A goal of our field is to develop a complete molecular and physiological model of intracellular oxidative stress. We want to know how superoxide and hydrogen peroxide are formed inside cells, which biomolecules they damage, and the strategies by which cells defend themselves against them. We are currently trying to solve these problems in Escherichia coli. This model organism provides unique experimental advantages, including the ability to generate and culture defenseless mutants in the absence of oxygen. In the past 15 years enormous strides were made in understanding superoxide stress, largely from the analysis of an E. coli mutant that lacked superoxide dismutase. We have recently generated an analogous strain that cannot scavenge hydrogen peroxide. This mutant has allowed us to quantify the rate of intracellular H202 formation and to detect the growth and survival defects that this H202 causes. In this application we propose to exploit this and other mutants, as well as a battery of experimental methodologies that were developed for E. coli, in order to nail down the details of oxidative stress mechanisms and defenses. (1) We will pinpoint the sites at which H202 is formed in the cytosol and 02- is formed in the eriplasm of intact, aerobic cells.(2) We will determine the mechanisms by which H202 inhibits biosynthesis in log-phase cells and kills early-stationary-phase cells.(3) We will identify mechanisms by which Dps protects DNA and an undefined factor protects the iron-sulfur cluster of aconitase A. We believe that knowledge of oxidative damage and defense in E. coli will provide a blueprint for efforts to solve key issues in oxidative stress: obligate anaerobiosis, the killing mechanism of) hagocytes, and endogenous oxidative stress in higher organisms.
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