Abstract
Bacteriophages of the class Caudoviricetes represent an exceptionally important component of the global virome. Bacteriophages infect and kill bacteria; accordingly, phages are an important component of global ecology as well as a powerful therapeutic when rationally applied to treatment of bacterial infections. Current models of bacteriophage structure and infectious function have been primarily focused on phages from the T odd and even collection. While such studies have been invaluable in furthering our understanding of phage infection, they have focused on a minority of the broad taxonomic abundance observed in bacteriophages. During my PhD I used cryogenic electron microscopy (cryo-EM) to study the complete virion structure of two long tailed contractile myophages: PhiTE and Bas63, and one short tailed podophage PhiM1. We aimed to contrast the structure of these phages to model bacteriophages to further our understanding of phage structural and functional diversity, and to identify deep evolutionary relationships between viruses, which are often only interpretable at the structural level.
Firstly, we explore the virion structure of the myophage PhiTE, infectious agent of agricultural pathogen Pectobacterium atrosepticum. Through this exploration we first describe an alternate topology in proteins that connect the capsid to the tail, two alternate stoichiometries for the tail length determining tape measure protein (TMP). We then describe a novel connectivity between tail fibre and baseplate proteins that likely acts as a novel trigger for tail contraction. Finally, we deposited a composite PDB structure of the PhiTE virion, which represents the 2nd largest model (by residue number) in the PDB out of 225,946 experimentally derived PDB structures as of the 15th of October 2024.
We then explore the virion structure of Escherichia myophage phage Bas63 (formally "phageJohannRWettstein"), a representative of the new BASEL phage collection and a member of the Felixounavirus genus. We first identify a novel pseudo symmetric capsid decoration protein putatively related to the phage T4 highly immunogenic outer capsid protein, and a collar/whisker assembly with the closest structural homology to a distantly related podophage tail fibre assembly. We also observe three-fold and six-fold TMP regions similar to PhiTE, and a system of long tail fibres that represent a simplified single gene product version of the intricate T4 long tail fibre.
Finally, we investigate the virion structure phage PhiM1, another infectious agent against Pectobacterium atrosepticum. Unlike the two former long tailed phages, PhiM1 utilizes a scarcely understood internally packaged structure known as the ejectosome that is embedded into the host cell wall during infection. Using cryo-EM, we produced the complete virion structure of PhiM1 including the intricate ejectosome, which represents the first T7-like ejectosome described (omitting T7 itself). We further describe several unique capsid decoration proteins, we show putative DNA density spiralling the ejectosome (not observed in phage T7), and we identify the presence of multiple non-identical isomeric virion sub-populations that complicates the process of bacteriophage asymmetric reconstruction.
Combing the outputs of the projects described in this thesis, we have modelled 50 unique proteins (120 chains if we include conformational variants), 28 of which are currently deposited in the protein data bank.