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

Nature Water (2025)Cite this article

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Abstract

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.

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Fig. 1: H2S drives the conjugative transfer of plasmid RP4 within wastewater microbiota.
Fig. 2: H2S drives plasmid conjugation by intensifying RP4-specific glutamine hijacking.
Fig. 3: H2S drives plasmid RP4 conjugation by de-repressing transfer genes and supplying glutamine-derived dNTPs.
Fig. 4: Upf32.8 is essential for H2S-driven plasmid conjugation.
Fig. 5: The AA sequence and protein structure of Upf32.8.
Fig. 6: GlsS32.8 is a conserved backbone protein within globally distributed IncP-1α plasmids.
Fig. 7: Proposed plasmid–host interactions in the presence and absence of sulfide.

Data availability

The RNA sequencing and 16 s rRNA sequencing data have been deposited in the National Center for Biotechnology Information (NCBI) Short-Read Archive (SRA) under accession no. PRJNA1242992 and PRJNA1242997, respectively. In addition, the raw data for the mass spectrometry proteomics and metabolome have been deposited to the ProteomeXchange Consortium via the iProX partner repository with the dataset identifier PXD062398 and the National Genomics Data Center (identifier no. PRJCA037937), respectively. Source data are available via figshare at https://doi.org/10.6084/m9.figshare.29533577 (ref. 51).

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Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (grant nos. 52470056 to H.H., 52470053 to Y.C. and 52100059 to H.H.) and the special fund of State Key Laboratory of Regional Environment and Sustainability (grant no. 25K05REST to H.H.). We thank B.F. Smets from Aarhus University, as well as Y. Luo and X. Wang from Nanjing University, for generously providing the GFP-tagged plasmid RP4.

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Authors and Affiliations

  1. State Key Laboratory of Water Pollution Control and Green Resource Recycling, School of Environmental Science and Engineering, Tongji University, Shanghai, China

    Haining Huang, Lin Lin, Qimeng Liu, Xinyi Li, Lei Dong, Leiyu Feng, Xiong Zheng & Yinguang Chen

  2. Australian Centre for Water and Environmental Biotechnology, The University of Queensland, Brisbane, Queensland, Australia

    Junqi Liao & Jianhua Guo

  3. Shanghai Municipal Engineering Design Institute (Group) Co. Ltd., Shanghai, China

    Lei Dong

  4. State Key Joint Laboratory of Environmental Simulation and Pollution Control, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China

    Junya Zhang

  5. Shanghai Engineering Research Center of Biotransformation of Organic Solid Waste, School of Ecological and Environmental Sciences, East China Normal University, Shanghai, China

    Yinglong Su

  6. Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, Ministry of Education, College of Environment, Hohai University, Nanjing, China

    Jingyang Luo

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Contributions

Y.C. and J.G. supervised the project and revised the paper. H.H. conceived of the study, designed and performed the experiments, prepared the figures and wrote the original draft. L.L., Q.L. and X.L. assisted with data analysis. J. Liao and J.Z. contributed to plasmid database searching and analysis. Y.S. and J. Luo helped with drawing the schematics and edited the paper. L.D., L.F. and X.Z. contributed to the experimental procedures and provided feedback on the paper. All authors discussed the results, contributed to their interpretation and approved the final version.

Corresponding authors

Correspondence to Jianhua Guo or Yinguang Chen.

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Huang, H., Lin, L., Liu, Q. et al. Hydrogen sulfide drives horizontal transfer of plasmid-borne antibiotic resistance genes in wastewater ecosystems. Nat Water (2025). https://doi.org/10.1038/s44221-025-00523-7

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