RUI: Acid and Base Stress in Escherichia coli

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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.