RUI: Acid and Base Stress in Escherichia coli

细菌可以在很宽的pH值范围内生长。 在pH值变化剧烈的土壤和水生环境中，耐酸碱性对生存起着重要作用。 嗜中性生物如大肠杆菌是特别令人感兴趣的，因为它们在比其细胞内pH更酸性（低至pH 4.5）或更碱性（高至pH 9.2）的pH值下生长期间可以维持内部pH稳态（pH 7.3-7.8）。大肠杆菌可以在静止的非生长状态下保持数小时后的活力;这种现象分别称为耐酸性或耐碱性。 许多遗传系统使E.大肠杆菌和相关的肠道微生物，以维持内部pH值和逆转外部酸化。 氨基酸脱羧酶产生碱性胺来中和酸性。 Na/H逆向转运蛋白在碱性pH值下将钠离子交换为水合氢离子。酸和碱抗性系统与对其他应激条件（如厌氧、氧化应激和固定相）的响应相关联。黄石国家公园（YNP）代表了一个独特的环境，其中发生了重大的地热活动。 这些热区域包括水生系统以及土壤，并且在温度、化学和物理性质方面变化显著。 有一个特殊的机会来观察，跟踪和量化土壤微生物种群的变化，以应对温度升高。 通常，作为嗜热菌研究重点的环境是成熟的、已建立的地热特征（相对于人类经验和已知记录）。 这项先发制人的研究将利用自然发生的温度梯度，最近出现在整个景观在一个特定的位置在YNP。 这些最近的变化提供了一个难得的机会，从而嗜热菌和/或嗜热菌群落的发展可以在真实的时间进行研究。 与后勤上难以进入的深海喷口等其他深海环境不同，研究地点很容易进入和取样。 使用分子和培养技术相结合，随着时间的推移，在这个不断变化的热环境中的微生物群落正在研究，与明显的变化，在社区结构与土壤温度和化学性质的变化。 几个E.先前不知道是pH依赖性的大肠杆菌蛋白最近已显示在二维电泳凝胶（2-D凝胶）中表现出pH依赖性表达。 在这个项目中，这些蛋白质的pH值的响应，以及它们在极端pH值下的生存作用，将在遗传上进行表征，使用乳糖融合报告基因和无效突变体构建的等位基因置换。 一种仅在酸中表达的蛋白质是YfiD，其是丙酮酸甲酸裂解酶的同系物。 将观察yfiD：：lac的表达作为pH和抑制内部pH的渗透酸的函数。将测试yfiD无效突变体在极端酸中的存活。 由碱诱导的蛋白质是TnaA，色氨酸脱氨酶，在高pH下成为细胞中最丰富的蛋白质之一。这一观察结果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。 谷氨酸脱羧酶（GadA，GadB）和烷基过氧化氢还原酶（AhpC）的pH响应也将进行研究。 该项目还将继续对pH应激、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，这些联系很少被研究。 将使用高分辨率2-D凝胶分离在这些应激条件的各种组合下表达的蛋白质。 显示pH依赖性反应的蛋白质将通过N-末端序列鉴定并与E. coli基因组序列。 该项目将提高我们对细菌对pH反应的不同机制的理解，以及细菌保持自身pH稳态和控制其环境外部pH的方式。 该项目将通过继续一个成功的涉及本科生的研究计划，为国家的人力资源做出贡献，鼓励他们中的许多人从事科学事业。

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.