Hi‐C Links Reveal Viral Activity and Infection Within the Free‐Living Microbial Community of a Secondary Chlorophyll Maximum in the Eastern Tropical North Pacific

Oxygen-deficient zones (ODZs) influence global nitrogen cycling as key sites for the removal of bioavailable nitrogen through denitrification and anammox. Despite their importance, many microbes and viruses in ODZs remain uncultivated, limiting our understanding of their ecological roles. This study employed Hi-C proximity linkages, combined with long and short read metagenomic sequencing to characterise active viral interactions in the prokaryotic community at a secondary chlorophyll maximum in the Eastern Tropical North Pacific ODZ. Among the identified 861 assembled viral contigs over 10 kb, 75 showed significant links to microbial genomes. Virus-host linkages indicated 19 novel virus-microbe pairs that were likely infectious, and which conventional in silico host prediction methods largely missed. The virus-host relationships involved nine distinct microbial phyla, with previously unrecorded viral infections of Planctomycetes, Chloroflexota, Alphaproteobacteria, Gammaproteobactera, Myxococcota and Verrucomicrobia. Most hosts carried the genomic potential for denitrification. Phylogenetic analysis of the terminase large subunit (terL) genes from linked viruses suggested that many active phages resemble known temperate phages, indicating that lysogeny may be an ecological strategy in ODZs. Our comprehensive metagenomic approach offers new insights into viral-host interactions in this ecosystem, highlighting the importance of including proximity methods in viral ecology studies of uncultivated microbial populations.