RUI: Acid and Base Stress in Escherichia coli

RUI：大肠杆菌中的酸和碱胁迫

• 批准号: 9982437
• 负责人: Joan Slonczewski
• 金额: $ 37万
• 依托单位: Kenyon College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9982437&HistoricalAwards=false
• 关键词: RUI; Acid; Base; Stress; Escherichia

Bacteria can grow at a wide range of pH values. Acid and base resistance play important roles in survival in soil and aquatic environments, where pH may vary drastically. Neutrophiles such as Escherichia coli are particularly interesting because they can maintain internal pH homeostasis (pH 7.3-7.8) during growth at pH values either more acidic (as low as pH 4.5) or more alkaline (as high as pH 9.2) than their intracellular pH. At even greater extremes of acid (pH 2) or base (pH 10), E. coli can retain viability after many hours in a stationary, non-growing state; this phenomenon is termed acid resistance, or base resistance, respectively. A number of genetic systems enable E. coli and related enteric organisms to maintain internal pH and reverse external acidification. The amino acid decarboxylases produce basic amines to neutralize acidity. The Na/H antiporter exchanges sodium ion for hydronium ion at alkaline pH. Acid and base resistance systems connect with responses to other stress conditions such as anaerobiosis, oxidative stress, and stationary phase. Yellowstone National Park (YNP) represents a unique setting wherein significant geothermal activity occurs. These thermal areas include aquatic systems as well as soils, and vary significantly with respect to temperature, chemistry, and physical properties. There is an exceptional opportunity to observe, follow, and quantify changes in a soil microbial population that occur in response to elevated temperature. Typically, environments that are the focus of thermophile investigations are mature, established geothermal features (relative to the human experience and known records). This preemptive study will take advantage of naturally occurring temperature gradients that have recently surfaced across the landscape at one specific location in YNP. These recent changes provide a rare opportunity, whereby thermophiles and/or the development of thermophile communities can be studied in real time. As opposed to other neothermal environments such as deep sea vents which are logistically difficult to access, the research site is easily accessible and sampled. Using a combination of molecular and culturing techniques, the microbial community in this evolving thermal environment is being studied over time, with apparent alterations in community structure being correlated with changes in soil temperature and chemical properties. Several E. coli proteins not previously known to be pH-dependent have recently been shown to exhibit pH-dependent expression in two-dimensional electrophoretic gels (2-D gels). In this project, the response of these proteins to pH, and their role in survival at extreme pH, will be characterized genetically, using lac fusion reporters and null mutants constructed by allelic replacement. One protein expressed only in acid is YfiD, a homolog of pyruvate formate-lyase. The expression of yfiD::lac will be observed as a function of pH and of permeant acids which depress internal pH. A yfiD null mutant will be tested for survival in extreme acid. A protein induced by base is TnaA, tryptophan deaminase, becoming one of the most abundant proteins of the cell at high pH. This observation confirms the prediction that amino acid deaminases are induced to help neutralize alkaline growth media. Expression of tnaA::lac, and of other amino acid deaminases, will be tested for pH dependence. The role of tnaA in neutralizing growth media will be tested. The pH responses of the glutamate decarboxylase (GadA, GadB) and of alkyl hydroperoxide reductase (AhpC) will also be investigated. The project will also continue proteomic investigation of the connections between pH stress, anaerobiosis and stationary phase. The connections between pH and other stresses are known, but until recently these connections have been little studied. Proteins expressed under various combinations of these stress conditions will be separated using high-resolution 2-D gels. Proteins showing pH-dependent responses will be identified by N-terminal sequence and matched to the E. coli genomic sequence. This project will enhance our understanding of the diverse mechanisms of bacterial response to pH, and the ways in which bacteria both maintain their own pH homeostasis and control the external pH of their environment. The project will contribute to the nation's human resources by continuing a successful research program involving undergraduates, many of whom are encouraged to pursue careers in science.

