Molecular and cellular biology of the phage nucleus and spindle

项目摘要 建立和维持亚细胞组织的能力是生物学的基本设计原则。 系统.我们最近发现，许多巨型噬菌体建立了令人惊讶的复杂亚细胞组织 包括在宿主内分隔噬菌体复制的核样结构或“噬菌体核 细胞质噬菌体核执行许多以前只归因于真核细胞核的功能 包括分离关键过程（DNA复制和修复、mRNA合成、蛋白质合成和 代谢）和选择性蛋白质输入和mRNA输出。然而，噬菌体核在结构上不同， 真核细胞核，主要由一层噬菌体编码的蛋白质组成， 与细胞质交换的明显开口，而不是含有突出的双层脂质膜 孔隙复合体。我们已经确定了构成噬菌体核壳的关键蛋白质;然而， 组装全功能噬菌体核所需的组分及其在这种多用途结构中的作用 仍然未知。在这里，我们的目标是确定和表征额外的蛋白质，需要构建一个 全功能噬菌体核，以获得更深入的了解这些未充分研究的病毒的生物学和 亚细胞区室化的基本原理以前几乎只在真核生物中观察到。 我们将使用一种综合的方法，结合遗传学，细胞生物学，生物化学和原位和体外 利用结构生物学方法研究假单胞菌和大肠杆菌中的巨型噬菌体。杆菌在一起，知识 从这个项目中获得的知识将提供对亚细胞组织基本原理的深入了解。

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.