c Formation of nonnative disulfide bonds in the cytoplasm, so-called disulfide stress, is an integral component of oxidative stress. Quantification of the extent of disulfide bond formation in the cytoplasm of Escherichia coli revealed that disulfide stress is associated with oxidative stress caused by hydrogen peroxide, paraquat, and cadmium. To separate the impact of disulfide bond formation from unrelated effects of these oxidative stressors in subsequent experiments, we worked with two complementary approaches. We triggered disulfide stress either chemically by diamide treatment of cells or genetically in a mutant strain lacking the major disulfide-reducing systems TrxB and Gor. Studying the proteomic response of E. coli exposed to disulfide stress, we found that intracellular disulfide bond formation is a particularly strong inducer of the heat shock response. Real-time quantitative PCR experiments showed that disulfide stress induces the heat shock response in E. coli 32 dependently. However, unlike heat shock treatment, which induces these genes transiently, transcripts of 32 -dependent genes accumulated over time in disulfide stress-treated cells. Analyzing the stability of 32 , we found that this constant induction can be attributed to an increase of the half-life of 32 upon disulfide stress. This is concomitant with aggregation of E. coli proteins treated with diamide. We conclude that oxidative stress triggers the heat shock response in E. coli 32 dependently. The component of oxidative stress responsible for the induction of heat shock genes is disulfide stress. Nonnative disulfide bond formation in the cytoplasm causes protein unfolding. This stabilizes 32 by preventing its DnaK-and FtsH-dependent degradation.O xidative stress is defined as an imbalance between the generation of reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide, and hydroxyl radicals, and their detoxification by cellular antioxidant systems. ROS have the potential to attack virtually any cellular macromolecule, including DNA, lipids, and proteins. To counteract the damaging effects, microorganisms respond to oxidative stress in various ways. The classical oxidative stress response is regulated by transcription factors, such as OxyR from Escherichia coli, OhrR from Bacillus subtilis, and Yap1 from Saccharomyces cerevisiae, which all act as reversible redox switches (1-3). Activated by the oxidation of highly conserved cysteines, these regulators induce the expression of antioxidant enzymes, such as peroxiredoxins, glutaredoxins, and thioredoxins.A separate class of transcription factors which specifically sense disulfide stress has been reported in a number of organisms. These regulators are activated when the thiol-disulfide balance is perturbed, resulting in the formation of inter-and intramolecular disulfides within proteins or thiolation of proteinogenic amino acids with small-molecule thiols such as glutathione. This condition may be caused by an accumulation of naturally occurring electrophiles, such as...