Role of DNA modifications in Mycoplasma gallisepticum

Tatiana A. Semashko; Alexander A. Arzamasov; Daria V. Evsyutina; Irina A. Garanina; Daria S. Matyushkina; Valentina G. Ladygina; Olga V. Pobeguts; Gleb Y. Fisunov; Vadim M. Govorun

doi:10.1371/journal.pone.0277819

Peer Review History

Original SubmissionJune 17, 2022
17 Aug 2022 Decision Letter - Mary Bryk, Editor PONE-D-22-17373Role of DNA modifications in Mycoplasma gallisepticum PLOS ONE Dear Dr. Semashko, 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. 1. Please address comments made by the reviewer and myself. Most comments ask for clarification of statements made in the manuscript. 2. Reviewer 1 requests additional informatics work to address the probability of finding a methylation motif in a promoter region normally, given the GC content of the organism. Because this information is important to support arguments made in the manuscript, this analysis should be performed and the results included in your revised manuscript. 3. Include additional evidence to support your suggestion that the RM system was acquired by horizontal gene transfer in your revised manuscript. Please submit your revised manuscript by Oct 01 2022 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. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. 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Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Additional Editor Comments: This is a solid manuscript that is well written. I identified a few points that require clarification. Lines 176-177. The m6A modification and DNA modification motif ANCNNNNCCT were identified only in the WT strain, and the ΔS.MgaS6I strain lacked them. I do not understand why the DNA modification motif ANCNNNNCCT was not identified in the ΔS.MgaS6I strain. I did not understand the meaning of the statement in lines 177-178, "This indicates that the RM system common for M. gallisepticum strains is nonfunctional, unless in the S6 strain." Are you suggesting that the common RM system is functional in the presence of the unique RM system in MgaS6I? [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: In this work, the authors investigate genome methylation within Mycoplasma gallisepticum. The work itself is mostly solid (though I take issue with the “underrepresentation” part), so it’s rather unfortunate that most of the results are negative. The authors find one methylation motif and associate it with the one predicted functional RM system in the genome. They show this RM system basically does what RM systems are known to do, and the system has no other real impact on the physiology of the organism. It’s not transformative research, but I can’t blame the authors for that. Major comments It is my understanding that SMRT sequencing only is able to detect m6A modifications on native DNA, and special procedures have to be performed to identify m5C methylations. The authors should comment on whether they performed these procedures, otherwise we don’t know if they missed m5C modifications or not. One of the pieces of data the authors present is the general lack of methylation sites within promoter regions. However, it’s not clear if that has occurred randomly or it has been selected for. I would have liked to have seen some informatic work to give an indication on what the probability of finding a methylation motif in a promoter region would be normally, given the GC content of the organism. Is the actual occurrence less than expected by random chance? Or is it that the genome has reduced so much and the motif itself is not simple enough where just randomly the motif doesn’t occur in those spots. Basically, I want to know if nature has selected for removal of methylation motifs from promoters because it could be disruptive. Without this analysis the authors cannot state that methylation sites are “underrepresented” in these places as they do later in the manuscript. They could only say they were not detected. We need to know what the actual expected representation would be to know if the sites are actually underrepresented. As a side note, I appreciate that the authors did not try to over-extend their transcriptional change data. It would have been easy to present some of those transcriptional changes as significant in the figure, but they relied on the statistics and showed that most genes showed no significant change in gene expression. That’s good science. Line 320 – the authors state the RM system was acquired by horizontal gene transfer. That’s a big statement to make. I would make sure to provide all the evidence that supports it. The only thing that comes to mind right now is that it’s present in the S6 strain and none of the others. But there are things that could be done to back up that statement. Line 328 – honestly, I just don’t understand what the authors are talking about in this paragraph. Please clarify. Minor comments: Line 16-17 – poor grammar, should be “investigated DNA modifications of the model” Line 178-179 – I’m confused by this sentence. The authors state that the RM system common to the different strains of M. gallisepticum is non-functional, which makes sense based on the mutations in this system, but then include the clause “unless in the S6 strain”. It’s this last part I don’t understand. I thought the point was that the unique system is responsible for the methylation, but that clause indicates the common system is responsible for the methylation. Line 195 – I believe there is a typo, and it should be MgaS6I instead of MhaS6I ****** 6. 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Revision 1
