Evolution of two metabolic genes involved in nucleotide and amino acid metabolism in Pseudomonas aeruginosa

Yutong Wu; Yuqi Shi; Xiaohui Liang

doi:10.1371/journal.pone.0315931

Peer Review History

Original SubmissionAugust 6, 2024
30 Oct 2024 Decision Letter - Rajesh P. Shastry, Editor PONE-D-24-33220Evolution of two metabolic genes involved in nucleotide and amino acid metabolism in Pseudomonas aeruginosaPLOS ONE Dear Dr. Liang, 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. Please submit your revised manuscript by Dec 14 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments (if provided): [Note: HTML markup is below. Please do not edit.] 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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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 ****** 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 ****** 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 research article from Wu, Shi, and Liang presents an interesting look into how a guanidine deaminase and a branch chain amino acid transporter evolved in the priority pathogen Pseudomonas aeruginosa. By understanding how P. aeruginosa and in turn other bacteria, we can gain an understanding of how the bacterium maintains homeostasis of the nucleotide pool within the cell. This article presents a comprehensive analysis on the evolution and acquisition of these important genes. The discussion and conclusions within this research are impeccable, I found myself writing down questions while reading through the results which were subsequently comprehensively answered within the discussion. I would like to thank the authors for their attention to detail within this manuscript, it was enjoyable to read. I have some questions and very minor corrections I believe are critical to evaluate and attempt to address to improve this manuscript. These are presented in the attached documentation. Reviewer #2: 1. The manuscript relies heavily on correlative data without providing strong experimental evidence to support the conclusions drawn. For example, the functional roles of the co-expressed genes related to infection and antibiotic resistance are not adequately substantiated. More rigorous experimental validation is necessary to strengthen the claims made. 2. The interpretation of the dN/dS ratios and their implications for functional divergence is not sufficiently rigorous. The authors should provide a more critical analysis of how their findings relate to the broader context of evolutionary biology in P. aeruginosa. 3. The discussion does not adequately address the limitations of the study or the potential implications of the findings. 4. In the discussion authors should provide a stronger rationale for why these findings significantly advance our understanding of P. aeruginosa's metabolic versatility compared to previous studies. 5. The two hypotheses presented regarding the evolutionary source of BraZ are not adequately substantiated. While the authors provide some evidence for both, the discussion tends to be speculative, please justify. 6. The manuscript does not sufficiently engage with recent literature that could provide additional context for the findings. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. 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 #1: Yes: Dr Samuel Wardell Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: PONE-D-24-33220_reviewer_comments.pdf https://doi.org/10.1371/journal.pone.0315931.r001
Revision 1
14 Nov 2024 Author Response Xiaohui Liang, Ph. D. Department of Critical Care Medicine, Nanjing Drum Tower Hospital Nanjing 210008, P. R. of China Cell phone: +86 19852837758 439397013@qq.com November 1, 2024 Dear Editor, We thank you very much for giving us an opportunity to revise our manuscript and we appreciate you and reviewers very much for your comments and suggestions on our manuscript entitled “Evolution of two metabolic genes involved in nucleotide and amino acid metabolism in Pseudomonas aeruginosa” (PONE-D-24-33220). We have carefully read the reviews' comments and tried our best to revise our manuscript according to the reviewer's questions and suggestions. The detailed explanation of revision in response to the reviewers' concerns is given point by point in the following pages, and the part we have modified were marked in red in the revised manuscript. We greatly appreciate your encouragement, and thank you for consideration. Looking forward to hearing from you. Sincerely yours, Xiaohui Liang Reviewer 1 Questions: Can the authors comment on the possibility of a gene duplication events occurring in P. aeruginosa ancestors to acquire new genes which can then diversified and co-evolved, compared acquisition of these genes from external means. Is it more or less likely to occur via external ancestors like shown here? Answers: Thank you for your questions. It is a very interesting suggestion. According to our literature research, gene duplication events are common in organisms with cellular structures, including animals, plants, and microorganisms (Kuzmin et al., 2022). Previous studies have also found gene duplication-amplification (GDA) event or gene duplication event in P. aeruginosa. For example, Toussaint et al (2017) identified a gene