细菌可以在很宽的 pH 值范围内生长。 耐酸碱能力对于土壤和水生环境中的生存起着重要作用，因为土壤和水生环境的 pH 值可能变化很大。 大肠杆菌等中性粒细胞特别令人感兴趣，因为它们在生长过程中可以在比细胞内 pH 值更酸性（低至 4.5）或更碱性（高达 pH 9.2）的 pH 值下维持内部 pH 稳态（pH 7.3-7.8）。 在更极端的酸（pH 2）或碱（pH 10）条件下，大肠杆菌可以在静止、非生长状态数小时后保持活力；这种现象分别称为耐酸或耐碱。 许多遗传系统使大肠杆菌和相关肠道生物能够维持内部 pH 值并逆转外部酸化。 氨基酸脱羧酶产生碱性胺以中和酸性。 Na/H 反向转运蛋白在碱性 pH 值下将钠离子交换为水合氢离子。 耐酸和耐碱系统与对其他应激条件（例如厌氧、氧化应激和稳定期）的反应有关。黄石国家公园 (YNP) 具有独特的环境，这里发生着显着的地热活动。 这些热区包括水生系统和土壤，并且在温度、化学和物理特性方面差异很大。 这是观察、跟踪和量化土壤微生物种群因温度升高而发生的变化的绝佳机会。 通常，嗜热研究的重点环境是成熟的、已确定的地热特征（相对于人类经验和已知记录）。 这项先发制人的研究将利用最近在 YNP 某个特定位置的景观中自然发生的温度梯度。 这些最近的变化提供了一个难得的机会，可以实时研究嗜热菌和/或嗜热菌群落的发展。 与其他新热环境（例如在后勤上难以进入的深海喷口）相反，该研究地点很容易进入和取样。 结合分子和培养技术，随着时间的推移，人们正在研究这种不断变化的热环境中的微生物群落，群落结构的明显变化与土壤温度和化学性质的变化相关。 最近发现，一些以前不知道具有 pH 依赖性的大肠杆菌蛋白在二维电泳凝胶（2-D 凝胶）中表现出 pH 依赖性表达。 在该项目中，将使用 lac 融合报告基因和通过等位基因替换构建的无效突变体，从遗传学上表征这些蛋白质对 pH 的响应以及它们在极端 pH 下生存的作用。 YfiD 是一种仅在酸中表达的蛋白质，它是丙酮酸甲酸裂解酶的同源物。 yfiD::lac 的表达将被观察为 pH 值和降低内部 pH 值的渗透酸的函数。 将测试 yfiD 无效突变体在极端酸中的存活情况。 碱诱导的蛋白质是 TnaA（色氨酸脱氨酶），在高 pH 条件下成为细胞中最丰富的蛋白质之一。 这一观察证实了氨基酸脱氨酶被诱导以帮助中和碱性生长培养基的预测。将测试 tnaA::lac 和其他氨基酸脱氨酶的表达的 pH 依赖性。 将测试 tnaA 在中和生长培养基中的作用。 还将研究谷氨酸脱羧酶（GadA、GadB）和烷基氢过氧化物还原酶（AhpC）的 pH 响应。 该项目还将继续对 pH 胁迫、厌氧和稳定期之间的联系进行蛋白质组学研究。 pH 值和其他压力之间的联系是已知的，但直到最近这些联系还很少被研究。 在这些应激条件的各种组合下表达的蛋白质将使用高分辨率二维凝胶进行分离。 显示 pH 依赖性反应的蛋白质将通过 N 端序列进行鉴定，并与大肠杆菌基因组序列进行匹配。 该项目将加深我们对细菌对 pH 值反应的多种机制，以及细菌维持自身 pH 稳态和控制其环境外部 pH 值的方式的理解。 该项目将通过继续一项涉及本科生的成功研究计划，为国家人力资源做出贡献，其中许多人被鼓励从事科学事业。