Diverse microbial metal resistance and novel metal cycling organisms in copper/nickel mine tailings

Mine tailings contribute to environmental heavy metal contamination through the formation of acid mine drainage (AMD). Microbially-mediated processes such as iron and sulfur redox cycling influence metal mobility. Here, we applied an integrated metagenomic and metaproteomic approach to profile microbial communities across vertical geochemical gradients in legacy copper/nickel tailings in Sudbury, Ontario, Canada. From 43 samples, we recovered 454 non-redundant metagenome-assembled genomes (MAGs), revealing diverse populations within the Actinobacteriota, Desulfobacterota, and uncultured lineages such as Candidatus Eremiobacterota and SZUA-79. Functional profiling identified 301 putative iron- and sulfur-cycling MAGs, including those within the Ca. Eremiobacterota and SZUA-79 phyla. Metal resistance genes were widespread and diverse, with abundances that did not correlate with measured metal concentrations. Proteomic data confirmed in situ expression of selected metal resistance genes and iron/sulfur metabolism genes, despite limited protein recovery from this challenging matrix. Our findings highlight both the depth of microbial diversity in metal resistance and metal biogeochemical cycling in mining waste, as well as the technical challenges that currently limit genomic and proteomic sequencing coverage in low-biomass, metal-rich matrices. This work also presents new protocols for multi-omics data capture and analysis from metal contaminated environments, including new protein extraction and bioinformatic gene annotation tools. Highlights Metagenomics recovered 454 non-redundant MAGs from copper/nickel mine tailings, revealing high taxonomic novelty including uncultured Ca. Eremiobacterota and SZUA-79 lineages. A custom HMM-based pipeline targeting metal resistance genes uncovered a widespread and diverse set of metal resistance genes across MAGs in tailings, which did not correlate with metal concentrations. Metaproteomics validated in situ expression of both iron/sulfur cycling genes and metal resistance proteins, although low biomass and contamination limited proteomic sequencing coverage.