We recently described the discovery of a nucleus like structure assembled by phage 201?2-1 after infection of Pseudomonas chlororaphis. The phage nucleus is composed of a protein shell (GP105) that segregates phage and host bacterial proteins according to function, with metabolic enzymes and ribosomes in the cytoplasm and proteins related to DNA and RNA processing inside the phage nucleus. This compartment is centered by a bipolar spindle composed of the phage-encoded tubulin PhuZ. Capsids assemble on the bacterial membrane and then migrate to and dock on the surface of the phage nucleus where phage DNA is packaged. Ultimately, capsids assemble with tails to create mature particles and the cell lyses. The GP105 shell appears in our preliminary cryoEM as an irregularly shaped 5 nm wide border that encloses phage DNA. Remarkably, this shell allows selective entry or retention of specific proteins. This work raises a number of questions such as: Is the GP105 shell essential for phage replication? Are other proteins required for shell assembly? What is the structural organization of GP105 within the shell and how does it assemble and incorporate new subunits as it grows? Does it contain pores that allow diffusion of mRNA, proteins and small molecules in and out of the structure? How is the PhuZ spindle organized over time as it pushes the growing GP105 to midcell? Does the spindle participate in other aspects of phage development such as capsid movement? Here, we propose to use a combination of genetics, biochemistry, structural biology, and cell biology to study the nucleus like structure assembled by GP105 and the spindle assembled by PhuZ and determine how these two structures participate in viral lytic growth.
This study focuses on understanding the mechanisms by which a phage nucleus like structure and a tubulin based spindle participate in viral lytic growth. The work proposed here will expand our understanding of the mechanisms by which proteins can assemble to form subcellular compartments and how tubulin based spindles can transport intracellular structures.
