Chen, M.; Gregoire, D. S.; Bain, J. G. S.; Blowes, D. W.; Hug, L. A.

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.