Prophages, dormant bacteriophage genomes integrated within the bacterial chromosome, play pivotal roles in shaping microbial communities when awakened. Our current understanding of prophage activation is largely shaped by a narrow set of traditional DNA-damaging inducers, such as mitomycin C and ciprofloxacin, which trigger the bacterial SOS response. This study employed high-throughput screening of 3,921 compounds to identify novel prophage inducers using model lambdoid prophage HK97. We identified multiple new inducers across diverse pharmacological classes, including dietary supplements and therapeutics. Despite the variety in compounds, all acted through SOS-dependent pathways. However, bleomycin, an antineoplastic antibiotic, demonstrated broad-spectrum and potent prophage induction exceeding standard inducers, with activity validated across multiple phage-host pairings. These findings expand the repertoire of prophage inducers into commonly ingested xenobiotics and introduce bleomycin as a powerful, cost-effective tool for prophage research.
IMPORTANCE: Around 75% of bacteria carry within them dormant viruses (prophages), which can awaken when the bacterium is stressed, killing the bacterium. Historically, this has been done using DNA-damaging antibiotics, but increasingly, more such signals have been discovered. Here, through a high-throughput screen, we identify phage-waking activity in several commonly consumed compounds, such as the SSRI Prozac, as well as a new DNA-damaging agent that is much more effective in waking phages than the previous gold standard.