Hydrogenase-mediated metabolic functions contribute to formation of bacterial antibiotic tolerance through regulating redox homeostasis and maintaining membrane potential.

Nutrient limitation imposes significant challenges to bacterial survival, entailing activation of the stringent response and other stress defense pathways that confer phenotypic tolerance to multiple environmental stresses. Hydrogen metabolism mediated by [NiFe] hydrogenases plays a critical role in microbial redox homeostasis and stress defense; however, whether this function also contributes to the onset and maintenance of antibiotic tolerance during nutrient deprivation remains unexplored. Here, we investigated the role of the Hyd-1, Hyd-2, and Hyd-3 hydrogenases in development of a sustainable antibiotic tolerance phenotype in bacteria subjected to nutrient starvation. Mutants unable to synthesize components of these enzymes (hyaD, hybA, hybB, hybE, hybF, hycE, hycG, and hycI) were found to exhibit significantly reduced survival rates when exposed to antibiotics under nutrient-limiting conditions. Our findings establish hydrogenases as key determinants of bacterial antibiotic tolerance during starvation, highlighting their important role in proton motive force maintenance, activation of efflux function, oxidative stress defense and energy generation in the tolerant cells. This study highlights the intricate relationship between redox imbalance and antibiotic killing when bacteria are nutritionally restricted, providing a theoretical basis for novel antibacterial strategies that act by targeting the metabolic-oxidative defense cross-pathways.