Bacteriophages of class Caudoviricetes represent a highly anthropologically relevant group of viruses. During infection, bacteriophages use high DNA packaging density for genomic ejection, creating a capsid to genome size ratio that is evolutionarily constrained. In headful packing phages, this ratio is maintained through nonspecific genomic overpacking, such that termini contain redundant DNA. This introduces a dynamic where mutations that increase capsid volume also increase redundant DNA, allowing integration of novel genes. This “evolutionary ratchet” mechanism was used to explain triangulation variation between bacteriophage species. We expand on this ratcheting concept to include alternate capsid expansion mechanisms that might provide evolutionary advantages over changes in triangulation. We structurally analysed the headful packaging phage PhiTE using CryoEM. Phage PhiTE internal volume was found to be larger than other T=13 bacteriophages. The large capsid size of PhiTE appears mostly caused by formation of large hexamers. Increased hexamer size results in a capsid with ~20-25% greater internal volume relative to other T=13 phages, allowing packaging of the large PhiTE genome.
Our cryoEM analysis also offers a series of important insights into understanding general principles of phage architecture. Firstly, we describe two novel decorations proteins: the DEC protein, and the PAGODA protein. The DEC shows homology to the beta-tulip class of decorations, while the PAGODA protein likely binds to hexamer centres as a trimer and appears structurally unrelated to other known decorations. Secondly, we identify and model all structural elements in the PhiTE connector, highlighting a minimalist arrangement of structural proteins that may contribute to a ‘sheath slipping” phenotype observed in CryoEM micrographs. Thirdly, we reconstruct the helical tail connector and tube in contracted and native states, identifying changes in helical parameters and peptide orientations that occur during contraction. Finally, we describe the FTE baseplate, which features a similarly minimalist architecture, and a unique “claw-like” fibre network.