Surface protein glycosylation conserved in the human pathogen Mycoplasma genitalium and retained in the synthetic organism JCVI-Syn3A

John William Sanford; James Mobley; Kevin Dybvig; Thomas Prescott Atkinson; James Michael Daubenspeck

doi:10.1371/journal.pone.0329506

Peer Review History

Original SubmissionApril 28, 2025
4 Jun 2025 Decision Letter - Chih-Horng Kuo, Editor Dear Dr. Daubenspeck, 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 Jul 19 2025 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. 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'. 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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.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: Two experts in mycoplasmology found the study well-conceived, clearly presented, and of interest to the field. Please revise according to their comments and suggestions. [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? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: N/A Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: Sanford et al. have described hexosylation of membrane proteins in Mycoplasma genitalium, JCVI.Syn3A (a synthetically minimized organism), and Mycoplasma mycoides subsp. capri, the naturally occurring parent of the latter. This extends observations of similar modifications in two other species of mycoplasma. The modification target amino acids are non-canonical, including not only the usual amino acids but also aspartic acid and glutamic acid. Evidence is presented that the modification is enzymatic, using available hexoses perhaps in relation to their availability in the medium as components of oligosaccharides, as opposed to glycation. The mechanism, including the identity of the enzyme that catalyzes the transfer, is unidentified. The manuscript is very well-written and the experiments are appropriate to draw the conclusions that the authors have reached, although a small number of improvements can be made to enhance clarity. Major issues: 1) Please describe why the specific bands in Figure 1 were selected for analysis. 2) Please indicate on all figures with spectra (including supplemental figures) the relationship between the peak and the identity of the modification it represents. Minor issues: 3) Line 14: Is there a reason to say "may expand" instead of "expands"? 4) Line 31: "arthritidis" 5) Line 37: Although the phrase "smallest genome... capable of self-replication" is often thrown around, I honestly don't know what it means. All organisms need externally-derived material to replicate; they don't "know" whether that material is provided by a living cell or something else. The distinction is not "self-replication"; the distinction is that they can be grown without other living cells. 6) Line 39: Biofilms are structures composed of cells as well as material they secrete or capture. Biofilms cannot be described as being secreted. 7) Figure 7 panels A-C are commonly known structures. It is unclear to me what their inclusion adds. 8) Line 292: "but" instead of "however." Reviewer #2: This manuscript reports on protein glycosylation in three Mycoplasma species with reduced genomes. The study has been conducted in a well-organized manner, and sufficient data have been presented to support the presence of glycosylation in the three Mycoplasmas. The fact that glycosylation is observed even in Syn3A, which possesses a minimal genome, is highly interesting, and the role of glycosylation in maintaining Mycoplasma cells is of great interest. The discussion is also highly engaging and is considered to provide valuable insights for future research. Minor comments: Line 44 It is stated that the strain is “a highly virulent pathogen”; however, Syn3A was derived from Mycoplasma mycoides subsp. capri (Mmc), which is known to cause mild symptoms. Mmc differs from Mycoplasma mycoides subsp. mycoides small colony (Mmm), the causative agent of contagious bovine pleuropneumonia. Therefore, the term “highly” should be removed. Line 65 It is recommended that the reasons for adding 3 mg/mL of maltose be briefly explained here. Although the reasons are clearly described in the Discussion section, it is not apparent when reading the Methods section alone. Lines 70–71 It may be helpful to explain the reasons for this overnight step. This step is presumed to serve the purpose of inactivating protease activity; however, the need for such prolonged treatment may be due to irreversible inhibition or the requirement for the reagent to penetrate into the cells. Including such a description would be informative for readers and would aid in better understanding. Line 162 The reasons for selecting bands A and B from among lots of protein bands should be provided. Lines 165–166 and Figure 2 In Figure 2, is it possible to add labels on the peaks corresponding to glucose and mannose? Tables 2 and 3 Regarding the comparison between Syn3A and its parental strain Mmc, many of the glycosylated proteins identified are different. As Syn3A is a genome-reduced strain, there are almost no additional genes compared to Mmc. Therefore, it would be expected that glycosylation detected in Syn3A should also be detectable in Mmc. The reasons for these differences should be discussed. Could this be due to the detection sensitivity or threshold settings in the MS analysis? Figures 3–6 The resolution of the figures is low and the font size is too small, making it difficult to read the text in the figures. Please improve the figures by increasing both the font size and resolution. As figure readability also depends on the final published size, the font size and resolution should be adjusted accordingly. ****** 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 ******** [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 https://doi.org/10.1371/journal.pone.0329506.r001
