Supporting information：Ampicillin exposure and glutathione deficiency synergistically promote conjugative transfer of plasmid-borne antibiotic resistance genes

Plasmid-mediated conjugation is an important pathway for the spread of antibiotic resistance genes (ARGs), posing a significant risk to global public health. It has been reported that conjugative transfer of ARGs could be enhanced by oxidative stress. Whether endogenous glutathione (GSH), a major non-protein thiol compound involved in the cellular redox homeostasis, influences the conjugative transfer is unknown. In this study, we show that deletion of GSH biosynthesis gene gshA and ampicillin exposure synergistically promoted the conjugative transfer of plasmid RP4 bearing multiple ARGs from the soil bacterium Enterobacter sp. CZ-1 to Escherichia coli S17-1λπ in co-culture experiments and to diverse soil bacteria belonging to eight phyla, including some potential human pathogens, in a soil incubation experiment. Deletion of gshA increased ROS generation and cell membrane permeability, and upregulated expression of the genes involved in the intracellular oxidative stress regulation, membrane permeability, plasmid replication and SOS response process, especially under ampicillin exposure. These results suggest that endogenous GSH is an important factor affecting the spread of plasmid-borne ARGs. Exposure to antibiotics and environmental stresses that cause a depletion of endogenous GSH in vivo are likely to increase the risk of ARGs dissemination in the environment.