Systematic mapping of bacteriophage gene essentiality with HIDEN-SEQ

The 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 large amount 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 (hidden Acr-enabled transposon-insertion sequencing), 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. Our method is easily portable across diverse non-model phages and reveals conditionally essential genes in multiple bacterial hosts and growth conditions, including previously unknown antidefence factors that we matched to specific antiviral defences. We anticipate that HIDEN-SEQ will be leveraged to reveal functions of viral genes with direct relevance for microbial ecology, biotechnology and phage therapy.