Revision 1
9 Jul 2025 Author Response July 8, 2025 Editor, PLoS ONE 1160 Battery Street Koshland Building East, Suite 100 San Francisco, CA 94111 Dear Sir/Ms: We thank the reviewers for their careful review of our manuscript. Please find below the detailed responses to their critiques: Reviewer #1: Major issues: 1) Please describe why the specific bands in Figure 1 were selected for analysis. We have expanded the sentence starting at line 184 to say: “Two sets of bands from gel regions that have been previously shown to contain modified proteins in M. arthritidis and M. pulmonis were excised from the Coomassie-stained gel from each organism.” 2) Please indicate on all figures with spectra (including supplemental figures) the relationship between the peak and the identity of the modification it represents. We have adjusted our figure’s visual design to more clearly show which peaks represent the modified amino acid. Minor issues: 3) Line 14: Is there a reason to say "may expand" instead of "expands"? We have removed ‘may’ from the sentence. 4) Line 31: "arthritidis" We have corrected that spelling mistake and appreciate the reviewer noting it. 5) Line 37: Although the phrase "smallest genome... capable of self-replication" is often thrown around, I honestly don't know what it means. All organisms need externally-derived material to replicate; they don't "know" whether that material is provided by a living cell or something else. The distinction is not "self-replication"; the distinction is that they can be grown without other living cells. We concur that this is a difficult phrase to word appropriately. We have changed it to “Mycoplasma genitalium (Mgen) is a human urogenital pathogen that has streamlined its 580-kbp genome under natural selective pressures resulting in the smallest genome of any identified organism capable of axenic culture.” 6) Line 39: Biofilms are structures composed of cells as well as material they secrete or capture. Biofilms cannot be described as being secreted. To improve the accuracy of our wording, we have changed it to “produces extracellular polymeric substances to form biofilms.” 7) Figure 7 panels A-C are commonly known structures. It is unclear to me what their inclusion adds. We concur that panels A-C were superfluous and have streamlined figure 7 to just show the hypothesized mechanism. 8) Line 292: "but" instead of "however." We have incorporated this change. Reviewer #2: Minor comments: 1) Line 44: It is stated that the strain is “a highly virulent pathogen”; however, Syn3A was derived from Mycoplasma mycoides subsp. capri (Mmc), which is known to cause mild symptoms. Mmc differs from Mycoplasma mycoides subsp. mycoides small colony (Mmm), the causative agent of contagious bovine pleuropneumonia. Therefore, the term “highly” should be removed. We appreciate the reviewer suggesting this phrasing implies Syn3A descends from Mmm, we have therefore removed ‘highly’ as suggested. Line 65: It is recommended that the reasons for adding 3 mg/mL of maltose be briefly explained here. Although the reasons are clearly described in the Discussion section, it is not apparent when reading the Methods section alone. We have expanded the sentence to include “3 mg/mL of maltose added to the medium to act as the required oligosaccharide substrate for mycoplasma hexosylation” to improve readability. Lines 70–71: It may be helpful to explain the reasons for this overnight step. This step is presumed to serve the purpose of inactivating protease activity; however, the need for such prolonged treatment may be due to irreversible inhibition or the requirement for the reagent to penetrate into the cells. Including such a description would be informative for readers and would aid in better understanding. To clarify the purpose of this step, we have changed the wording of the paragraph starting at line 71. We thank the reviewer for noting the ambiguity of this section of the methods section and hope this has clarified the purpose of the overnight equilibration step. Line 162: The reasons for selecting bands A and B from among lots of protein bands should be provided. We have expanded the sentence starting at line 173 to say: “Two sets of bands from gel regions that have been previously shown to contain modified proteins in M. arthritidis and M. pulmonis were excised from the Coomassie-stained gel from each organism.” Lines 165–166 and Figure 2: In Figure 2, is it possible to add labels on the peaks corresponding to glucose and mannose? We have added labels to show which peaks are which glycans on Figure 2, we thank the reviewer for improving the clarity of our figure. Tables 2 and 3: Regarding the comparison between Syn3A and its parental strain Mmc, many of the glycosylated proteins identified are different. As Syn3A is a genome-reduced strain, there are almost no additional genes compared to Mmc. Therefore, it would be expected that glycosylation detected in Syn3A should also be detectable in Mmc. The reasons for these differences