# The physiology of oxidative stress in Escherichia coli

> **NIH NIH R01** · UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN · 2020 · $550,135

## Abstract

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 centra...

## Key facts

- **NIH application ID:** 9961608
- **Project number:** 5R01GM049640-27
- **Recipient organization:** UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
- **Principal Investigator:** JAMES A. IMLAY
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $550,135
- **Award type:** 5
- **Project period:** 1994-05-01 → 2021-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9961608

## Citation

> US National Institutes of Health, RePORTER application 9961608, The physiology of oxidative stress in Escherichia coli (5R01GM049640-27). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9961608. Licensed CC0.

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