The Hidden Virosphere likely drives the microbial community and their functions in the pristine groundwater

The ocean contains 1010 virus-like particles (VLP) per liter, vastly outnumbering host cells. Viruses significantly affect nutrient cycling by lysing 20-40% of bacteria daily, reprogramming host cells and promoting horizontal gene transfer (1029 genes/day). Playing a vital role in carbon fluxes, including carbon transport to the deep sea, contributing to marine ecosystem stability amid climate change. Pristine groundwater is a vital drinking water source, yet we lack an understanding of its viral ecosystem. We examine virus diversity, ecological importance, and functional interactions in a two-year study of seven wells within a pristine groundwater system along a hillslope transect. From ~1.3 terabases of metagenomics data, we identified >7 million virus contigs, resulting in 257,814 and 82,807 virus operational taxonomic units (vOTUs) of ≥5 kb or ≥10 kb, respectively, representing a ~22-fold increase compared to publicly available groundwater vOTUs (n=3,584, ≥10 kb). Taxonomic analysis revealed that 99% of Hainich groundwater viruses were species-level unique, even when compared to a global ocean dataset. Approximately 81% could be taxonomically classified, with 99% belonging to the Caudoviricetes class (tailed phages). Ecological analysis using read mapping demonstrated site-specific endemism in virus communities, evidenced by strong grouping based on the sampled groundwater wells (p-value <0.001). Our host prediction analysis found that 88% of viruses infect 78% of microbes (1,275 MAGs) from the same sample, including ecologically important groups like Pastecibacteria (81%) and Proteobacteria (74%). Additionally, about 5% of viruses may reprogram ~38% of host pathways through auxiliary metabolic genes (5,883 AMGs found, 24 times more than the GOV v.1). For instance, GAPDH (K00134), which breaks down glucose for energy and carbon. Furthermore, additionally Nitrite Reductase (K15876) and Sulfate Adenylyltransferase (K00957) help groundwater microbes thrive in oxygen-depleted conditions. Overall, this research offers valuable insights into groundwater virus communities and the mitigation of human impacts on groundwater resources.