known because it is the main MDR pump in this bacterium, which is an important model organism, gut commensal, pathogen, and is widely used in biotechnology. This pump was first studied because of its role in conferring antibiotic resistance by effluxing different antibiotics. Recent findings indicate that this pump also plays a central role in regulating gene expression and bacterial physiology; however, how this pump controls these functions remains unknown. By identifying the molecules internally produced by E. coli that are effluxed by the AcrAB-TolC pump, and how this pump and its regulators control gene expression and physiology, this project will make multiple significant contributions to science and the overall well-being of society. More specifically, this project will advance our understanding of how bacteria maintain their internal balance and adapt to new environments, and offer novel insights into how to bioengineer bacteria to improve the production of biofuels and other compounds of industrial interest. The broader outcomes of this project stem from its integration into a re-designed Microbial Physiology course at California State University, Northridge (CSUN). CSUN is a primarily undergraduate and minority-serving institution. Thus, this project will expand the number of students from diverse backgrounds that have access to hands-on research, which is essential to train and inspire the next generation of scientist and engineers.MDR pumps regulate gene expression, metabolism, stress responses, bacterial motility, and other physiological processes. However, the molecular mechanisms that allow these pumps to control all these processes remain unknown. Especially intriguing is the question of how MDR pumps, which are located in the cell envelope, can regulate gene expression. Our central hypothesis is that the main function of the AcrAB-TolC pump is to efflux cellular metabolites, whereas the transcriptional repressor AcrR senses the concentrations of different pump substrates and then acts as a global regulator of gene expression. This hypothesis is based on recent findings from the PI’s laboratory and other groups. To attain the overall objective, we will pursue two specific aims: 1) Identify the genes directly regulated by AcrR and the metabolites that control AcrR function; and 2) Identify the metabolites that bind to AcrAB-TolC and the effect of AcrB-AcrZ interactions in modulating the function of this pump. We will accomplish these aims by employing bioinformatics and electrophoretic mobility shift assays to identify genes directly regulated by AcrR; as well as fluorescence polarization binding assays to identify the cellular metabolites effluxed by AcrAB-TolC, determine the effect of the small protein AcrZ on substrate recognition by this pump, and identify which pump substrates bind to and regulate AcrR function. Ultimately, this project will uncover the functional link between pump function and gene regulation by identifying which cellular metabolites are both pump substrates and AcrR ligands.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.