PONE-D-21-22171Genomic diversity of bacteriophages infecting Rhodobacter capsulatus and their relatedness to its gene transfer agent RcGTAPLOS ONE

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Reviewer #1: Yes

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Reviewer #1: N/A

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Reviewer #1: Yes

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Reviewer #1: Yes

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5. Review Comments to the Author

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Reviewer #1: In the study by Rapala and colleagues, the authors describe the isolation and characterisation of 26 phages infecting Rhodobacter capsulatus, a bacterial model for photosynthesis and nitrogen fixation for which phage discovery has been neglected for decades. The authors put particular emphasis on the genomic characterisation of the phage collection, placing the genomic sequences in the context of other R. capsulatus phages and the transfer agent RcGTA.

The study presents a detailed analysis of new phage genomes, increasing the known diversity for R. capsulatus and providing information valuable for phage researchers interested in this bacterial model. Yet, several passages of the manuscript lack information relevant to understanding the context or the methods behind the results presented, thus affecting the reproducibility of the work. Below, I have listed a series of points that I consider require addressing.

Major comments

Line (L) 26. The authors claim the report of 26 new bacteriophages; however, only 21 genome accessions are provided and from Table 3, the Results and Methods sections, it is not clear which phages are reported and sequenced in this study. The authors should clarify this in a revised version of the manuscript in all relevant sections (including the abstract) and Figure/Table legends.

L120-124. Indicate in the Table legend the number of virions measured. Likewise, as the table contains average values, include a value representing the level of variation amongst the measurements in parentheses.

L128. Why/how the potential host strains were chosen? Are they representative of the species diversity?

The methods lack critical information and precision, including

Versions and settings of the software used (even if they were run on default settings this has to be indicated)

Relevant cut-off values used to determine the sequence clusters and during the capsid and terminase BLASTp searches.

The bioinformatics strategy to predict phage life style and determining the type of genome end. The latter being particularly important to support genome completeness.

L178-180. The authors should consider repeating the search restricting the search set to viral sequences (e.g. Viruses (taxid:10239) or Caudovirales (taxid:28883)) in cases where no matches against phage genomes were found in the list of top 100 hits, and amend the relevant table and text if required.

L182. In table 4, add the % of coverage and sequence identity of the reported matches.

L190. “Very low numbers” is ambiguous, indicate the range of shared genes for singletons and in methods clarify what was the cut-off value to define them as such.

Figures 3 and 4 are redundant regarding the primary information they provide. I suggest the authors keeping Figure 4 and sending Figure 3 to supplementary material.

The manuscript contains an excessive number of genome comparisons as main figures, 7 in total (Figures 5-10 and 13). I encourage the authors to send these per-group comparisons to the supplementary material and compiling one main figure with the genome maps of the representatives of each group plus those of the singletons. Figure 13 displays relationships between some of the phages and RcGTA and can therefore stay as part of the main text.

The authors should ensure that all Figures are provided in the optimal resolution before publication, as the current ones do not allow close inspection when zooming in. This is especially relevant for the text and numbers embedded in the genome maps.

The authors report a diversity of GC content on the phage genomes sequenced. How do these different values compare to the GC content of the host genome? I recommend briefly discussing this topic in the relevant section of the manuscript.

L600. What do the authors mean by “It was not able to be further cultivated”? No new plaques were generated on a bacterial lawn from a previous plaque? Please clarify.

L649. Clarify in the figure legend what components and colours of the figure correspond to nucleotide or protein similarity and the minimum similarity value.

L634. It is quite interesting that the authors did not find many homologs between RcGTA and the phages reported. Has RcGTA been previously compared to other phages? Briefly discuss this in the relevant section (e.g. L684-685).

To ensure experiments reproducibility, it is essential that the authors provide information missing in the Methods section.

In the growth and Isolation sub-section: Add how long you grew the phage hosts in both liquid and solid cultures, including the number of propagation steps.

In the Lysogen testing sub-section: Indicate which host strains were used in these experiments; L729 - What PFU a “high titer lysate” corresponds to?; L733 and L736 - "After several days of incubation" and “several days until sufficiently grown” is ambiguous, state what the incubation period was and what sufficiently grown means (e.g. OD).

In the Sequencing and annotation sub-section: L758 - Clarify the origin and titer of the phage sample; L767 - Was the Illumina library paired-end? Clarify. Indicate the range of genome coverage achieved.

Minor comments

Considering that R. capsulatus is a model for photosynthesis, did the authors search for phage-encoded genes potentially involved in this or other cellular processes important for the host? Such genes have been previously described in phages; hence, a brief sentence in the discussion would be of value to the reader interested in this topic.

L101. Remove “gross”

L116-118. In Figure 1, it can be implied that the phage cluster is indicated above the phage name, but it would be clearer if this is indicated in the figure legend.

L131. Plaque morphology can be affected by several factors, and similar morphology does not guarantee sequence similarity. Thus, I recommend the authors focus this sentence on the patterns of plaque formation observed.

L142. If the authors are referring to plaques that are not clear, “turbid” is a more standard term than “cloudy”.

L157. Colour shades in the table are different from those used in figures. Overall, I consider the colours in the table unnecessary as the phage cluster is indicated.

L195. In Figure 2, it would be useful to mark (e.g. with an asterisk) which genomes were available prior to this study to easily distinguish them from the ones reported in the paper.

L220. Add the reference for Gepard in the figure legend.

L226. What does DSMZ stand for?

L231. Remove “phylogenomic” and clarify that the comparison was made at the nucleotide level (BLASTn).

Indicate the minimum level of pairwise sequence similarity in the legend of figures displaying comparative genome maps.

L285. Add a note indicating what “nkf” stands for.

L473. Replace “lysogens” for “prophages”

L521. “Myoviridae” shouldn’t be underscored.

L625. Remove “:”

L630. In Figure 13, both tail proteins are annotated as minor tail proteins.

L645. Indicate similarity levels at nucleotide or protein level rather than stating “substantial similarity”.

L663. What does the RMA and DWB abbreviations mean?

L681. Add a reference for “a pattern commonly seen with studies of phages”

L688-690. What is the role of the “peptidase”?

L704-706. Add an appropriate reference to this statement.

L712. Clarify how many phages reported in the study were isolated by the students.

L772-775. This paragraph should be moved into the Genome comparisons section.

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Reviewer #1: No

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Genomic diversity of bacteriophages infecting Rhodobacter capsulatus and their relatedness to its gene transfer agent RcGTA

PONE-D-21-22171R1

Dear Dr. Alvey,

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Gabriel Moreno-Hagelsieb

Academic Editor

PLOS ONE

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