PONE-D-23-12094

Pan-genome analysis of six complete Paracoccus type strain genomes from hybrid next generation sequencing

PLOS ONE

Dear Dr. Daniel,

I apologize for the delay in getting back to you with a first decision on your work.

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

As you can see below, both reviewers have highlighted the importance of your study for the field. However, there are several major concerns that will require your attention before the manuscript can be considered further.

The first point is the conclusions the authors have arrived at based on the comparison of only six genomes from type specimens, which was highlighted by both reviewers. Both reviewers provide very helpful suggestions for the authors to move forward (comparison of free-living and host-associated genomes within the same species, inclusion of more genomes for comparison, or the construction a phylogenomic tree using all available 21 genomes and then further selection and analysis of diverse Paracoccus strains based on their phylogenomic positions.

Reviewer 1 further suggested adding a functional enrichment analysis to identify differentially present or absent genes in the COG/KEGG categories, along with further characterization of free-living vs. host-associated features between the species (detailed below in the major comments raised by the reviewer), with which I wholeheartedly agree. Finally, I would like to encourage the authors to pay specific attention to the clarity of their figures, as highlighted by reviewer 2. 

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PLOS 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: Partly

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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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

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 study presents a comprehensive analysis of the globally distributed bacteria genus Paracoccus, which occupies diverse ecological niches. The authors conducted phylogenomic analysis to classify previously unclassified Paracoccus strains and identified misclassifications. Additionally, pan-genome analysis was performed on Paracoccus-type strains isolated from various environments, including soils, sediments, and host associations, to investigate the role of lifestyle, adaptation potential, and genomic characteristics. However, there are some conclusions in this study that cannot be directly inferred from the results. Further analysis could be conducted to delve deeper into the comparison of genomes.

Major comments:

1. The authors compared only six complete genomes of different Paracoccus species to examine the differences between free-living and host-associated genomes. However, this result does not convincingly demonstrate the impact of lifestyle or association, as the genomes belong to different species. It is suggested that the authors compare free-living and associated genomes within the same species or include more genomes for comparison.

2. The comparison of COG classification alone does not reveal significant differences of free-living group and associated group. It is recommended that the authors perform functional enrichment analysis on the genomes, specifically identifying differentially present/absent genes in COG and KEGG categories. This analysis can be conducted using anvi’o tool(Eren, A. Murat, et al., Nature microbiology, 2021, ).

3. The authors discuss the genetic differences between free-living and host-associated Paracoccus species. It would be valuable to explore the differences in genes related to cell attachment, chemotaxis, polysaccharide synthesis, and flagella, as well as genes associated with host substance exchange, signaling molecules, and secondary metabolism. The potential functions of Paracoccus-host interaction should also be discussed in the discussion section.

Minor comments:

1. The authors could discuss the relationship between Paracoccus and its hosts, particularly in the context of genome functions.

2. For the re-sequenced four Paracoccus type strains, it would be helpful to compare their genomes with previous sequencing results. Could this information be presented in a table format?

3. In Table 1, consider adding a column to indicate the circularity or strand of contigs.

4. From lines 104-109, the authors could try using the toolkit gtdbtk(Chaumeil, Pierre-Alain, et al. 2020) for classification, as GTDB has an extensive database of genome information. This approach would assign the genomes to the species or genus level.

5. In line 144, please clarify that the reference states the boundary as 95-96%.

6. The order of Figure 4 and Figure 5 appears to be incorrect.

Reviewer #2: Title: Pan-genome analysis of six complete Paracoccus type strain genomes from hybrid next generation sequencing.

Summary: The manuscript by Hollensteiner et al. studied Paracoccus, a globally distributed genus that exhibits high taxonomic, metabolic, and lifestyle diversity, enabling it to thrive in various marine and terrestrial ecological niches. Despite its high taxonomic, metabolic, and lifestyle diversity, little was known about the adaptation strategies of Paracoccus. In this study, the genus was analyzed phylogenomically using 160 strains, resulting in the classification of 16 previously unclassified Paracoccus strains and the identification of five misclassifications. The researchers also performed a pan-genome analysis of Paracoccus strains from different ecological niches, including soils, tidal flat sediment, and host associations. By assembling six complete Paracoccus genomes, the study expanded the collection of high-quality genomes from 15 to 21. They found that Paracoccus genomes possess a high level of genomic plasticity, shaped by various mobile genetic elements, and free-living genomes tend to have larger genomes and more extra-chromosomal elements compared to host-associated strains. The presence of a vast number of adaptive genes enables Paracoccus to rapidly adapt to changing environmental conditions.

General comment: The manuscript by Hollensteiner et al. raises significant concerns regarding the selection of only six Paracoccus type strains for the overall analysis, despite the availability of 21 complete genomes (15 from previous studies and 6 from this study). The authors' rationale for choosing these specific strains based on their various environments and different lifestyles does not appear sufficient, as a pan-core genome analysis of all the complete genomes would provide a better understanding of the Paracoccus genus. Additionally, the exclusion of Paracoccus denitrificans (isolated from soil), the type species of the Paracoccus genus, is questionable since the paper focuses on the pan-genome analysis of type strains. To address these issues, the authors could have either included all 21 complete genomes in their analysis or constructed a phylogenomic tree using the 21 genomes and selected diverse Paracoccus strains based on their phylogenomic positions for further analysis. Alternatively, they could have focused solely on the six Paracoccus strains analyzed without discussing the overall genus status and type strains. Despite these concerns, the computational methods employed in this study were robust. The clarity of certain figures should be enhanced.

