Oxidative stress has a large imprint upon biology: It determines the structure of microbial communities, is central to the action of the innate immune system, and is suspected of underlying a variety of human pathologies. Several basic questions frame the field: How are oxidants formed, and in what quantities? What are the specific biomolecules that they damage most rapidly? How do cells defend themselves? Why does oxidant sensitivity differ among organisms? Mechanistic details have primarily emerged from studies of the model bacterium E. coli. This application proposes to deepen and broaden the understanding. To date the reactive oxygen species (ROS) O2 and H2O2 are known to disrupt growth primarily by inactivating cluster-dependent dehydratases and non- - redox mononuclear iron enzymes. Dehydratases are vulnerable in virtually all organisms. However, it appears that the mononuclear enzymes of some aerobes acquired resistance by employing divalent metals other than iron. Such an evolutionary adaptation would be fascinating, as it would recapitulate a tactic that E. coli invokes during oxidative stress.
Aim One will test whether this substitution is driven by changes in the intracellular metal pools or by modifications of the enzymes, and it will probe whether the shift in metallation exerts a cost in catalytic efficiency.
Aim Two investigates the hypothesis that radical SAM enzymes comprise a novel third class of ROS- sensitive enzyme. Circumstantial data support the idea: they employ over-oxidizable peripheral iron-sulfur clusters, and E. coli responds to stress by replacing one of these enzymes with a cluster-free analogue. Yet those clusters might plausibly be protected in vivo either by bound SAM or by rapid re-reduction via their native electron donor. If this enzyme family is ROS-sensitive, then stress will affect a broad range of cell processes. Transcriptomic analyses have revealed surprising strategies by which E. coli copes with ROS, and Aim Three probes two newly discovered ones. First, the ClpSA and ClpX unfoldases somehow stabilize branched- chain biosynthesis during periods of H2O2 stress. The mechanism may be that by acting as chaperones the Clp proteins protect apo-dehydratases from aggregation and/or proteolysis. Thus damaged clusters can be re- built. Second, the data reveal that H2O2-stressed cells induce exonuclease III, a key enzyme that is specifically required to repair oxidative DNA lesions. The work will determine how its expression is controlled, whether other repair enzymes are co-regulated, and which enzymes are needed to allow growth in the face of environmental H2O2. Finally, Aim Four extends prior investigations of the molecular basis of obligate anaerobiosis. Previous work identified two key enzymes that are poisoned upon aeration of a model anaerobe; the next step is to resolve the underlying mechanisms. As proof of principle, this Aim will culminate in an effort to construct a derivative strain whose central metabolism is not blocked by oxygen. These projects are selected to fill in key pieces of the puzzle of oxidative stress. They are closely interconnected, with the mechanistic theme being the incompatibility of oxygen and iron-centered metabolism.
Each Aim derives directly from data obtained during the current funding period.

Public Health Relevance

. Oxidative stress is a source of human pathology, but its molecular nature is not fully understood. Using model bacteria, we will identify the principle cell processes that oxidants disrupt and the defenses that permit aerobic life. Such information may contribute to our ability to manipulate microbial communities and to foster drugs that improve human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM049640-25
Application #
9514997
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Barski, Oleg
Project Start
1994-05-01
Project End
2021-06-30
Budget Start
2018-07-01
Budget End
2019-06-30
Support Year
25
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Illinois Urbana-Champaign
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
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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