James A. Imlay

Dihydrodichlorofluorescein derivatives have been employed as reporters of intracellular reactive oxygen species (ROS) in innumerable studies. Their use in bacteria has led investigators to conclude that a wide array of cellular stresses owe their toxicity to the formation of ROS. However, in the present study we find that this dye reacts too poorly with physiological concentrations of superoxide or hydrogen peroxide to signal their presence, and that lethal doses of hydroxyl radicals are dispersed among too many intracellular targets for the dye to be detectably oxidized. Instead, common redox moieties inside the cell—including flavins, quinones, and metal centers—capably oxidize the dye. Indeed, the dye is oxidized as rapidly inside anaerobic cells as inside aerobic ones, refuting any involvement of ROS. The ability of diverse stresses to increase the dye signal derives from the fact that both the reduced and oxidized forms of the dye are continuously exported by protonmotive force-driven pumps. Any stress or growth condition that diminishes the cellular energy charge has the effect of slowing these pumps and thereby causing an increase in the intracellular dye signal. Therefore, the unfortunate combination of ROS-independent dye oxidation plus stress-driven metabolic slowdowns have led investigators to conclude incorrectly that diverse stressors generate ROS. Those conclusions, including ones involving clinical antibiotics, must be reconsidered.

Molecular oxygen, superoxide, and hydrogen peroxide are related oxidants that can each impair the growth of microorganisms. Strikingly, these species exhibit large differences in their abilities to cross biological membranes. This Perspective explains the basis of those differences, and it describes natural situations in which the permeability of membranes to oxidants determines the amount of stress that a bacterium experiences.

A wide variety of stresses have been proposed to exert killing effects upon bacteria by stimulating the intracellular formation of reactive oxygen species (ROS). A key part of the supporting evidence has often been the ability of antioxidant compounds to protect the cells. In this study, some of the most‐used antioxidants—thiourea, glutathione, N‐acetylcysteine, and ascorbate—have been examined. Their ability to quench superoxide and hydrogen peroxide was verified in vitro, but the rate constants were orders of magnitude too slow for them to have an impact upon superoxide and peroxide concentrations in vivo, where these species are already scavenged by highly active enzymes. Indeed, the antioxidants were unable to protect the growth and ROS‐sensitive enzymes of E. coli strains experiencing authentic oxidative stress. Similar logic posits that antioxidants cannot substantially quench hydroxyl radicals inside cells, which contain abundant biomolecules that react with them at diffusion‐limited rates. Indeed, antioxidants were able to protect cells from DNA damage only if they were applied at concentrations that slow metabolism and growth. This protective effect was apparent even under anoxic conditions, when ROS could not possibly be involved, and it was replicated when growth was similarly slowed by other means. Experimenters should discard the use of antioxidants as a way of detecting intracellular oxidative stress and should revisit conclusions that have been based upon such experiments. The notable exception is that these compounds can effectively degrade hydrogen peroxide from environmental sources before it enters cells.

The oxidizability of cysteine residues is exploited in redox chemistry and as a source of stabilizing disulfide bonds, but it also raises the possibility that these side chains will be oxidized when they should not be. It has often been suggested that intracellular oxidative stress from hydrogen peroxide or superoxide may result in the oxidation of the cysteine residues of cytoplasmic proteins. That view seemed to be supported by the discovery that one cellular response to hydrogen peroxide is the induction of glutaredoxin 1 and thioredoxin 2. In this study we used model compounds as well as alkaline phosphatase to test this idea. Our results indicate that molecular oxygen, superoxide, and hydrogen peroxide are very poor oxidants of N-acetylcysteine and of the protein thiols of alkaline phosphatase in vitro. Copper could accelerate thiol oxidation, but iron did not. When alkaline phosphatase was engineered to remain in the cytoplasm of live cells, unnaturally high concentrations of hydrogen peroxide were required to oxidize it to its active, disulfide-dependent form, and toxic levels of superoxide had no effect. At the same time, far lower concentrations of these oxidants were sufficient to poison key metalloenzymes. The elimination of glutaredoxin 1 and thioredoxin 2 did not change these results, raising the question of why E. coli induces them during peroxide stress. In fact, when catalase/peroxidase mutants were chronically stressed with hydrogen peroxide, the absence of glutaredoxin 1 and thioredoxin 2 did not impair growth at all, even in a minimal medium over many generations. We conclude that physiological levels of reduced oxygen species are not potent oxidants of typical protein thiols. Glutaredoxin and thioredoxin must either have an alternative purpose or else play a role under culture conditions that differ from the ones we tested.