should be discussed. Could this be due to the detection sensitivity or threshold settings in the MS analysis? We appreciate the reviewer asking this astute question. We believe the variability between the modified proteins in both organisms is due to our current sample size detecting few hexosylation events. We propose that with a large enough sample, we would see more overlap between proteins conserved between the two organisms with variability in glycosylation sites, assuming our model is correct. We have added a paragraph starting at line 403 conjecturing why this is the case. Figures 3–6 The resolution of the figures is low and the font size is too small, making it difficult to read the text in the figures. Please improve the figures by increasing both the font size and resolution. As figure readability also depends on the final published size, the font size and resolution should be adjusted accordingly. We have increased the resolution and adjusted the fonts and visual design of our slides to be significantly more readable. We hope this is much easier to read and follow. Attachments Attachment Submitted filename: Response to Reviewers.pdf https://doi.org/10.1371/journal.pone.0329506.r002
16 Jul 2025 Decision Letter - Chih-Horng Kuo, Editor Dear Dr. Daubenspeck, 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 Aug 30 2025 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. 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'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Chih-Horng Kuo, Ph.D. Academic Editor PLOS ONE Journal Requirements: 1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. 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: In the abstract, the sentence referring to the “smallest genome… capable of self-replication” is inconsistent between the Response and the Main Text File. There is no need to emphasize the phrase “smallest genome”, as this point is not central to the current study and may be revised as new organisms with smaller genomes are discovered in the future. In addition, the version in the Response describes Mycoplasma genitalium as “a human urogenital pathogen that has streamlined its 580-kbp genome under natural selective pressures”. I do not agree with this use of the active voice, which incorrectly implies agency in an evolutionary process. I suggest revising the sentence as follows: “We report here that this surface protein hexosylation system is conserved in Mycoplasma genitalium, a human urogenital pathogen with a 580-kbp genome that can be cultured axenically.” This manuscript can be accepted pending this final revision. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: (No Response) Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: N/A ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: My concerns have all been addressed, except that the "self-replicating" statement still appears in the abstract. Reviewer #2: (No Response) ****** 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 ******** [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 https://doi.org/10.1371/journal.pone.0329506.r003
Revision 2
16 Jul 2025 Author Response Editor, PLoS ONE 1160 Battery Street Koshland Building East, Suite 100 San Francisco, CA 94111 Dear Sir/Ms: We thank the reviewers for their careful review of our manuscript’s revisions. Please find below the detailed responses to their critiques: Reviewer #1: Major issues: In the abstract, the sentence referring to the “smallest genome… capable of self-replication” is inconsistent between the Response and the Main Text File. There is no need to emphasize the phrase “smallest genome”, as this point is not central to the current study and may be revised as new organisms with smaller genomes are discovered in the future. In addition, the version in the Response describes Mycoplasma genitalium as “a human urogenital pathogen that has streamlined its 580-kbp genome under natural selective pressures”. I do not agree with this use of the active voice, which incorrectly implies agency in an evolutionary process. I suggest revising the sentence as follows: “We report here that this surface protein hexosylation system is conserved in Mycoplasma genitalium, a human urogenital pathogen with a 580-kbp genome that can be cultured axenically.” This manuscript can be accepted pending this final revision. We sincerely appreciate Reviewer 1’s attention to detail and mindfulness of clear language through both rounds of peer review and have therefore incorporated their suggested sentence into the abstract. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0329506.r004
18 Jul 2025 Decision Letter - Chih-Horng Kuo, Editor Surface protein glycosylation conserved in the human pathogen Mycoplasma genitalium and retained in the synthetic organism JCVI-Syn3A PONE-D-25-10974R2 Dear Dr. Daubenspeck, 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. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Chih-Horng Kuo, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0329506.r005
Formally Accepted
Acceptance Letter - Chih-Horng Kuo, Editor PONE-D-25-10974R2 PLOS ONE Dear Dr. Daubenspeck, 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 You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. 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. Chih-Horng Kuo Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0329506.r006

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