15 Oct 2022 Author Response Dear Editor and Reviewer! Please accept the revised manuscript «Role of DNA modifications in Mycoplasma gallisepticum» of the following authors: Tatiana Semashko, Alexander Arzamasov, Daria Evsyutina, Irina Garanina, Daria Matyushkina, Valentina Ladygina, Olga Pobeguts, Gleb Fisunov and Vadim Govorun for consideration as a publication in PLOS ONE journal. Thank you for your time and for your appreciation of our work. We have corrected our manuscript in accordance with all your comments and suggestions. We hope you find our manuscript suitable for publication. Response to Editor. 1. Please address comments made by the reviewer and myself. Most comments ask for clarification of statements made in the manuscript. 2. Reviewer 1 requests additional informatics work to address the probability of finding a methylation motif in a promoter region normally, given the GC content of the organism. Because this information is important to support arguments made in the manuscript, this analysis should be performed and the results included in your revised manuscript. We have performed the requested calculations and corrected the text of the manuscript in accordance with results. 3. Include additional evidence to support your suggestion that the RM system was acquired by horizontal gene transfer in your revised manuscript. We included supplementary figure with comparing genomes of different strains, extended paragraph “RM systems in M. gallisepticum and their functionality” in the Results and extended the reasons of our suggestion that the RM system was acquired by horizontal gene transfer in the Discussion. Additional Editor Comments: Lines 176-177. The m6A modification and DNA modification motif ANCNNNNCCT were identified only in the WT strain, and the ΔS.MgaS6I strain lacked them. I do not understand why the DNA modification motif ANCNNNNCCT was not identified in the ΔS.MgaS6I strain. I did not understand the meaning of the statement in lines 177-178, "This indicates that the RM system common for M. gallisepticum strains is nonfunctional, unless in the S6 strain." Are you suggesting that the common RM system is functional in the presence of the unique RM system in MgaS6I? Thank you for your comments. Both these statements are the results of mistakes. We fixed them. Response to Reviewer. Major comments 1. It is my understanding that SMRT sequencing only is able to detect m6A modifications on native DNA, and special procedures have to be performed to identify m5C methylations. The authors should comment on whether they performed these procedures, otherwise we don’t know if they missed m5C modifications or not. Yes, you are right. For identification of DNA modification using SMRT sequencing without additional special procedures, 25x sequencing fold coverage is required for the identification of 4-methylcytosine, glucosylated 5-hydroxymethylcytosine, 6-methyladenine and 8-oxoguanine; 250x sequencing fold coverage is required for the identification of 5-methylcytosine and 5-hydroxymethylcytosine [https://www.pacb.com/wp-content/uploads/2015/09/WP_Detecting_DNA_Base_Modifications_Using_SMRT_Sequencing.pdf]. In our study the coverage of the genome per strand was approximately 90× for WT M. gallisepticum S6 and approximately 110× for the ΔS.MgaS6I strain. We didn’t find annotated m5C-methyltransferases in M. gallisepticum genome and didn’t identify m5C modifications. But if m5C-methyltransferases were present, we would have seen a not very significant signal for m5C modifications in our experiment. 2. One of the pieces of data the authors present is the general lack of methylation sites within promoter regions. However, it’s not clear if that has occurred randomly or it has been selected for. I would have liked to have seen some informatic work to give an indication on what the probability of finding a methylation motif in a promoter region would be normally, given the GC content of the organism. Is the actual occurrence less than expected by random chance? Or is it that the genome has reduced so much and the motif itself is not simple enough where just randomly the motif doesn’t occur in those spots. Basically, I want to know if nature has selected for removal of methylation motifs from promoters because it could be disruptive. Without this analysis the authors cannot state that methylation sites are “underrepresented” in these places as they do later in the manuscript. They could only say they were not detected. We need to know what the actual expected representation would be to know if the sites are actually underrepresented. We thank the reviewer for pointing this out. In previous version of manuscript we used two-tailed Fisher’s exact test to validate the observed number of ANCNNNNCCT motif in promoter’s regions compared to occurrence it across whole genome. The promoter sequences in M. gallisepticum S6 were determined in our previous work [Fisunov GY et. al. Reconstruction of Transcription Control Networks in Mollicutes by High-Throughput Identification of Promoters. Front Microbiol. 