Exposure to Environmentally Relevant Concentrations of Antibiotics Increases N₂O Emissions and Delays Nitrate Removal: New Insights into Bacteriostatic Antibiotics at the Cellular Level

Antibiotics are being increasingly detected in the environment, threatening to perturb microbe-mediated nitrogen cycling. However, the cellular mechanisms through which subinhibitory concentrations of ribosome-targeting antibiotics affect denitrification and nitrous oxide (N2O) emissions remain inadequately understood. This study aimed to elucidate how tetracycline (TC), a representative ribosome-targeting antibiotic, interferes with denitrification pathways in Paracoccus denitrificans. We evaluated the effects of environmentally relevant concentration (ERC) of TC on nitrate removal kinetics, N2O production, cellular physiology, and transcription. The results indicated that TC delays nitrate removal and significantly increases N2O emissions without causing cell death. Enzyme activity assays revealed nonspecific inhibition of denitrification enzymes, whereas assessment of physiological responses and transcriptomic analyses uncovered disruptions in carbon metabolism and electron transport. TC inhibited protein synthesis, suppresses glucose utilization, and impairs electron transport, leading to intracellular redox imbalance and reactive oxygen species accumulation. Notably, this oxidative stress preferentially suppressed the activity of N2O reductase, a critical factor driving N2O accumulation. Collectively, our findings unveil a dual interference mechanism of TC at ERC, involving suppression of enzyme expression and enhancement of oxidative stress. This study has significant implications for understanding the microbial mechanisms underlying N2O emissions.