Genetic and Structural Implications of Bacteriophage P1 Assembly and Infection

Abstract

Coliphage P1 has been involved in major advances of molecular and microbiological tools. Nonetheless, modern molecular and microscopy applications have not been applied to further understand bacteriophage P1. Structural work to understand the P1 virion was conducted in the 1980’s but has not been pursued since then. Furthermore, while the defense against restriction system of P1 has been observed to be packaged into the P1 virion in the stepwise manner, little is known about the genetic underpinnings of the anti-restriction system. In this work, mass-spectrometry was used to better understand P1 structural proteins. A combination of mass-spectrometry and bioinformatics suggested an appropriate role for P1 proteins identified by mass-spectrometry. I also report on the effects genetic manipulation have on the defense against restriction system, and to identify key motifs that are responsible for the anti-restriction activity. In addition, I report on the capsid localization signal of a key anti-restriction enzyme and demonstrate the capsid localization signal is sufficient to package mCherry into the P1 capsid. Finally, I report on a novel prong protein located on the P1 central spike. This prong protein is encoded by upfB and the lack of the prongs from the P1 virion result in an observable difference in infection of V. cholerae and particular Enterobacteriaceae. This work provides a foundation for the P1 structural identity as well as a basis to explore the defense against restriction system in further detail. Additionally, the identification of the P1 prong and its open reading frame can be used to identify homologous genes in other bacteriophage backgrounds.