A genome-resolved atlas reveals lineage-specific activity patterns of prophages in cultured human gut bacteria

Abstract

Prophages dominate the gut virome, yet their basal activity landscapes remain elusive. Here, we developed ACTIVE (Activity Comparison Test for Induced Viral Elements), a robust coverage-based framework to accurately profile prophage replication activity based on sequencing data of individual bacteria. Applying ACTIVE across 31,150 gut bacterial isolates (875 species), we compiled these data into GPAC (Gut Prophage Activity Catalog) and found that 5.4% of prophages are detectably active under standard culture, with pronounced enrichment in Bacillota. Moreover, we uncovered distinct active viral lineages with broad host distributions. Comparative analyses identified 46 viral families significantly enriched for activity (SA-vFAMs). Notably, SA-vFAM7 represents a hyper-prevalent Candidatus Heliusvirales lineage whose host distribution expands into Bacteroidota (Tannerellaceae). Furthermore, we propose Candidatus Mubacviridae (SA-vFAM1), an active, hankyphage-inclusive transposable phage family associated with Bacteroidales, characterized by conserved MuA/B operons, diversity-generating retroelements, and multi-genus host distribution. Together, these findings establish GPAC as a resource linking prophage activity to host taxonomy and viral evolutionary lineages, providing a foundation for investigating prophage induction, host-phage interactions, and ecological dynamics of the human gut microbiome.