RUI: Acid and Base Stress in Escherichia coli

摘要酸碱环境对细菌在pH变化剧烈的土壤和水生环境中生存至关重要，在人类宿主中，pH变化有助于毒力的产生。酸和碱诱导多种耐药基因。肠道中ph依赖性代谢产生酸和多胺，这些渗透碱的摄取促进肿瘤发生。在发酵工业中，pH值对生长和生产起着至关重要的作用。氢化酶的pH调节对氢燃料的生物生产具有重要意义。据推测，其表达响应pH变化的基因通过几种机制有助于pH稳态和极端pH生存。该项目将描述一些假定的pH胁迫反应，这些反应来自DNA阵列研究，比较有氧和厌氧培养的全球pH胁迫。结果显示，在酸或碱中有一组“核心pH基因”上调，其中30个在酸移实验中表现出快速反应。细胞膜蛋白基因yagU就是一个例子。对于yagU和其他核心pH基因，将筛选敲除突变体和过量生产克隆，以便在中等酸性或碱性（pH 5或pH 8.5）中厌氧生长，以及在极端酸性或极端碱性（pH 2.0或pH 9.8）中存活。另一类依赖ph值的重要基因是氢化酶，它在厌氧环境下在酸性环境中被上调，而在曝气环境下在碱性环境中上调。假设氢化酶在高pH下将H2转化为2H+，并通过逆反应去除酸性。氢气的生产和消耗，在低pH值和高pH值，将通过微呼吸测定法进行测试。氢化酶突变体将测试在低pH和高pH下对生长和存活的影响。突变体将使用一种新的细菌pH测量方法来表征pH稳态，该方法是由目前美国国家科学基金会资助的项目设计的，基于pH可滴定的YFP和GFP报告基因融合。更广泛的影响。该项目将继续由大学生开展的创新研究项目，为国家的人力资源做出贡献。本科生撰写自己的小型提案，设计并进行实验，并参与撰写报告以供发表。大多数被当前项目吸引的本科生决定从事科学事业。在更广泛的社区，PI为中学教师举办科学教育讲习班，并向民间组织介绍微生物学的最新进展。

AbstractGrowth in acid and base is important for bacteria to survive in soil and aquatic environments, where pH varies drastically, as well as in the human host where pH change contributes to virulence. Acid and base induce numerous multidrug resistance genes. pH-dependent metabolism in the gut generates acids as well as polyamines, permeant bases whose uptake promotes tumorigenesis. In the fermentation industry, pH plays critical roles in growth and production. pH regulation of hydrogenase enzymes has implications for the biological production of hydrogen fuel.It is hypothesized that genes whose expression responds to pH change contribute to pH homeostasis and extreme-pH survival, by several mechanisms. The project will characterize a number of putative pH stress responses arising from DNA array studies of global pH stress comparing aerobic and anaerobic cultures. The arrays revealed a set of "core pH genes" up-regulated in acid or base, of which thirty showed rapid response in an acid-shift experiment. An example is the inner membrane protein gene yagU. For yagU and other core pH genes, knockout mutants and overproducing clones will be screened for anaerobic growth in moderate acid or base (pH 5 or pH 8.5), and for survival in extreme acid or extreme base (pH 2.0 or pH 9.8). Another important class of pH-dependent genes is the hydrogenases, which are up-regulated in acid with anaerobiosis, but up in base with aeration. It is hypothesized that hydrogenases convert H2 to 2H+ at high pH, and that they may remove acidity by the reverse reaction. H2 production and consumption, at low pH vs. high pH, will be tested by microrespirometry. Hydrogenase mutants will be tested for effects on growth and survival at low pH vs. high pH. Mutants will be characterized for pH homeostasis using a new method of bacterial pH measurement devised through the current NSF-funded project, based on pH-titratable YFP and GFP reporter gene fusions.Broader impacts. The project will contribute to the nation's human resources by continuing an innovative research program run by undergraduates. Undergraduates write their own mini-proposals, design and conduct the experiments, and participate in writing up the reports for publication. Most of the undergraduates attracted to work on the current project decide to pursue careers in science. In the wider community, the PI conducts middle-school teacher workshops on science education and addresses civic organizations on current advances in microbiology.