PONE-D-22-03108Genomic comparison of Planktothrix agardhii isolates from a Lake Erie embaymentPLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This paper focuses on the function and metabolic behavior of Planktothrix agardhii through comparative genomics research methods. As a common genus for forming harmful cyanobacterial blooms, people pay less attention to Planktothrix genus than Microcystis. The information conveyed by this article will help us better understand the role of Planktothrix in the formation of harmful algal blooms. This is the greatest value of this article. However, I find that this manuscript could be improved further. Generally, the logic of this article is not so clear. There are also many writing format problems in this article. The quality of the graphs and tables also needs to be improved.

The specific details are as follows:

1.Page 3 Line60 "P. agardhii harmful algal blooms (HABs) typically produce a suite of secondary..." may be revised to "Harmful algal blooms (HABs) typically produce a suite of secondary...".

2.Page 3 Line73 "...all of which are also found in other P. agardhii cultures across the globe (Kurmayer et al)" should add a comma.

3.There is too much space to introduce the MC. The second and third paragraphs may be better combined.

4.Page 9 Line 248 "(Supplemental figure 1)" to "(Supplemental Figure 1)".

5.Page 9 Line 253-255 "P. agardhii, Planktothrix" needs to be italicized.

6.Page 10 Line 276 "Supplementary table 3" may be revised to " Supplementary Table 3".

7.Page 14 Line 408-409 "P. agardhii " needs to be italicized.

8.Page 14 Line 409-410 "P. agardhii " needs to be italicized.

9.Page 26, Table1 is missing a title, and a three-line table format is recommended.

10.It is recommended to add a color legend to Figure 1.

11.The phylogenetic tree of Figure2, Figure3 and Figure5 is a bit rough and can be beautified again.

12.The Figure7 requires a uniform strain writing format.

13.The format of the figures and tables in the supplemental materials should be further checked.

Reviewer #2: Overall comments:

The authors have sequenced the genomes of 20 new Planktothrix strains and performed comparative genomic analysis on them. At first glance, the manuscript appeared to present interesting results on the ecology and genomics of Planktothrix. The CRISPR-Cas system analysis in this manuscript was particularly impressive. However, some of the experimental methods are questionable, and the reliability of some of the results and discussions are also questionable. Some deficiencies were found in the descriptions regarding Figures and Tables.

Specific comments:

1) Lines 43-128. Regarding the Introduction, I felt that background and purpose of this manuscript were not well organized. For example, there is no need to indicate which strain produces which secondary metabolite, and no need to elaborate on the antifungal activity of unknown metabolite, which does not appear in the Results or Discussion at all.

2) Lines 156-159. If the strains are non-axenic, it should be shown that the assembled genome sequences have no contaminations from other microorganisms. This point is very important to interpret the results.

3) Lines 176-191. If my understanding is correct, the authors have mapped the small contigs generated by de novo assembly to the reference sequence for the generation of scaffolds and large contigs. Do the sequences produced in this way reflect the true genomic arrangement? If not, how should I interpret the results of the subsequent analysis of genome rearrangement by mauve and the Average Nucleotide Identity and Alignment Percentage?

4) Regarding Figure 2, how did authors make this phylogenetic tree from the AP and ANI analysis. What does the bar with the number 0.250 mean? I could not understand Lines 235-238.

5) Regarding Figure 3 and Lines 252-263, why did the authors choose the UPGMA method rather than other methods such as the maximum likelihood method to infer phylogenetic tree? It would be better to estimate the phylogenetic tree using the maximum likelihood method and show the reliability of the branches using the bootstrap method. In addition, few genes were used to estimate the phylogenetic tree, and therefore the resolution of the phylogenetic tree is very low. The authors should add more genes and increase the resolution. What does the bar with the number 0.001 in the Figure mean? Please describe it in the Figure legend.

6) Lines 265-289. Why did the authors identify the genes of interest by the annotation name? Gene annotations are sometimes wrong by several reasons. If the authors discuss whether the genes exist or not in the genomes, sequence similarity search such as BLAST would be better than the gene annotation name search. It is better to explain the validity of their gene search strategy or re-examine the gene search by sequence similarity.

7) Figure 2 and Figure 3. Topologies of the trees in Figure 2 and 3 are different. The reasons must be explained in the text.