2016 Dec 6;7:1977]. Now according your suggestion we tried to estimate motif underrepresentation in the promoter regions through ratio of observed over expected motif counts. There are several methods to estimate the expected frequency of short oligonucleotides in the genome. The simplest ones are based on the frequencies of mono- or dinucleotides in the genome. More complex methods use the maximal order Markov model or method by Samuel Karlin [Karlin S, Cardon LR. Computational DNA sequence analysis. Annu Rev Microbiol. 1994;48:619-54.], [Elhai J. Determination of bias in the relative abundance of oligonucleotides in DNA sequences. J Comput Biol. 2001;8(2):151-75.]. We calculated ratio of observed over expected motif through frequencies of each nucleotide in M. gallisepticum S6 genome and promoter regions. For whole genome, ratio was 1.29; for promoter regions - 0.2. It means there is no ANCNNNNCCT avoidance in whole genome of M. gallisepticum S6, but it probably exists in promoter regions. We also estimated enrichment ratio of ANCNNNNCCT in set of promoter sequences and in 100 sets of random generated sequences with the same length and GC content of the M. gallisepticum S6. In this case enrichment ratio was 0.49±0.11. 3. As a side note, I appreciate that the authors did not try to over-extend their transcriptional change data. It would have been easy to present some of those transcriptional changes as significant in the figure, but they relied on the statistics and showed that most genes showed no significant change in gene expression. That’s good science. Thank you! 4. Line 320 – the authors state the RM system was acquired by horizontal gene transfer. That’s a big statement to make. I would make sure to provide all the evidence that supports it. The only thing that comes to mind right now is that it’s present in the S6 strain and none of the others. But there are things that could be done to back up that statement. The MgaS6I system does not have close homologs in other M. gallisepticum strains. Additionally, it has a unique location in a genomic context compared to other strains. We added figure S1 with alignments of genomes of M. gallisepticum different strains. We extended of arguments for our suggestion that the RM system was acquired by horizontal gene transfer in text of the manuscript: in the paragraph “RM systems in M. gallisepticum and their functionality” in the Results and in the second paragraph of the Discussion. 5. Line 328 – honestly, I just don’t understand what the authors are talking about in this paragraph. Please clarify. In this paragraph we discuss underrepresentation of recognition sites in whole genome of M. gallisepticum S6. Sites are not underrepresented comparing with genomes of other strains. We have rewritten this paragraph. Minor comments: Line 16-17 – poor grammar, should be “investigated DNA modifications of the model” Thank you, we fixed it. Line 178-179 – I’m confused by this sentence. The authors state that the RM system common to the different strains of M. gallisepticum is non-functional, which makes sense based on the mutations in this system, but then include the clause “unless in the S6 strain”. It’s this last part I don’t understand. I thought the point was that the unique system is responsible for the methylation, but that clause indicates the common system is responsible for the methylation. Thank you, we fixed this typo. Line 195 – I believe there is a typo, and it should be MgaS6I instead of MhaS6I Thank you, we fixed it. Best regards, Tatiana Semashko Address: Nauchnyj proezd 18, Moscow, Russia Email: t.semashko@gmail.com Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0277819.r002
4 Nov 2022 Decision Letter - Mary Bryk, Editor Role of DNA modifications in Mycoplasma gallisepticum PONE-D-22-17373R1 Dear Dr. Semashko, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. 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. Kind regards, Mary Bryk, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Thank you for addressing the major and minor comments thoughtfully. I found three minor errors that you will want to correct. Line 216 “was” should be “were”. Line 361 “of its own operon” should be “in its own operon”. Line 388 “this data” should be “these data”. https://doi.org/10.1371/journal.pone.0277819.r003
Formally Accepted
14 Nov 2022 Acceptance Letter - Mary Bryk, Editor PONE-D-22-17373R1 Role of DNA modifications in Mycoplasma gallisepticum Dear Dr. Semashko: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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. Mary Bryk Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0277819.r004

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