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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Ikeda, Richard A
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University of Illinois Urbana-Champaign
Schools of Arts and Sciences
United States
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Lu, Zheng; Sethu, Ramakrishnan; Imlay, James A (2018) Endogenous superoxide is a key effector of the oxygen sensitivity of a model obligate anaerobe. Proc Natl Acad Sci U S A 115:E3266-E3275
Li, Xin; Imlay, James A (2018) Improved measurements of scant hydrogen peroxide enable experiments that define its threshold of toxicity for Escherichia coli. Free Radic Biol Med 120:217-227
Khademian, Maryam; Imlay, James A (2017) Escherichia coli cytochrome c peroxidase is a respiratory oxidase that enables the use of hydrogen peroxide as a terminal electron acceptor. Proc Natl Acad Sci U S A 114:E6922-E6931
Lu, Zheng; Imlay, James A (2017) The Fumarate Reductase of Bacteroides thetaiotaomicron, unlike That of Escherichia coli, Is Configured so that It Does Not Generate Reactive Oxygen Species. MBio 8:
Imlay, James A (2015) Transcription Factors That Defend Bacteria Against Reactive Oxygen Species. Annu Rev Microbiol 69:93-108
Mancini, Stefano; Imlay, James A (2015) Bacterial Porphyrin Extraction and Quantification by LC/MS/MS Analysis. Bio Protoc 5:
Mancini, Stefano; Imlay, James A (2015) The induction of two biosynthetic enzymes helps Escherichia coli sustain heme synthesis and activate catalase during hydrogen peroxide stress. Mol Microbiol 96:744-63
Imlay, James A (2015) Diagnosing oxidative stress in bacteria: not as easy as you might think. Curr Opin Microbiol 24:124-31
Imlay, James A (2014) The mismetallation of enzymes during oxidative stress. J Biol Chem 289:28121-8
Sobota, Jason M; Gu, Mianzhi; Imlay, James A (2014) Intracellular hydrogen peroxide and superoxide poison 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase, the first committed enzyme in the aromatic biosynthetic pathway of Escherichia coli. J Bacteriol 196:1980-91

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