Adventitious disulfide bond formation in Escherichia coli and the involvement of thioredoxin and glutaredoxin in suppressing oxidative stress

Adventitious disulfide bond formation has long been believed to be a stress which bacteria have to deal with during their life cycle. The formation of accidental disulfide bonds can lead to the inactivation of enzymes with active site cysteine ligands and to toxic protein aggregates which physically impair the cell. With the advent of oxygen in the atmosphere a couple of billion years ago, the thiol groups of microbes were threatened by both oxidative stress and the copper that was released by oxygen from sediments. Researchers have long suggested that the accidental formation of disulfide bonds by ROS and copper is a real threat for bacteria. However, evidence of their in vivo toxicity towards thiol groups has been missing from the literature. This work aimed to fix this – copper and ROS have been evaluated as natural sources of adventitious disulfide bond formation. Further, we investigated the roles of Glutaredoxin 1 (Grx1) and Thioredoxin 2 (Trx2) during H2O2 stress. E. coli uses efflux systems to keep copper out of its cytoplasm as well as the periplasm. A main route of copper toxicity is through damage of Fe-S clusters of dehydratases. Yet, a growth phenotype persists when this damage is circumvented by the addition of branched-chain amino acids in the growth medium. Adventitious thiol oxidation of cytoplasmic proteins has long been hypothesized to comprise an additional mechanism of copper toxicity. Copper is redox-active and thiophilic and therefore has the properties needed to catalyze the chemistry. Nonetheless, this turned out not to be the case. Copper oxidized protein thiols in the periplasm but not in the cytoplasm. Our data suggest that the E. coli cytoplasm is too thiol-rich compared to the periplasm, and cytoplasmic copper is trapped in an inert Cu(I)-thiyl-radical complex. Oxygen, superoxide, and hydrogen peroxide have been investigated as candidates for adventitious disulfide bond formation in the cytoplasm. The transcription factor OxyR responds to hydrogen peroxide via disulfide bond formation, and the NADH peroxidase AhpC scavenges hydrogen peroxide through thiol redox that results in a disulfide bond. These examples indicate that hydrogen peroxide is a plausible candidate to create disulfide stress. Further, OxyR turns on two of the redoxins, Grx1 and Trx2. The role of the redoxins during H2O2 stress has yet to be fully elucidated. Three hypotheses have been experimentally investigated: the involvement that Grx1 and Trx2 reduce accidental disulfide bonds in cytoplasmic proteins; that they participate in the repair process of mononuclear iron enzymes; and that they help to repair Fe-S clusters. Our results argue against each of these hypotheses. We infer that these redoxins either have a distinct, undiscovered role, or else that they are needed under growth conditions other than the ones that we tested.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2023

Author dc:creator

• Eben, Stefanie Susanne

Contributors dc:contributor

• Imlay, James
• Cronan, John
• Vanderpool, Carin
• Kehl-Fie, Thomas

• Oxidative Stress
• Disulfide Stress
• Bacteria
• Thioredoxin
• Glutaredoxin

Statement dc:rights

• Copyright 2023 Stefanie Eben

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/122207

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F122207