Cytoskeletal proteins are of ancient origin, predating the divergence of prokaryotes and eukaryotes. Although these proteins play key roles in a variety of cellular processes, the proteins that make up the prokaryotic cytoskeleton are still poorly defined. In bacteria, only a few distinct families of tubulin have been characterized: FtsZ, a widely distributed protein critical for cell division, TubZ, involved in plasmid segregation and BtubA/BtubB, whose functions are still unknown. We recently discovered a divergent tubulin-like cytoskeletal protein, PhuZ, encoded by the very large (317 kb) Pseudomonas chlororaphis bacteriophage, 201?2-1. By expressing a GFP-tagged PhuZ at low levels in Pseudomonas, we could observe filament formation during lytic phage infection. We solved the structure of PhuZ to 1.67A resolution, and found a conserved tubulin fold with a novel, extended C-terminus that we showed to be critical for polymerization both in vitro and in vivo. Surprisingly, we found that PhuZ assembles a dynamic spindle that positions a single large complex of phage DNA at the center of the cell during lytic growth. Moreover, using PhuZ mutants designed from our structure, we could show that the dynamic nature of PhuZ filaments is required for phage centering. Bacterial viral particles appear to assemble around the periphery of this central DNA mass, creating a corona-like structure similar to the replication factories of herpes viruses, whic are distantly related to dsDNA bacteriophage. This is the first example of a prokaryotic spindle that performs a genome centering function analogous to the role of microtubule-based spindles of eukaryotes. Here, we propose to elucidate the biochemical, structural, and genetic basis of the ability of PhuZ to center DNA and the underlying mechanisms by which the polymer participates in viral lytic growth. Plausible roles for the polymer and centering include: defininga site to coordinate replication and packaging, facilitating phage head and or tail assembly, and facilitating cell lysis. Not only will we seek to answer these questions, but our work will also provide new insights into how tubulin family polymers can participate in such divergent functions as cell division, separation of plasmid DNA and organizing DNA into replication factories. Specifically, we propose the following research aims: 1. Examine the role of PhuZ in viral lytic growth. 2. Examine the possible connections between PhuZ assembly and DNA replication and movement, phage assembly and cell lysis in vivo. 3. Structurally and functionally characterize the mechanism and properties of PhuZ filaments assembled in vitro. 4. Identify phage and host proteins that interact with PhuZ and determine if they affect PhuZ polymerization, localization or other aspects of function. 5. Perform electron tomography and cryoTomography at various stages of infection to gain high resolution insights into the structural organization of PhuZ and viral capsids assembled in vivo during lytic growth.
This study focuses on understanding the mechanisms by which the phage cytoskeletal protein, PhuZ, participates in viral lytic growth. The work proposed here will expand our understanding of tubulins in general, provide structural insights into how different tubulin sequences can lead to different filament morphologies and uncover the completely unexpected linkage between cytoskeletal proteins and phage replication. Since herpes virus and double stranded bacteriophage are distantly evolutionarily related, this work may also have implications for understanding conserved mechanisms involved in the assembly and function of viral replication factories.
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