Investigating the early stages of infection of nucleus-forming jumbo phage.

Abstract

Bacteriophages or simply “phages” - viruses that infect bacteria- are in a constant arms race. Recently, it has become evident that bacteria have evolved a plethora of immune systems to combat phage infection. On the other hand, phages have developed various mechanisms to protect themselves against host defenses. A novel mechanism used by some jumbo phages creates a protective nucleus shell around its genome. We found that the nucleus shell is mainly formed by a single phage-encoded 70-kDa protein we called chimallin. A puzzling question remains: How do jumbo phages protect their genome against DNA-targeting host defense systems at early stages of infection, prior to the nucleus shell formation? At this time, the single injected phage genome is at its most vulnerable to host defenses, and a shell would have to form swiftly after infection. Using cryo-electron tomography, we study the early stage of infection of the nucleus-forming Goslar phage that infects Escherichia coli by using a mutant strain that produces ∼0.5-μm daughter minicells. The use of minicells allows us to overcome a technically challenging step we have used in the past to study infection, i.e., cryo-focused-ion beam milling of cells to prepare thin samples amenable to cryo-ET. Minicells are thin enough to be directly imaged by cryo-ET and yield more data for analysis. Our previous work showed a potential pre-nucleus structure that protects the injected phage genome before the phage nucleus is formed, different than the mature nuclear shell. Our data can discern between two models: is the pre-nucleus formed by invagination of the inner membrane followed by chimallin decoration, or does it form swiftly after infection?