Unlocking the genomic repertoire of a cultivated megaphage

Megaphages are bacteriophages (i.e., phages) with exceptionally large genomes that have been discovered computationally across the globe. To date, all have evaded cultivation except phage G, which we examined with multiomics and artificial intelligence (AI) to resolve its 50-year cultivated history. Phage G is one of the largest phages with a size of >0.6 µm, about half the width of the host cell, and a 499 kbp, non-permuted, linear genome that has, uniquely among known phages, two pairs of ends. Phage G has >650 protein-coding open reading frames (ORFs), with >65% being hypothetical proteins, with the expansive repertoire of auxiliary metabolic genes (AMGs) acquired from its bacterial host, manipulate host sporulation (sspD, RsfA, spoK), and antiviral escape genes (e.g., anti-CBass nuclease and Anti-Pycsar protein). Our study represents a doorway into the complexity of the genomic repertoire of the only cultivated megaphage.