The concentration of atmospheric methane continues to increase with microbial communities controlling soil-atmosphere fluxes. While there is substantial knowledge of the diversity and function of organisms regulating methane production and consumption, the frequency and impact of interactions with viruses on their activity in soil is unknown. Metagenomic sequencing of soil microbial communities has enabled identification of linkages between viruses and hosts. However, determining host-virus linkages through sequencing does not determine whether a virus or a host are active. In this study, we identified active individual interactions in situ by following the transfer of assimilated carbon from active hosts to virus. Using DNA stable-isotope probing combined with metagenomic analyses, we characterized methane-fueled microbial networks in acidic and neutral pH soils, specifically primary and secondary utilizers of carbon, together with the recent transfer of methane-derived carbon to viruses. Sixty-three percent of viral contigs from replicated soil incubations contained genes associated with known methanotrophic and methylotrophic bacteria. Genomic sequences from 13C-enriched viruses were present in clustered regularly interspaced short palindromic repeats (CRISPR) arrays of multiple, closely-related Methylocystis populations, revealing differences in their history of viral interaction. Viruses infecting non-methanotrophic methylotrophs and heterotrophic predatory bacteria were also identified through the analysis of shared homologous genes, demonstrating that carbon is transferred to a diverse range of viruses associated with methane-fueled microbial food networks.

Viruses are an important top-down control on microbial communities, yet their direct 11 study in natural environments has been hindered by culture limitations [1][2][3] . The advance of 12 sequencing and bioinformatics over the last decade enabled the cultivation independent 13 study of viruses. Many studies focus on assembling new viral genomes 4-6 and studying viral 14 diversity using marker genes amplified from free viruses with POLA being the most impacted and SPOT resembling open ocean conditions. In all of these 33 sites free virus-like particles outnumber bacteria and archaea roughly 10:1 (sup. fig. 1). We 34 examined only the 0.2-1 µm size-fraction, which includes most bacteria, archaea and some 35 picoeukaryotes. Via assembly of metatranscriptomes, we obtained 1455 contigs longer than 5 kb 36 of which 57 (3.9%) were characterized as viral using virSorter and virFinder (see methods). 37Additionally, a cross-assembly of the metatranscriptomic viral contigs with metagenomes of the 38 same samples (N=12) yielded 9 more contigs (mean length 26,563 bp) characterized as viral. 39Most of the contigs represent dsDNA viruses (N= 65) as apparent from their presence in 40 metagenomes, but one appears to be an RNA virus possibly infecting a eukaryotic host. This 41 contig contained an RNA-dependent-RNA-polymerase whose nearest match in NCBI non-42 redundant database was marine Antarctic phytoplankton RNA virus PAL_E4 9 . These 66 viral 43 contigs revealed varied patterns of presence (in metagenomes) and activity (in 44 metatranscriptomes) in the three sites over a year (fig.