Bacteriophage genome-wide transposon mutagenesis

Bacteriophage genomes are densely packed with coding sequences and frequently encode genes of unknown function. Unbiased phage functional genomics approaches are therefore needed, particularly for large lytic phages. Here, we harness the mariner transposase to develop phage transposon mutagenesis and sequencing (phage TnSeq), which enables pooled sequencing to identify both fitness-conferring and dispensable genes. Using the Pseudomonas aeruginosa-infecting nucleus-forming jumbo phage ΦKZ (280,334 bp; 371 predicted genes), we show that ∼110 genes are fitness-conferring via phage TnSeq, identifying many known and previously unknown essential genes. Moreover, this phage harbors ∼261 non-essential genes, including some capsid and tail proteins, many of which are important for fitness across different clinical isolates or conditions. Phage TnSeq was also extended to a base-modified phage. Together, phage TnSeq is a scalable technology that can identify essential phage genes, generate knockouts in all non-essential genes, and sensitively assign the quantitative fitness contributions of every gene in parallel.