Abstract Large-scale metagenomic and data mining efforts have uncovered an expansive diversity of bacteriophages (phages) within the human gut 1–3 . These insights include broader phage populational dynamics such as temporal stability 4 , interindividual uniqueness 5,6 and potential associations to specific disease states 7,8 . However, the functional understanding of phage-host interactions and their impacts within this complex ecosystem have been limited due to a lack of cultured isolates for experimental validation. Here we characterise 125 active prophages originating from 252 diverse human gut bacterial isolates using seven different induction conditions to substantially expand the experimentally validated temperate phage-host pairs originating from the human gut. Importantly, only 17% of computationally predicted prophages were induced with common induction agents and these exhibited distinct gene patterns compared to non-induced predictions. Active Bacteroidota prophages were among the most prevalent members of the gut virome, with extensive use of diversity generating retroelements and exhibiting broad host ranges. Moreover, active polylysogeny was present in 52% of studied gut lysogens and led to coordinated prophage induction across diverse conditions. This study represents a substantial expansion of experimentally validated gut prophages, providing key insights into their diversity and genetics, including a genetic pathway for prophage domestication and demonstration that differential induction was complex and influenced by divergent prophage integration sites. More broadly, it highlights the importance of experimental validation alongside genomic based computational prediction to enable further functional understanding of these commensal viruses within the human gut.

Temperate gut phages are prevalent, diverse, and predominantly inactive