E. coli prophages encode an arsenal of defense systems to protect against temperate phages.

In recent years, dozens of anti-phage defense systems have been identified. However, efforts to find these systems have focused predominantly on lytic phages, leaving defense against temperate phages poorly understood. Here, we isolated 33 temperate phages from a diverse collection of E. coli to create a library of single lysogens, which were tested for defense against the same set of temperate phages. We found that the majority of lysogens offer protection against at least one additional phage from the collection, often displaying broad defense against various phages. Defense efficacy varies based on growth media and host background, suggesting that some systems are context dependent. Using an iterative deletion-based strategy, we identify 17 systems responsible for the prophage-encoded defense, including 5 toxin-antitoxin modules. Collectively, our work uncovers a diverse array of phage-phage interactions and indicates that temperate phages encode a previously unrecognized arsenal of anti-phage defense systems.