Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species. This notion has been supported by many groups but has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H 2 O 2 sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress. Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H 2 O 2 . We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality.reactive oxygen species | DNA repair | mutagenesis T he increasing incidence of antibiotic-resistant infections coupled with a declining antibiotic pipeline has created a global public health threat (1-6). Therefore there is a pressing need to expand our conceptual understanding of how antibiotics act and to use insights gained from such efforts to enhance our antibiotic arsenal. It has been proposed that different classes of bactericidal antibiotics, regardless of their drug-target interactions, generate varying levels of deleterious reactive oxygen species (ROS) that contribute to cell killing (7,8). This unanticipated notion, built upon important prior work (9-11), has been extended and supported by multiple laboratories investigating wide-ranging drug classes (e.g., β-lactams, aminoglycosides, and fluoroquinolones) and bacterial species (e.g., Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Mycobacterium tuberculosis, Bacillus subtilis, Staphylococcus aureus, Acinetobacter baumannii, Burkholderia cepecia, Streptococcus pneumonia, Enterococcus faecalis) using independent lines of evidence (12-39). Importantly,...

Decades of study have revealed more than 100 ribonucleoside structures incorporated as post-transcriptional modifications mainly in tRNA and rRNA, yet the larger functional dynamics of this conserved system are unclear. To this end, we developed a highly precise mass spectrometric method to quantify tRNA modifications in Saccharomyces cerevisiae. Our approach revealed several novel biosynthetic pathways for RNA modifications and led to the discovery of signature changes in the spectrum of tRNA modifications in the damage response to mechanistically different toxicants. This is illustrated with the RNA modifications Cm, m5C, and m2 2G, which increase following hydrogen peroxide exposure but decrease or are unaffected by exposure to methylmethane sulfonate, arsenite, and hypochlorite. Cytotoxic hypersensitivity to hydrogen peroxide is conferred by loss of enzymes catalyzing the formation of Cm, m5C, and m2 2G, which demonstrates that tRNA modifications are critical features of the cellular stress response. The results of our study support a general model of dynamic control of tRNA modifications in cellular response pathways and add to the growing repertoire of mechanisms controlling translational responses in cells.

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