Review 1: "Sequential Membrane- and Protein-bound Organelles Compartmentalize Genomes During Phage Infection"

RR:C19 Evidence Scale rating by reviewer:

• Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review: 

In 2017, researchers first identified that several giant bacteriophages form a unique spherical structure at the center of the cell during host infection. This structure, surrounded by a protein shell, was of great interest. Subsequent studies revealed that only phage DNA and specific enzymes associated with the replication and transcription of the phage genome are located within this structure, while mRNA is transported out of this structure for ribosomal translation. Given the striking resemblance of this structure to the nucleus of a eukaryotic cell, it has been named the "phage nucleus". Additionally, it's been found that this protein shell casing protects the phage DNA from bacterial defense mechanisms that target dsDNA.

This preprint could be separated into two core parts. The first one meticulously underscores the pivotal role of ChmA, the primary protein of the phage nucleus shell, in the progression of phage infection. This revelation was elegantly achieved using the CRISPRi-ART target RNA interference approach. This methodology proved impeccable for addressing intricacies associated with phages from the Chimalliviridae family, especially considering the challenges posed by the genome's encapsulation within the phage nucleus. The data unambiguously shows that without the phage nucleus's formation, DNA replication does not occur. And there is no development of phage infection in the cells lacking ChmA synthesis.

The second part of the article delves into the early stages of E.coli cell infection by the Goslar phage. Drawing parallels with earlier observations in bacteriophages phiKZ, SPN3US, and 201phi2-1, researchers detected spherical compartments in cells during the initial phases of Goslar phage infection. Utilizing cryo-electron tomography, it was revealed that the compartment boundaries likely present a bilipid layer, indicative of a membranous compartment formation during the initial infection moments. The authors termed these compartments early phage infection (EPI) vesicles. The hypothesis presented in the article suggests that these vesicles might correlate with the DAPI-stained punctum observed within the cell early in the infection. Previous studies on phiKZ and 201phi2-1 phages have aligned the appearance of DAPI-stained puncta at infection onset with phage DNA using FISH techniques. The authors postulate the phage DNA content inside EPI vesicles based on the equal observation frequency of DAPI punctum by fluorescent microscopy and these vesicles using cryo-electron tomography in the samples from the same culture. However, this evidence seems circumstantial, only alluding to the possibility that EPI vesicles are characteristic of infected cells. Concrete evidence confirming the presence of phage DNA within these compartments would strengthen their claim. Furthermore, the authors' brief mention of polysomes likely adjacent to these membranous compartments is exciting. However, with just a solitary image hinting at polysomes in proximity to vesicles among the entire preprint and supplementary materials, one yearns for more extensive data. Considering the implications of polysomes presence (indicating transcriptional activity), any supplementary evidence the authors can provide would significantly enhance the depth of their conclusions.