RUI: Acid and Base Stress in Escherichia coli

细菌可以在广泛的pH值范围内生长。在土壤和水环境中，耐酸和耐碱对生存起着重要作用，在这些环境中，pH可能会有很大的变化。中性粒细胞，如大肠杆菌，特别有趣，因为它们可以在比细胞内pH更酸性(低至pH 4.5)或更碱性(高至pH 9.2)的pH值下生长过程中保持内部pH稳态(pH 7.3-7.8)。在更极端的酸(PH 2)或碱(PH 10)中，大肠杆菌在静止、不生长的状态下仍能在数小时后保持活力；这种现象分别称为耐酸性或耐碱性。许多基因系统使大肠杆菌和相关的肠道生物能够维持内部pH并逆转外部酸化。氨基酸脱羧酶产生碱性胺来中和酸性。在碱性条件下，Na/H逆向转运体将钠离子交换为氢离子。耐酸和耐碱系统与对其他应激条件的反应有关，如厌氧、氧化应激和静止期。黄石国家公园(YNP)代表了一个独特的环境，在那里发生了重大的地热活动。这些热区包括水生系统和土壤，在温度、化学和物理性质方面差异很大。有一个特殊的机会来观察、跟踪和量化土壤微生物种群因温度升高而发生的变化。通常，作为嗜热者调查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YNP一个特定地点最近出现的自然温度梯度。最近的这些变化提供了一个难得的机会，借此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，如深海喷口在后勤上难以进入相反，研究地点很容易进入和采样。利用分子和培养技术的结合，随着时间的推移，人们正在研究这种不断演变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，一些未知的pH依赖的大肠杆菌蛋白在双向凝胶(2-D Gel)中表现出pH依赖的表达。在这个项目中，这些蛋白质对pH的反应，以及它们在极端pH下生存中的作用，将使用Lac融合报告和通过等位基因替换构建的零突变来进行遗传学表征。一种只在酸中表达的蛋白质是YfiD，它是丙酮酸甲酸裂解酶的同源物。YfiD：：Lac的表达将被观察到作为pH和降低内部pH的指导酸的函数。将测试一个yfiD缺失突变体在极端酸性环境中的存活情况。碱诱导的蛋白质是TNAA，色氨酸脱氨酶，在高pH条件下成为细胞中含量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介质的预测。TNAA：：Lac和其他氨基酸脱氨酶的表达将进行pH依赖性测试。将测试TNAA在中和生长介质中的作用。谷氨酸脱羧酶(GADA，GADB)和烷基过氧化氢还原酶(AhpC)的pH响应也将被研究。该项目还将继续对pH应激、厌氧和静止相之间的联系进行蛋白质组学研究。PH和其他压力之间的联系是已知的，但直到最近，对这些联系的研究还很少。在这些胁迫条件的不同组合下表达的蛋白质将使用高分辨率的二维凝胶进行分离。具有pH依赖性反应的蛋白质将通过N末端序列进行鉴定，并与大肠杆菌基因组序列相匹配。这个项目将加深我们对细菌对pH反应的不同机制的理解，以及细菌如何维持自己的pH动态平衡和控制环境的外部pH的方式。该项目将通过继续一个涉及本科生的成功研究项目来为国家的人力资源做出贡献，其中许多本科生被鼓励从事科学事业。

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.