RUI: Acid and Base Stress in Escherichia coli

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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.