Reductive Stress Boosts the Horizontal Transfer of Plasmid-Borne Antibiotic Resistance Genes: The Neglected Side of the Intracellular Redox Spectrum

The dissemination of plasmid-borne antibiotic resistance genes (ARGs) among bacteria is becoming a global challenge to the "One Health" concept. During conjugation, the donor/recipient usually encounter diverse stresses induced by the surrounding environment. Previous studies mainly focused on the effects of oxidative stress on plasmid conjugation, but ignored the potential contribution of reductive stress (RS), the other side of the intracellular redox spectrum. Herein, we demonstrated for the first time that RS induced by dithiothreitol could significantly boost the horizontal transfer of plasmid RP4 from Escherichia coli K12 to different recipients (E. coli HB101, Salmonella Typhimurium, and Pseudomonas putida KT2440). Phenotypic and genotypic tests confirmed that RS upregulated genes encoding the transfer apparatus of plasmid RP4, which was attributed to the promoted consumption of intracellular glutamine in the donor rather than the widely reported SOS response. Moreover, RS was verified to benefit ATP supply by activating glycolysis (e.g., GAPDH) and the respiratory chain (e.g., appBC), triggering the deficiency of intracellular free Mg²⁺ by promoting its binding, and reducing membrane permeability by stimulating cardiolipin biosynthesis, all of which were beneficial to the functioning of transfer apparatus. Overall, our findings uncovered the neglected risks of RS in ARG spreading and updated the regulatory mechanism of plasmid conjugation.