duplication-amplification (GDA) event covering several genes, including the quorum-regulated nucleoside hydrolase gene, nuh, and PA0148, encoding an adenine deaminase in P. aeruginosa. And Gorecki et al (2020) revealed an ancient gene duplication as the origin of the MdtABC efflux pump in P. aeruginosa using phylogenetic analysis, and conformed that this gene duplication was an ancient event which occurred before the split of Proteobacteria into Alpha-, Beta- and Gamma- classes. Therefore, gene duplication events occurring in P. aeruginosa ancestors are possible. The main sources of new genes are gene replication events or horizontal gene transfer events. As the reviewer 1 pointed out that new genes could diversified or co-evolved, both for these two types of evolutionary events. Based on the presence patterns of new genes in related species, the evolutionary origin of new genes can be clarified. It will be an interesting evolutionary research to compare new genes obtained from gene replication with those obtained from external means. Whereas, this work aimed at revealing the evolutionary scenario of metabolic genes with two copies unique to P. aeruginosa, possibly via the horizontal gene transfer events. Minor revisions: General comment/ Please ensure throughout the nomenclature for genes and proteins is correct and consistent. For example, guaD (gene, italics) and GuaD (protein, non-italics capitalised first letter). Answers: Thank you for your questions and sorry for our unscientific writing about the nomenclature for genes and proteins. Here, we have carefully checked the manuscript and corrected the writing errors to ensure throughout the nomenclature for genes and proteins is correct and consistent in the revised manuscript. General comment/ I find the introduction is lacking somewhat. More information could be included about what is currently known about the function of guanidine deaminase and BCAA specific transporters, and why they would be important to P. aeruginosa metabolism and evolution. (i.e., What do they do and why?) Answers: Thank you for your suggestion and sorry for lacking the detailed descriptions about the function of guanidine deaminase and BCAA specific transporters. Here, we have carefully added more information about what do they and why they would be important to P. aeruginosa metabolism and evolution in the revised manuscript (line74-81, page 4). Ln 32/ “Little is known about the evolution of these two metabolic genes”. I understand you are referring to guaD and the BCAA transporter, however you only give the name of guaD. Please rephrase to refer to proteins or include the BCAA transporter gene name. Answers: Thank you for your suggestion and sorry for the unclear descriptions. Here, we have carefully corrected the corresponding descriptions in a clearer form (line32-33, page 2). About the BCAA transporter gene name, we did not discover one scientific name based on our literature research. Notably, the names of two BCAA transporters, BraB and BraZ, were unlike with that of guanine deaminase (guaD). Based on BraB and BraZ, we named the transporter specific to BCAAs as BraT by combining the transport characteristic with the previous names (Bra) for ease of reading and understanding in the revised manuscript (line31, page 2). Ln 57/ Citation 14 is incorrect, this is not the primary source for the WHO’s classification of carbapenem resistant P. aeruginosa as a priority pathogen. Please update with the official citation directly from the WHO. Answers: Thank you for your question and sorry for the unscientific citation. Here, we have carefully checked and corrected the corresponding citation in the revised manuscript (line533-537, page 24). Ln 60/ “has presented key challenges for this pathogen to adapt to environmental shifts including clinical antibiotic therapy and immune stress” Answers: Thank you for your suggestion and sorry for the unclear descriptions. Here, we have carefully corrected the corresponding descriptions in a clearer form according to your comments in the revised manuscript (line61, page 3). Ln89-99/ I appreciate the authors having included specific version of the databases and software utilized, could you please include the citations for Pfam and TIGRFAM, and the version for MUSCLE, Faops, trimA1, IQ-TREE, and iTOL (if they exists) Answers: Thank you for your question and sorry for missing the specific versions of the databases and software utilized. Here, we have carefully checked and added the corresponding descriptions in the revised manuscript (line104-106, page 5). Significantly, no specific version information of Faops was found. Ln103/ Please cite PANTHER. Answers: Thank you for your suggestion. Here, we have carefully checked and added the corresponding descriptions in the revised manuscript (line111, 115, page 6). Ln110/ I trust the BLASTP was used from BLAST+ via command line. Could you please provide version and citation for this. Answers: Thank you for your suggestion. The BLASTP was indeed used from BLAST+ via command line. Here, we have carefully checked and added the corresponding descriptions in the revised manuscript (line118, page 6). L111/ I am unsure what this sentence is trying to say. Thank you for your suggestion. The description of “Stop analyzing when the number