Copper avidly binds thiols and is redox active, and it follows that one element of copper toxicity may be the generation of undesirable disulfide bonds in proteins. In the present study, copper oxidized the model thiol N‐acetylcysteine in vitro. Alkaline phosphatase (AP) requires disulfide bonds for activity, and copper activated reduced AP both in vitro and when it was expressed in the periplasm of mutants lacking their native disulfide‐generating system. However, AP was not activated when it was expressed in the cytoplasm of copper‐overloaded cells. Similarly, this copper stress failed to activate OxyR, a transcription factor that responds to the creation of a disulfide bond. The elimination of cellular disulfide‐reducing systems did not change these results. Nevertheless, in these cells, the cytoplasmic copper concentration was high enough to impair growth and completely inactivate enzymes with solvent‐exposed [4Fe‐4S] clusters. Experiments with N‐acetylcysteine determined that the efficiency of thiol oxidation is limited by the sluggish pace at which oxygen regenerates copper(II) through oxidation of the thiyl radical–Cu(I) complex. We conclude that this slow step makes copper too inefficient a catalyst to create disulfide stress in the thiol‐rich cytoplasm, but it can still impact the few thiol‐containing proteins in the periplasm. It also ensures that copper accumulates intracellularly in the Cu(I) valence.

Cells regularly experience fluid flow in natural systems. However, most experimental systems rely on batch cell culture and fail to consider the effect of flow-driven dynamics on cell physiology. Using microfluidics and single-cell imaging, we discover that the interplay of physical shear rate (a measure of fluid flow) and chemical stress trigger a transcriptional response in the human pathogen Pseudomonas aeruginosa . In batch cell culture, cells protect themselves by quickly scavenging the ubiquitous chemical stressor hydrogen peroxide (H ₂ O ₂ ) from the media. In microfluidic conditions, we observe that cell scavenging generates spatial gradients of H ₂ O ₂ . High shear rates replenish H ₂ O ₂ , abolish gradients, and generate a stress response. Combining mathematical simulations and biophysical experiments, we find that cells in flow are sensitive to a H ₂ O ₂ concentration that is 100-1000 times lower than traditionally studied in batch cell culture. Surprisingly, the shear rate and H ₂ O ₂ concentration required to trigger a transcriptional response closely match their respective values in the human bloodstream. Thus, our results explain a long-standing discrepancy between H ₂ O ₂ levels in experimental and natural systems. Finally, we demonstrate that the shear rate and H ₂ O ₂ concentration found in the human bloodstream trigger gene expression in the blood-relevant human pathogen Staphylococcus aureus , suggesting that flow sensitizes bacteria to chemical stress in natural environments.

Cysteine is a singularly reactive amino acid; in high concentrations, it can disrupt cytoplasmic metabolism. This phenomenon prompted the view that the cyuPA operon of Escherichia coli serves to detoxify cysteine by degrading it.

E. coli responds to hydrogen peroxide (H₂O₂) by inducing defenses that protect H₂O₂-sensitive enzymes. DNA is believed to be another important target of oxidation, and E. coli contains enzymes that can repair oxidative lesions in vitro. However, those enzymes are not known to be induced by H₂O₂, and experiments have indicated that they are not necessary for the cell to withstand natural (low-micromolar) concentrations. In this study we used H₂O₂-scavenging mutants to impose controlled doses of H₂O₂ for extended time. Transcriptomic analysis revealed that in the presence of 1 µM cytoplasmic H₂O₂, the OxyR transcription factor induced xthA, encoding exonuclease III. The xthA mutants survived a conventional 15-minute exposure to even 100 times this level of H₂O₂. However, when these mutants were exposed to 1 µM H₂O₂ for hours, they accumulated DNA lesions, failed to propagate, and eventually died. Although endonuclease III (nth) was not induced, nth mutants struggled to grow. Low-grade H₂O₂ stress also activated the SOS regulon, and when this induction was blocked, cell replication stopped. Collectively, these data indicate that physiological levels of H₂O₂ are a real threat to DNA, and the engagement of the base-excision-repair and SOS systems is necessary to enable propagation during protracted stress.