Hydrogen sulfide drives horizontal transfer of plasmid-borne antibiotic resistance genes in wastewater ecosystems

Antibiotic resistance genes (ARGs), which spread via horizontal gene transfer, pose a significant threat to global health. Although exogenous stressors are known to promote conjugation, the role of endogenous microbial metabolites remains poorly understood. Here we report that hydrogen sulfide (H₂S)—a ubiquitous metabolite in wastewater—acts as a potent, yet overlooked, promoter of plasmid conjugation, enhancing conjugation frequency and broadening the recipient range of plasmid RP4 within wastewater microbiota. We elucidate a plasmid-autonomous activation mechanism distinct from the classic SOS response. Specifically, plasmid RP4 uses its encoded protein Upf32.8 (renamed GlsS32.8) to sense intracellular glutamine levels, trigger plasmid de-repression and enhance RP4-specific glutamine-centric metabolic hijacking from the host, thereby facilitating conjugation under H₂S. Notably, evolutionary analysis shows that GlsS32.8 is conserved across globally prevalent IncP-1α plasmids, underscoring a universal risk of ARG spread in H₂S-rich environments. Our findings redefine microbial metabolites as critical players in resistance spread and provide valuable insights into plasmid–host interactions for combatting ARG dissemination.