Most bacterial pathogens are polylysogens, harbouring multiple vertically transmitted prophages1–3. These prophages enhance bacterial pathogenicity and survival by encoding virulence factors and anti-phage defence systems while retaining the capacity for horizontal transfer. Thus, prophage-encoded anti-phage defences must block propagation of external phages without inhibiting the spread of the prophages that encode them. Here we identify HepS—an abortive infection system encoded on the Gifsy-1 prophage constituted of a single HEPN domain protein—which restricts phages of the Siphoviridae family. We demonstrate that in its native host context of Salmonella enterica serovar Typhimurium, HepS both senses phage infection and enacts abortive infection. Structures of HepS reveal a tetrameric nuclease complex that undergoes allosteric activation upon recognition of Siphoviridae tail tip proteins during production of new phage particles. Once activated, HepS cleaves specific transfer RNA anticodon loops and arrests phage replication. Gifsy-1, a Siphoviridae itself, evades self-targeting by expressing a tail tip variant that does not trigger HepS, as do co-resident Siphoviridae prophages Gifsy-2 and Gifsy-3. This evasion permits Gifsy-1 to spread despite encoding HepS. These findings reveal a mechanism by which a prophage defends the host while maintaining its propagation abilities.
