We observed the assembly of a nucleus-like structure in bacteria during viral infection. Using fluorescence microscopy and cryo-electron tomography, we showed that Pseudomonas chlororaphis phage 201ϕ2-1 assembled a compartment that separated viral DNA from the cytoplasm. The phage compartment was centered by a bipolar tubulin-based spindle, and it segregated phage and bacterial proteins according to function. Proteins involved in DNA replication and transcription localized inside the compartment, whereas proteins involved in translation and nucleotide synthesis localized outside. Later during infection, viral capsids assembled on the cytoplasmic membrane and moved to the surface of the compartment for DNA packaging. Ultimately, viral particles were released from the compartment and the cell lysed. These results demonstrate that phages have evolved a specialized structure to compartmentalize viral replication.

Summary We recently demonstrated that the large Pseudomonas chlororaphis bacteriophage 201φ2-1 assembles a nucleus-like structure that encloses phage DNA and segregates proteins according to function, with DNA processing proteins inside and metabolic enzymes and ribosomes outside the nucleus. Here we investigate the replication pathway of the Pseudomonas aeruginosa bacteriophages φKZ and φPA3. Bacteriophages φKZ and φPA3 encode a proteinaceous shell that assembles a nucleus-like structure that compartmentalizes proteins and DNA during viral infection. We show that the tubulin-like protein PhuZ encoded by each phage assembles a bipolar spindle that displays dynamic instability and positions the nucleus at midcell. Our results suggest that the phage spindle and nucleus play the same functional role in all three phages, 201φ2-1, φKZ and φPA3, demonstrating that these key structures are conserved among large Pseudomonas phages.

All viruses require strategies to inhibit or evade the immunity pathways of cells they infect. The viruses that infect bacteria, bacteriophages (phages), must avoid nucleic-acid targeting immune pathways such as CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) and restriction-modification (R-M) systems to replicate efficiently 1 . Here, we show that jumbo phage ΦKZ, infecting Pseudomonas aeruginosa, segregates its DNA from immunity nucleases by constructing a proteinaceous nucleus-like compartment. ΦKZ resists many DNA-targeting immune systems in vivo, including two CRISPR-Cas3 subtypes, Cas9, Cas12a, and the restriction enzymes HsdRMS and EcoRI. Cas and restriction enzymes are unable to access the phage DNA throughout the infection, but engineered re-localization of EcoRI inside the compartment enables phage targeting and cell protection. Moreover, ΦKZ is sensitive to the RNA targeting CRISPR-Cas enzyme, Cas13a, likely due to phage mRNA localizing to the cytoplasm. Collectively, we propose that Pseudomonas jumbo phages evade a broad spectrum of DNA-targeting nucleases through the assembly of a protein barrier around their genome.

Highlights d Capsids traffic along a viral encoded tubulin filament d Treadmilling of the filament provides the mechanism of capsid movement through the cell d Rotation of phage nucleus by the filament distributes capsids for efficient DNA packaging