Heavy metal (Cu(II)) stress alters lysogeny-lysis balance and drives phage-mediated transfer of co-resistance in the activated sludge process.

The co-selection of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) by heavy metals poses significant ecological risks. However, the contribution of bacteriophages (phages), particularly temperate phages, to this process via horizontal gene transfer (HGT) remains poorly understood. Here, we integrated metagenomics, metaviromics, and metatranscriptomics to investigate the impact of escalating Cu(II) concentrations (0.05-20.00 mg/L) on phage lifestyle dynamics and the dissemination of resistance genes in an activated sludge reactor. Our results revealed that phage-mediated HGT events of resistance genes were strongly threshold-dependent, predominantly occurring at high Cu(II) levels (10.00-20.00 mg/L). While the general temperate phage community shifted toward lysogeny to cope with stress, specific phages that mediated HGT of resistance genes exhibited higher lytic activity. Metatranscriptomic analysis further indicated upregulated transcriptional activity of HGT-associated MRGs under high Cu(II) stress, potentially conferring an adaptive advantage to hosts against metal toxicity. Notably, nearly all HGT events were associated with temperate phages, among which approximately 40% of the identified viral clusters (VCs) simultaneously harbored multiple resistance types, even in the absence of antibiotic selective pressure. Collectively, our findings highlight the important role of temperate phages in mediating resistance gene dissemination under Cu(II) stress and underscore the need to incorporate viral dynamics into resistance risk assessment in activated sludge systems.