Hi-C sequencing deciphers phage and plasmid host networks in wastewater biofilms

Mobile genetic elements (MGEs) such as bacteriophages and plasmids profoundly shape microbial community structure and drive horizontal gene transfer across ecosystems. Wastewater treatment systems, with their high cell densities, steep physicochemical gradients and close cell-to-cell contact, act as hotspots for MGE proliferation and exchange, yet the in situ assembly dynamics and host interaction networks of these elements have remained largely unresolved because conventional methods fail to establish direct MGE–host linkages in complex matrices. Here we show that an integrated framework combining metagenomics, metatranscriptomics, metaviromics, and Hi-C proximity ligation sequencing enables the efficient elucidation of DNA phage and plasmid assembly dynamics alongside their host interaction networks in biofilms. We reconstructed 17,672 viral operational taxonomic units and 11,454 high-confidence non-redundant plasmids, and established 529 phage–host and 5739 plasmid–host associations that link up to 52% of phages to 56% of prokaryotes and 70% of plasmids to 91% of prokaryotes, respectively. Hi-C substantially expanded and refined these networks, revealing taxon-specific and multi-host patterns. Host community composition and biofilm architecture emerge as primary drivers of MGE occurrence and abundance along the reactor flow path. Expression of auxiliary metabolic genes, antibiotic resistance genes and virulence factors carried by these MGEs demonstrates their active roles in modulating biogeochemical cycles and maintaining ecosystem stability. These findings establish a scalable, cultivation-independent framework for deciphering MGE–host networks in complex microbial ecosystems, and underscore the power of Hi-C sequencing to transform our mechanistic understanding of gene flow, resistome dissemination, and ecological resilience in engineered and natural microbiomes.