Project Summary The ability to establish and maintain subcellular organization is a fundamental design principle of biological systems. We recently discovered that many jumbo phage establish surprisingly complex subcellular organization involving a nucleus-like structure or “phage nucleus” that compartmentalizes phage replication within the host cytoplasm. The phage nucleus carries out many functions previously only attributed to the eukaryotic nucleus including segregation of key processes (DNA replication and repair, mRNA synthesis, protein synthesis and metabolism) and selective protein import and mRNA export. However, the phage nucleus is structurally dissimilar to the eukaryotic nucleus, being mainly composed of a single layer of a phage-encoded protein and lacking evident openings for exchange with the cytoplasm instead of a double-layer lipid membrane containing prominent pore complexes. We have identified the key protein that makes up the phage nucleus shell; however, the components required to assemble a fully functional phage nucleus and their roles in this multipurpose structure remain unknown. Here we aim to identify and characterize additional proteins that are required to construct a fully functional phage nucleus to gain greater insight into the biology of these understudied viruses and the underlying principles of subcellular compartmentalization previously observed almost exclusively in eukaryotes. We will use an integrative approach that combines genetics, cell biology, biochemistry and in situ and in vitro structural biology approaches to study the jumbo phage in Pseudomonas and E. coli. Together, the knowledge gained from this project will provide insight into fundamental principles underlying subcellular organization.