of all hits for each round remains the same.” was indeed redundant. We have carefully deleted the corresponding descriptions in the revised manuscript (line119-120, page 6). Ln118/ Please provide a version for Clinker if it exists. Answers: Thank you for your question. Significantly, no specific version information of Clinker was found in the corresponding reference. Ln120/ As above, for PPanGGOLiN and Gephi. Thank you for your question and sorry for missing the specific versions of the software utilized. Here, we have carefully checked and added the corresponding descriptions in the revised manuscript (line129-130, page 6). Ln126/ Please include ggplot2 version used. Thank you for your question and sorry for missing the specific versions of the software utilized. Here, we have carefully checked and added the corresponding descriptions in the revised manuscript (line136, page 7). Ln131/ likewise with affy. Thank you for your question. Significantly, no specific version information of affy was found in the corresponding reference. Ln140/ and with eggNOG. Thank you for your question and sorry for missing the specific versions of the software utilized. Here, we have carefully checked and added the corresponding descriptions in the revised manuscript (line150, page 7). L141/ Include version and citation for clusterProfiler. Thank you for your question and sorry for missing the specific versions of the software utilized. Here, we have carefully checked and added the corresponding descriptions in the revised manuscript (line152, page 7). Ln142/ Was this a corrected p value or raw p value as clusterProfiler can provide both. Thank you for your question and sorry for the unclear descriptions. Here, we have carefully checked and corrected the corresponding descriptions in the revised manuscript (line152, page 7). Ln150/ I believe alongside Sood et al, this would be a more appropriate citation (10.3389/fmicb.2015.01036). Thank you for your suggestion. We have carefully checked and changed the reference in the revised manuscript according to your comments (line161, page 8). Ln164/ Is there a mislabelling in Supplementary Figure 1, I cannot distinguish the two copies of BraZ, Additionally, in these supplementary figures, genes and loci are provided but when referring to bra, or gua, you label them with protein nomenclature, see ‘general comment’ above. Thank you for your question and sorry for the unclear descriptions. Here, we have carefully checked and corrected the corresponding descriptions in the Supplemental Figures. And we carefully corrected the writing errors to ensure throughout the nomenclature for genes and proteins is correct and consistent in the revised manuscript. Ln165/ “Genomic analysis of four commonly used reference isolates, namely …”. Thank you for your suggestion. We have carefully checked and changed the corresponding descriptions in the revised manuscript according to your comments (line177, page 9). Ln167/ should this read “flanking the two copies of the two metabolic genes, except…”. Thank you for your suggestion. We have carefully checked and changed the corresponding descriptions in the revised manuscript according to your comments (line179, page 9). Ln 175/ is it possible to include gene identification for the 5 flanking genes either side in figure 1C like shown in Supplementary figure 6B. I believe this will enhance clarity. Thank you for your suggestion. We have carefully checked and added the gene identification for the 5 flanking genes either side in figure 1C in the revised manuscript according to your comments. Ln243/ Version of CD-HIT. Thank you for your question and sorry for missing the specific versions of the software utilized. Here, we have carefully checked and added the corresponding descriptions in the revised manuscript (line256, page 12). Ln243-250/ This is borderline methods, perhaps this could be included in methods rather than in results section. Although I understand why the authors have done it this way. Thank you for your suggestion. As you pointed out, those corresponding descriptions are indeed borderline methods. Whereas, we tried transferring the corresponding descriptions in results section to the methods section according to your suggestion. Unfortunately, we cannot describe the corresponding analysis content concisely and logically due to the condition and logicality of subsequent analysis process. Therefore, we still suggest that the borderline methods should be included in results section for better understanding by readers. References: Kuzmin E, Taylor JS, Boone C. Retention of duplicated genes in evolution. Trends Genet. 2022,8(1):59-72. doi: 10.1016/j.tig.2021.06.016. Toussaint JP, Farrell-Sherman A, Feldman TP, Smalley NE, Schaefer AL, Greenberg EP, Dandekar AA. Gene duplication in Pseudomonas aeruginosa improves growth on adenosine. J Bacteriol. 