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Review 3: "Sequential Membrane- and Protein-bound Organelles Compartmentalize Genomes During Phage Infection"

Overall, the reviews provide critical scrutiny of the methodology and balanced perspective on the merits and limitations of this preprint's conclusions.

Published onNov 10, 2023
Review 3: "Sequential Membrane- and Protein-bound Organelles Compartmentalize Genomes During Phage Infection"

RR:C19 Evidence Scale rating by reviewer:

  • Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.



Some bacteriophages compartmentalize their genomes within bacterial cells during early infection, in manners reminiscent of eukaryotic viruses. These bacteriophages also form a nucleus-like protein shell around their genomic DNA at later stages of infection, which is required for viability. 

Armbruster and colleagues used Cas13-based genetic knockdown to study the role of nucleus formation in the Chimalliviridae, a family of nucleus-forming bacteriophages once considered to be genetically intractable. Using a combination of genetics and microscopy, the authors conclusively show that the formation of a proteinaceous nucleus-like structure is required for both infection and DNA replication in the E. coli phage Goslar. Multiple lines of evidence support these claims. Cas13-based depletion of the nuclear lattice protein ChmA prevented phage infection which could be complemented by additional ChmA expression in trans. Overexpression of a dominant-negative ChmA truncation also blocked phage infection. When examined by single-cell microscopy, these ChmA-deficient cells lacked the proteinaceous nucleus that is typically replete with DNA and found the cell midbody of bacteria infected by Chimalliviridae phages. Instead, a single, small punctum with co-localized ChmA and DNA was seen. When DNA was visualized with the fluorescent dye DAPI, this punctum was almost identical in size and intensity to the DNA found in extracellular phage capsids. This led the authors to speculate that the punctum represented phage DNA that was injected into a bacterial cell and failed to begin replication. Corroborating this idea, E. coli’s DNA polymerase, which is required for phage DNA replication, was excluded from this punctum in ChmA knockdown cells (but was present in the nucleus of ChmA-replete cells).

The authors looked more closely at phage puncta associated with ChmA knockdown by cryo-electron tomography. In these images, the authors noted an apparent lipid bilayer surrounding the un-replicated (presumably) phage DNA and named this structure the early phage infection (EPI) vesicle. Protruding from the EPI vesicle, a string of approximately 10 ribosomes was observed, which suggested that transcripts emerge from this structure to be translated in the bacterial cytosol. Previous work had established that a viral RNA polymerase was injected with phage genomic DNA and so the authors reasoned that the observed polysomes resulted from the expression of early phage genes from this vesicle. Mass spectrometry of phage-infected cells revealed many peptides from early phage genes in both ChmA-replete and ChmA-depleted samples, suggesting that the EPI vesicle was sufficient to trigger early transcription and translation, but not DNA replication. This vesicle could explain why nascently injected Goslar phage DNA appears to resist nuclease activity before nucleus formation, as it is compartmentalized. The compartmentalization of DNA replication and transcription away from host cell cytosol is analogous to many eukaryotic viruses but does not suggest shared homology between these systems.

This work builds upon many recent discoveries concerning Chimalliviridae biology from this group and others also studying this family of “Jumbo phages”. Similar nuclear structures and phage-derived puncta have been observed in other Chimalliviridae, suggesting that advances in this work will have broad applicability across Jumbo phages. There is strong evidence supporting the main claims of this manuscript and these findings should generally be considered actionable with little to no reservation. 

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