RUI: Acid and Base Stress in Escherichia coli

摘要酸和碱中的生长对细菌在土壤和水环境中的生存非常重要，在土壤和水环境中，pH变化会导致毒力的变化，在人类宿主中也是如此。酸、碱可诱导多种多药耐药基因。肠道中依赖于pH的新陈代谢会产生酸和多胺，也就是说碱，它们的吸收促进了肿瘤的发生。在发酵工业中，pH对生长和生产起着至关重要的作用。氢酶的pH调节对生物生产氢燃料具有重要意义。人们假设，表达对pH变化作出反应的基因通过几种机制促进pH动态平衡和极端pH生存。该项目将表征一些假定的pH应激反应，这些反应来自对全球pH应激的DNA阵列研究，比较了好氧和厌氧培养。这些阵列揭示了一组在酸或碱中上调的“核心pH基因”，其中30个在酸转移实验中表现出快速反应。内膜蛋白基因yagU就是一个例子。对于yagU和其他核心pH基因，将对敲除突变体和高产克隆进行筛选，以便在中等酸碱(pH 5或pH 8.5)中厌氧生长，以及在极端酸或极端碱(pH 2.0或pH 9.8)中生存。另一类重要的pH依赖基因是氢酶，在厌氧条件下，氢酶在酸性条件下上调，但在曝气条件下，氢酶在碱性条件下上调。假设氢酶在高pH条件下将H2转化为2H+，并可能通过反向反应去除酸性。氢的产生和消耗，在低pH和高pH的情况下，将通过微量呼吸测量法进行测试。氢酶突变株将被测试在低pH和高pH条件下对生长和存活的影响。通过当前NSF资助的项目，基于pH可滴定的YFP和GFP报告基因融合，将使用一种新的细菌pH测量方法来表征突变的pH动态平衡。该项目将通过继续由本科生开展的创新研究项目，为国家的人力资源做出贡献。本科生撰写他们自己的迷你提案，设计和进行实验，并参与撰写报告以供发表。大多数被当前项目工作所吸引的本科生决定从事科学职业。在更广泛的社区中，PI举办关于科学教育的中学教师讲习班，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.