Specific comments:

Title (Line numbers 1-2) - Sequencing or assembly method is not crucial in this context; so, remove “from hybrid next generation sequencing” from the title and consider incorporating significant information from the conclusion section, if applicable.

Line numbers 3-4 and 12-13: Is Jacqueline Hollensteiner a corresponding author? The asterisk (*) appears after both Jacqueline Hollensteiner and Rolf Daniel, indicating that they are co-corresponding authors. However, there seems to be an issue as only one corresponding email was provided. Rectify this discrepancy.

Line number 88: Please change the term “finished” to “complete”. In NCBI, the term "complete" is used to refer to a fully sequenced and completely assembled genome.

Line number 89: Change the term “finished” to “complete”.

Line number 98: Change "61.1" to "61.5" to accurately represent the lowest GC content for P. seriniphilus DSM 14827T, which is 61.5.

Line number 110: Under the 'Phylogeny of Paracoccus' section, a phylogenomic tree can be generated using 64 type strains, and the 21 complete genomes can be highlighted within that tree. Alternatively, a separate phylogenomic tree can be constructed based specifically on the 21 complete genomes. Either of these trees would provide valuable insights to readers regarding the diversity of the type strains or complete type strains within the Paracoccus genus.

Line numbers 119-122: Fig 2 legend - These methodologies have already been presented in the “Materials and Methods” section, so remove this part from here.

Line numbers 129-130: Table 2 - “New species classifications” serial number 21 (last row of the Table) - interchange the “Corrected Classification” and “Closest type/representative strain” data. This is a typographical error.

Line number 169: The statement “These results emphasize the highly adaptive genome” - this statement is not appropriate here according to the aforesaid results.

Line number 171: The statement "There is no correlation between lifestyle and cloud gene clusters" is not suitable in its current position. It would be more appropriate to include this statement after presenting the relevant results and findings.

Line number 175: “(Fig3 D)”- correct this typo.

Line number 297: Please search for "glycine betaine" clusters. Glycine betaine is known to assist bacteria in extreme conditions, exhibiting activities such as thermal endurance and salt tolerance. Therefore, conduct a literature search for studies on these activities (thermal endurance and salt tolerance) specifically related to the Paracoccus genus and consider including them in this section.

Line numbers 323-324: Fig 5 legend - These methodologies are already presented in the “Material and Methods” section, so remove this part from here.

Line numbers 360-362: Write the name of the medium as “Paracoccus alcaliphilus medium” and include the complete information as follows: “(DSM 772, https://www.dsmz.de/collection/catalogue/microorganisms/culture-technology/list-of-media for-microorganisms)” in parentheses, similar to the format provided above (Line numbers 357-359).

Line number 374: Remove the URL from the citation as the URL might change in the future, but the paper will remain accessible. Instead, cite the paper as follows: “Shifu Chen, Yanqing Zhou, Yaru Chen, Jia Gu; fastp - an ultra-fast all-in-one FASTQ preprocessor, Bioinformatics, Volume 34, Issue 17, 1 September 2018, Pages i884-i890”. The paper has already been cited in line number 378 as reference number 66.

Line numbers 375-376, 384-385: Please consider using common terms such as "quality value" (Phred score) ≥20 and "minimum length" ≥50 bp, etc. if possible. Employing these simplified terms will facilitate the readers' comprehension of the paper and enhance their study experience.

Line number 392: Format the date “20210104” like the style of line number 393, similar to “2023-03-03”.

Line number 392: The authors have already stated "Default parameters were used for all software unless otherwise specified" in line numbers 389-390. Therefore, remove "with default parameter" from line number 392.

Line number 420: Remove the URL as there is a chance of future alterations, but note that the paper will always be accessible. The authors have already cited a paper for the software (reference number 74).

Line number 455: Write the full form of SRA.

Lines numbers 649-651: Correct the incorrect URL for the cited paper.

Fig 1: “Complete (C) and duplicated” - the abbreviation "(D)" is missing here.

Fig 3: The authors should enhance the quality of this figure as it is currently of low clarity, making it difficult to interpret.

Fig 4: Improve the quality of the figure.

S1 Fig: Increase the font size of the “COG classification” section and the “Number of proteins” on the Y-axis.

S1 Table: Remove the reference URLs and cite the papers using the format “last name et al., year”.

S2 Table: Write the organism's name in the proper format (italics) and superscript the 'T' for type strains. Additionally, add the '%' sign in the completeness, contamination, and strain heterogeneity boxes.

S3 Table: Write the organisms' names in the proper format (italics) and superscript the 'T' for type strains.

S4 Table: Write the organisms' names in the proper format (italics) and include the letter 'T' for type strains.

S5 Table: Superscript the “T” for type strains.

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Reviewer #1: Yes: Cong Fei

Reviewer #2: No

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Pan-genome analysis of six Paracoccus type strain genomes reveal lifestyle traits

PONE-D-23-12094R1

Dear Dr. Daniel,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Gabriel Moreno-Hagelsieb

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The reviewer has noted that you addressed all of their comments. I agree. It is still be a very good idea to check the manuscript for missing commas and periods, since I found several sentences that ran too long without any punctuation.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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2. 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 #2: Partly

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

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

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6. 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 #2: (No Response)

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

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