Advisory Committee Chair
Terje Dokland
Advisory Committee Members
Gail E Christie
Peter E Prevelige
Jamil S Saad
David Schneider
Document Type
Dissertation
Date of Award
2011
Degree Name by School
Doctor of Philosophy (PhD) Heersink School of Medicine
Abstract
Staphylococcus aureus bacteriophage 80α is a temperate, double-stranded DNA phage that serves as a "helper" phage for the mobilization of several S. aureus pathogenicity islands (SaPIs), including SaPI1. When mobilized by 80α, SaPI1 genomes are packaged into smaller phage-like transducing particles composed of 80α capsid (gp47), scaffolding (gp46) and portal (gp42) proteins. In this dissertation, I utilize electron microscopy and biochemistry to tease apart the structural and genetic controls and consequences of SaPI1 as a molecular pirate. More specifically, I show that two SaPI1 proteins shift assembly from 63-nm, T = 7 icosahedral capsids to 47-nm, T = 4 capsids, commensurate with the smaller SaPI1 genome. Here, we demonstrate that co-expression of 80α gp46 and gp47 in S. aureus, but not E. coli, leads to normal N-terminal processing and the formation of procapsid-related structures, suggesting that a S. aureus protease and co-factor are required for assembly. Additionally, we find that co-expression of SaPI1 proteins gp6 and gp7 with 80α gp46 and gp47 in S. aureus promotes the formation of SaPI1-sized procapsids, identifying this as the minimal system for capsid size determination. I utilized cryo-electron microscopy and icosahedral reconstruction to demonstrate that the 80α procapsid and mature capsid have T = 7 icosahedral symmetry, while in a separate study, I found that SaPI1 capsids have T = 4 symmetry. The resulting model suggests that gp47 has an HK97-like fold and is organized into pentameric and hexameric clusters that interact by prominent trimeric densities, primarily mediated by a 22-residue ß hairpin, called the P loop, and are critical for capsid stability. Capsid expansion is associated with a conformational change in the Pro 132-induced "kink" in the α3 spine helix of gp47. Gain/loss of function mutants demonstrate unequivocally that gp6 and gp7 are necessary and sufficient, and function together synergistically as an efficient, capsid size determination mechanism. Furthermore, we show that small capsid assembly interferes with growth of 80α by preventing packaging of complete phage genomes. However, failure to form small capsids was not sufficient to alleviate phage interference, suggesting an additional role for gp6, gp7 and other SaPI1 proteins in 80α interference.
Recommended Citation
Spilman, Michael, "Molecular Piracy in the Mobilization of Staphylococcus aureus Pathogenicity Island 1" (2011). All ETDs from UAB. 3018.
https://digitalcommons.library.uab.edu/etd-collection/3018