2017, 199(21):e00261-17. doi: 10.1128/JB.00261-17. Górecki K, McEvoy MM. Phylogenetic analysis reveals an ancient gene duplication as the origin of the MdtABC efflux pump. PLoS One. 2020, 15(2):e0228877. doi: 10.1371/journal.pone.0228877. Reviewer 2 Questions: 1. The manuscript relies heavily on correlative data without providing strong experimental evidence to support the conclusions drawn. For example, the functional roles of the co-expressed genes related to infection and antibiotic resistance are not adequately substantiated. More rigorous experimental validation is necessary to strengthen the claims made. Answers: Thank you for your questions. Based on literature research, it has encountered bottlenecks to explore novel antibiotics from the perspective of antibiotic resistance genes or to discover alternative treatment strategies for drug-resistant bacterial infections. In recent years, several high-quality studies on conferring antibiotic resistance to bacteria via metabolic changes have been published (Lopatkin et al., 2021; Palomino et al., 2023). These works have provided us with new cut-in points for dealing with antibiotic resistance. Focusing on the evolution of metabolism-related genes, an initial survey revealed that 14 metabolism-related genes exhibited two copies in several typical strains of Pseudomonas aeruginosa, whereas only one copy was found in other investigated species. This study focuses on the evolution of two genes involved in material metabolism including guanine deaminase and transporters specific to BCAAs, and aims to reveal the evolutionary characteristics of these two metabolism genes at the population level of P. aeruginosa through multi-omics analysis. As pointed out by you, this study relies heavily on correlative data without providing strong experimental evidence to support the conclusions drawn. Firstly, the conclusion drawn from this study is mainly to illustrate the possible or speculative relationship between the existence of metabolic diversity and clinical adaptability. Secondly, we do not have the capability to directly conduct control experiments on highly pathogenic bacteria such as P. aeruginosa to demonstrate that the strains with two copies do indeed have stronger environmental adaptability than those strains with a single copy. As to transporters specific to BCAAs, functional differences of BraB and BraZ in P. aeruginosa have been proven by previous experimental evidence (Hoshino 1979; Hoshino and Kageyama 1979; Uratani et al., 1989; Hoshino et al., 1990; Hoshino et al., 1991). Given this, the presence of two copies can indeed enrich the metabolic diversity of P. aeruginosa, which may help it cope with environmental stress. Finally, the co-expressed genes identified at the transcriptional level were only used to illustrate different co-expression networks between the two copies, indirectly supporting the possibility that the presence of two copies may increase the metabolic diversity of P. aeruginosa. It is not the focus of this study that more rigorous experimental validation is conducted to strengthen the claims made. 2. The interpretation of the dN/dS ratios and their implications for functional divergence is not sufficiently rigorous. The authors should provide a more critical analysis of how their findings relate to the broader context of evolutionary biology in P. aeruginosa. Thank you for your suggestion. Firstly, this study infers the divergence of evolutionary paths between two copies based on the differences in distribution patterns of dN/dS ratios (line272-276, page 13). Secondly, this study provides possible inferences of functional divergence of two copies through evidence from multiple perspectives, including distribution patterns of dN/dS ratios, transcriptional profiles, co-expressed genes and their GO terms. Finally, as to two copies of transporters specific to BCAAs, functional differences of BraB and BraZ in P. aeruginosa have been proven by previous experimental evidence (Hoshino 1979; Hoshino and Kageyama 1979; Uratani et al., 1989; Hoshino et al., 1990; Hoshino et al., 1991). Although experimental studies on the two copies of guanine deaminase have not been reported, the results obtained from multiple perspectives in this study support th Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0315931.r002
4 Dec 2024 Decision Letter - Rajesh P. Shastry, Editor Evolution of two metabolic genes involved in nucleotide and amino acid metabolism in Pseudomonas aeruginosa PONE-D-24-33220R1 Dear Dr. Liang, 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. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. 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Reviewer #1: Yes Reviewer #2: Yes ****** 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 #1: Yes Reviewer #2: Yes ****** 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 #1: Yes Reviewer #2: Yes ****** 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 #1: I appreciate the authors attention to detail in addressing the comments from the reviewers, I have no additional comments to add Reviewer #2: Overall, it is a clear and well defined manuscript. all the queries are addressed well and included in the revised manuscript. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. 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 #1: No Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0315931.r003
Formally Accepted
5 Dec 2024 Acceptance Letter - Rajesh P. Shastry, Editor PONE-D-24-33220R1 PLOS ONE Dear Dr. Liang, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Rajesh P. Shastry Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0315931.r004

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