STRUCTURAL STUDIES OF THE PHAGE G CAPSID AND HELICAL TAIL SHEATH USING CRYO-EM

Abstract

Phages, viruses that infect bacteria, have been used for many studies in understanding fundamentals of molecular biology and taking advantage of their natural antimicrobial properties (Harper 2021). They are often noted for their overwhelming abundance and are recognized as the most abundant biological entities in the world (Harper 2021). The field has grown since the early 20th century, and now, there are several classes of phages that have been observed and characterized (Ackermann 2009). Within this abundant class of biological organisms, the order called Caudovirales, is the most populated group of phages to date (Harper 2021). In this order of viruses, the dsDNA genome phages have 2 main components, the icosahedral capsid, and a tail (Harper 2021). Though many tailed phages have been studied for many decades, new information about phages is still being found. Important findings such as the CRISPR gene editing tool adapted from phages in 2007 (Barrangou, Fremaux et al. 2007) have contributed to new biotechnology that impacts human health. For this reason, studies on phages have proven to be valuable in understanding fundamental biological questions and advancing basic research. In this dissertation, we investigated phage G, which has the largest capsid and genome of propagated phage studied to date (Donelli 1968, Sun and Serwer 1997, Pope 2011, Hua, Huet et al. 2017). By studying phage G, we may add to the knowledge of this relatively unexplored group of Jumbo phages with remarkably larger genomes (>200kbp) (Yuan and Gao 2017)to understand how their structure and function may be similar or different to the commonly studied, smaller bacteriophages, such as T4 and λ. For a majority of these studies, we outline how our structural biology insights of phage G using cryo-EM (cryo-Electron Microscopy) have shown it’s icosahedral capsid of ~ 180 nm in diameter at the 5-fold icosahedral vertex is composed of hexamer and pentamer proteins similar to what’s been discovered in other, smaller tailed phages (Gonzalez, Monroe et al. 2020). Our observations from microscopy data also show unique mechanistic properties in phage G’s tail that are inconsistent with current model of tail contraction within the Myoviridae family of tailed phages. Data suggest phage G’s structure and organization of its helical tail are still similar to contractile phages such as T4 (Amos and Klug 1975, Abuladze, Gingery et al. 1994) and phi812 (Novacek, Siborova et al. 2016), however, the mechanism of the tail sheath movement is inconsistent with the existing ideas of myophage function (Harper 2021).