UCSF

The ΦKZ-like family jumbo bacteriophage ΦPA3 infects the pathogenic bacterium Pseudomonas aeruginosa. Upon infection, the bacteriophage forms a dynamic, bipolar spindle within the host using a divergent tubulin family protein known as phage tubulin/FtsZ or PhuZ. This spindle is important for infectivity and centers the phage DNA. The phage DNA, along with the DNA replication and transcription machinery, is found inside of a proteinaceous shell, while the translation and nucleotide synthesis machinery is excluded. This partitioning of proteins according to function suggests a mechanism of selectivity, akin to that of the eukaryotic nucleus. This similarity inspired the naming of this proteinaceous, DNA-containing structure the phage nucleus. The phage nucleus is irregularly shaped and grows over the course of infection, reaching up to half the host cell size or about 0.5 μm in diameter. The dramatic phage nucleus growth is likely driven by DNA replication with which the protein shell must keep up by incorporating new subunits. The proteinaceous exterior of the ΦPA3 phage nucleus is constructed using the most highly expressed, non-virion protein, Gp53, which we name phage nuclear enclosure protein or PhuN. At the beginning of this research, PhuN had no previously characterized homologues. Since then, PhuN has been studied and observed in a series of jumbo phages spanning bacteria ranging from Escherichia coli to Serratia. Using an in vitro approach coupled with direct visualization using electron microscopy, the work presented in this dissertation begins to uncover the principles and mechanisms underlying the remarkable formation, growth, and function of the phage nucleus. In a series of collaborative work, we show that the phage nucleus shields the bacteriophage DNA from host defense systems, we utilize cryo-EM to show to 3.9 Å that the phage nucleus is enclosed in a proteinaceous 2D crystal, and, finally, I share unpublished observations worthy of further exploration. This work lays the foundations for future biochemical and structural investigation probing jumbo phage mechanisms including but not limited to phage nucleus subunit addition, selectivity, and capsid packaging.