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 H₂O₂ 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 H₂O₂. 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. Significance Substantial knowledge exists about how antibiotics interfere with core bacterial processes by binding to specific targets. Recently it has become appreciated that blocking these functions alters cellular redox state, and these perturbations may contribute to the lethality of antibiotics. In this work we explore whether antibiotic treatment of bacteria affects cellular oxidative stress and the role of such stress in antibiotic-mediated killing. We find that antibiotics dynamically alter cellular respiration and induce lethal levels of intracellular hydrogen peroxide. Antioxidants, including oxidative stress defense proteins, significantly reduce the killing by antibiotics, which is highly sensitive to the presence of molecular oxygen. These findings underscore the complex nature of antibiotic action and suggest practical approaches to enhancing our current antibiotic arsenal.

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 m²₂G, 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 m²₂G, 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. While the genetic code in DNA is read from four nucleobase structures, there are more than 100 ribonucleoside structures incorporated as post-transcriptional modifications mainly in tRNA and rRNA. These structures and their biosynthetic machinery are highly conserved, with 20–30 present in any one organism, yet the larger biological function of the modifications has eluded understanding. To this end, we developed a sensitive and precise mass spectrometric method to quantify 23 of the 25 ribonucleosides in the model eukaryotic yeast, Saccharomyces cerevisiae. We discovered that the spectrum of ribonucleosides shifts predictably when the cells are exposed to different toxic chemical stimulants, with these signature changes in the spectrum serving as part of the cellular survival response to these exposures. The method also revealed novel enzymatic pathways for the synthesis of several modified ribonucleosides. These results suggest a dynamic reprogramming of the tRNA and rRNA modifications during cellular responses to stimuli, with corresponding modifications working as part of a larger mechanism of translational control during the cellular stress response.

Selective translation of survival proteins is an important facet of the cellular stress response. We recently demonstrated that this translational control involves a stress-specific reprogramming of modified ribonucleosides in tRNA. Here we report the discovery of a step-wise translational control mechanism responsible for survival following oxidative stress. In yeast exposed to hydrogen peroxide, there is a Trm4 methyltransferase-dependent increase in the proportion of tRNA^Leu(CAA) containing m⁵C at the wobble position, which causes selective translation of mRNA from genes enriched in the TTG codon. Of these genes, oxidative stress increases protein expression from the TTG-enriched ribosomal protein gene RPL22A, but not its unenriched paralogue. Loss of either TRM4 or RPL22A confers hypersensitivity to oxidative stress. Proteomic analysis reveals that oxidative stress causes a significant translational bias towards proteins coded by TTG-enriched genes. These results point to stress-induced reprogramming of tRNA modifications and consequential reprogramming of ribosomes in translational control of cell survival.

Synthetic biology has expanded the availability of engineered bacterial systems for diverse applications and is now developing safeguards for their effective and secure use. The report of two synthetic gene circuit ‘kill switches’ provides new biocontainment mechanisms for engineered Escherichia coli. Biocontainment systems that couple environmental sensing with circuit-based control of cell viability could be used to prevent escape of genetically modified microbes into the environment. Here we present two engineered safeguard systems known as the 'Deadman' and 'Passcode' kill switches. The Deadman kill switch uses unbalanced reciprocal transcriptional repression to couple a specific input signal with cell survival. The Passcode kill switch uses a similar two-layered transcription design and incorporates hybrid LacI-GalR family transcription factors to provide diverse and complex environmental inputs to control circuit function. These synthetic gene circuits efficiently kill Escherichia coli and can be readily reprogrammed to change their environmental inputs, regulatory architecture and killing mechanism.

The increasing use of engineered organisms for industrial, clinical, and environmental applications poses a growing risk of spreading hazardous biological entities into the environment. To address this biosafety issue, significant effort has been invested in creating ways to confine these organisms and transgenic materials. Emerging technologies in synthetic biology involving genetic circuit engineering, genome editing, and gene expression regulation have led to the development of novel biocontainment systems. In this perspective, we highlight recent advances in biocontainment and suggest a number of approaches for future development, which may be applied to overcome remaining challenges in safeguard implementation.