8) Lines 373-375 and Figure 4E. For the microviridin biosynthesis, mvdE gene is essential because the gene encodes prepeptide, but this manuscript lack this gene. The gene clusters should be compared including the gene.

9) Lines 380-384. Why did the authors concatenate the secondary metabolite gene clusters? The length of each gene cluster is different and the mutation rate seems to be different. Moreover, some strains do not have these gene clusters. I felt that the authors have not clearly explained what the phylogenetic tree means. In addition, I could not understand the sentence "Particular isolates can be chosen at random within clusters (such as 1807, 1808, or 1809), but this analysis identifies two completely unique sequences in 1033 and 1813."

10) Line 506. "aeruginoside 102" production by P. agardhii NIES-204 is unclear form the gene cluster similarity, and the prediction is probably wrong. Generally, substrate for NRPS should be predicted from amino acid residues in a substrate-binding pocket of its adenylation domain. Some prediction programs, such as the NRPSpredictor2 (https://github.com/roettig/NRPSpredictor2) and PKS/NRPS Analysis Web-site (http://nrps.igs.umaryland.edu) have been developed. Indeed, in my test analysis using these programs, the aeruginosin gene cluster of P. agardhii NIES-204 appeared to produce a different structure of aeruginosin than "aeruginoside 102". Additionally, Shimura et al. (Harmful Algae, 101: 101942, 2021) have analyzed the gene cluster and stated as "In P. agardhii NIES-204, aerK, which is essential for aeruginosin biosynthesis, is present as aerK1 and aerK2, lacking part of the aerK sequence. Therefore, aerK may be inactive, and P. agardhii NIES-204 may not produce aeruginosins."

11) Line 65. " Dittmann et al. 1997" is not appropriate reference for the sentence because the paper only identified the microcystin gene for Microcystis. Review papers for microcystin biosynthesis may be appropriate.

12) Line 68. "P. rubescens" is italic.

13) Line 118. "dispersal or Ma-LMM01-like cyanophages" is probably a misspelling of "dispersal of Ma-LMM01-like cyanophages".

14) Line 137. Jaworski’s Medium needs a reference.

15) Line 280. Perhaps "Geneious Primer" is incorrect and "Geneious Prime" is correct.

16) Lines 292-295. Gene citations should be the papers in which the genes were originally identified from cyanobacteria. If possible, INSDC Accession numbers of Ref. genes should be appended for gene identification.

17) Line 470. "align" is maybe "aligned".

18) Line 471. "cluster" is maybe "clustered"

19) Line 473. "separate" is maybe "separated"

20) Line 475. "cluster" is maybe "clustered"

21) Lines 497-498. I could not understand "which consisted of several mutations different than the cluster found in P. agardhii NIVA-CYA 126/8,".

22) Lines 501-507. The two types of aeruginosin biosynthetic gene clusters in Planktothrix have been identified and reported by Shimura et al. (Harmful Algae, 101: 101942, 2021). The authors should cite the paper.

23) Figure 4. Could you explain what the box and line indicate and black and gray color means? Does the boxes represent presence of sequences and the lines represent sequence gaps? The width of the horizontal line is different in some areas. Please describe these points in the Figure legend.

24) Regarding Figure 5, description of phylogenetic analysis method is missing in the text.

25) Line 810. Description (title) of Table 1 is missing.

26) Regarding Table 1, I think the number in the "No. Contig" column is incorrect. I assume from Supplementary Table 1 that it is probably the total number of contigs and scaffolds. Also, the numbers for Plk1025, Plk1031, Plk1807, Plk1808, Plk1809, Plk1810, Plk1811, and Plk1813 are 16, 30, 67, 18, 14, 19, 15, and 23, as inferred from Supplement Table 1. Please check it. If possible, the completeness of the genome sequence should be shown in Table 1.

27) Line 832. Description (title) of Table 5 is missing.

28) Regarding Table 5, how did the authors compare amt1 and amt3 genes? These genes exist as different genes but represented in one column. Did the authors concatenate the genes before comparison? Please explain about that in the text. INSDC Accession numbers of Ref. genes should be appended.

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Reviewer #1: No

Reviewer #2: No

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Genomic comparison of Planktothrix agardhii isolates from a Lake Erie embayment

PONE-D-22-03108R1

Dear Dr. Bullerjahn,

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