Diversity and ecological roles of hidden viral players in groundwater microbiomes.

Groundwater ecosystems harbor diverse microbial communities adapted to energy-limited, light-deprived conditions, yet the role of viruses in these environments remains poorly understood. Here, we analyzed 1.24 terabases of metagenomic and metatranscriptomic data from seven wells in the Hainich Critical Zone Exploratory (CZE) to characterize groundwater viromes. We identified 257,252 viral operational taxonomic units (vOTUs) (≥ 5 kb), with 99% novel at order, family and genus levels against global ocean, freshwater and/or other publicly available datasets. In silico host predictions suggest that vOTUs primarily targeted Proteobacteria, Candidate Phyla Radiation (CPR) bacteria, and DPANN archaea, which reflects abundant and active groundwater microbial members. Patterns of virus-host abundance ratios, CRISPR-spacers, and prophage screening suggest the potential for multi-layer interactions involving CPR/DPANN lineages, their hosts, and viruses. Additionally, we identified 289 KEGG metabolic modules, 31.1% of which were targeted by 3378 vOTUs encoded auxiliary metabolic genes (AMGs) linked to carbon, nitrogen, and sulfur cycling. These findings provide a baseline for exploring how viruses influence microbial community dynamics, metabolic reprogramming and nutrient cycling in groundwater.