Systematic mapping of bacteriophage gene essentiality with HIDEN‑SEQ

The constant arms race of bacteriophages and their bacterial hosts has inspired major breakthroughs in biotechnology and shaped phages as fierce predators with great clinical potential to fight multidrug-resistant bacterial pathogens. However, the vast amount of genomic 'dark matter' composed of genes of unknown function in phage genomes remains a major obstacle for the molecular understanding of phage-host interactions. Here we present HIDEN-SEQ, a transposon-insertion sequencing method for phages that systematically links viral genes to selectable phenotypes. Using model phage T4, we show that HIDEN-SEQ readily reproduces the gene essentiality map established over decades of research. Subsequently, we show that our method is easily portable to different phages far beyond classical laboratory models. Across a panel of bacterial hosts and growth conditions, HIDEN-SEQ reveals many conditionally essential phage genes, including previously unknown viral anti-defense factors that we could match to specific antiviral defenses of the respective hosts. Compared to analogous techniques, HIDEN-SEQ provides unprecedented depth and near base-pair resolution as well as great ease of use and portability. We therefore anticipate that HIDEN-SEQ will accelerate discoveries in phage biology by uncovering functions of viral dark matter with direct relevance for microbial ecology, biotechnology, and improvements of phage therapy.