The Architecture of a Prototypical Bacterial Virus DNA-Ejectosome
Abstract
Bacterial viruses (or bacteriophages) represent nature’s most abundant form of life on earth. They play a vital role in the formation of the human microbiome and have ever-increasing applications in the food industry, biotechnology, and phage therapy. A key process in the life cycle of bacteriophages is the ejection of viral genomes through the host cell envelope, which is remarkably complex and hard to penetrate. Of the three main superfamilies of tailed bacteriophages on planet earth, Podoviridae are double-stranded DNA viruses characterized by a short, non-contractile tail that is not long enough to span the host cell envelope. When the prototypical T7 phage infects Escherichia coli, its tail fibers interact with receptors on the outer bacterial membrane triggering the release of three ejection proteins inside the host. These proteins, gp14, gp15, and gp16, are required for infectivity as they form a conduit for viral DNA to pass into the host cytoplasm. This process is accompanied by dramatic conformational changes in gp14, gp15, and gp16 that must partially unfold and pass through a narrow pore of the portal complex and then refold and assemble into a DNA-ejectosome, which, at low-resolution forms an elongated tube-like structure. This thesis focuses on the detailed structure of this multimeric machine and the mechanism underlying its assembly. Using a bottom-up biochemical approach, we purified all T7 ejection proteins and reconstituted complexes of the DNA-ejectosome, which we used for structural studies by cryo-electron microscopy single-particle analysis. We found that the smallest ejection protein, gp14 is an outer membrane protein that forms a constitutively open pore and is likely the first to be ejected from the capsid. Gp15 and the N-terminal portion of gp16 (gp16-N) form a hexameric tube-like assembly that spans the length of the periplasm connecting the outer membrane to the inner membrane. The C-terminal portion of gp16 (gp16-C) likely forms a transient pore in the inner membrane and projects a large cytoplasmic domain that may act as a motor for genome delivery. Altogether, this work describes the first high-resolution structural model for a prototypical DNA-ejectosome and lays the foundation for studying ejection proteins in other bacterial viruses of the Podoviridae family.
Subject Area
Biochemistry|Virology
Recommended Citation
Swanson, Nicholas A, "The Architecture of a Prototypical Bacterial Virus DNA-Ejectosome" (2021). ProQuest ETD Collection - Thomas Jefferson University. AAI28773981.
https://jdc.jefferson.edu/dissertations/AAI28773981
