PONE-D-24-26458Evidence for endogenous hydrogen peroxide production by E. coli fatty acyl-CoA dehydrogenasePLOS ONE

Dear Dr. Sirithanakorn,

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 Sep 13 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.

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'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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,

Vipin Chandra Kalia, FNASc, FAMI

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please update your submission to use the PLOS LaTeX template. The template and more information on our requirements for LaTeX submissions can be found at http://journals.plos.org/plosone/s/latex.

3. Thank you for stating in your Funding Statement: 

This study was financially supported by King Mongkut's Institute of Technology Ladkrabang (No. 2566-02-16-001) to C.S. and NIH grant GM49640 To J.A.I.

Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.  Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. 

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

4. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

We are grateful to John Cronan for providing guidance and resources throughout this investigation. This study was financially supported by King Mongkut's Institute of Technology Ladkrabang (No. 2566-02-16-001) to C.S. and NIH grant GM49640 To J.A.I

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

This study was financially supported by King Mongkut's Institute of Technology Ladkrabang (No. 2566-02-16-001) to C.S. and NIH grant GM49640 To J.A.I.

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

5. We note that your Data Availability Statement is currently as follows: All relevant data are within the manuscript and its Supporting Information files.

Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition).

For example, authors should submit the following data:

- The values behind the means, standard deviations and other measures reported;

- The values used to build graphs;

- The points extracted from images for analysis.

Authors do not need to submit their entire data set if only a portion of the data was used in the reported study.

If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories.

If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access.

6. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process.

7. 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:

Reviewers have reported that the study is interesting and needs minor revision.

You are requested to meticulously and carefully respond to the reviewers comments.

Reviewer's Comments 1:

Review comment on “Evidence for endogenous hydrogen peroxide production by E. coli fatty acyl-CoA dehydrogenase”

General comment: This is a nice piece of work on detail understanding on how superoxides and H2O2 are produced continuously in bacterial cells. Results presented in figures but not described elaborately, indeed results are given partially as in typical “results & Discussion” section, while this article has again a separate discussion section. So, it is actually better combine both sections as “Results and Discussion”, and describe a little of the Figures as a result component of the section.

Specific comment:

Lines22-23: “To avoid this problem cells maintain high levels of scavenging enzymes” provide names of a few important scavenging enzymes relevant to this study.

Lines115-116: “We observed that when the dehydrogenase, encoded by FadE, is strongly expressed, it elevates the rate of cellular H2O2 production.” Is there any information or did you try the rate of cellular H2O2 production in absence of FadE?

Line130-131: What is the source of dodecanoic acid? What concentration of ethanol was used to prepare dodecanoic acid/ and at what ratio to the LB medium ethanol was used (what would be final ethanol concentration in LB growth medium)? For H2O2 production studies in various genetic backgrounds, why you did not use WT cells along with media as controls?

Table1: write the full form of NEB. NO need to write “REF” just numbering is enough. Check for accurateness of the “Reference” column, which should have appropriate references/sources only.

Figure 6 legend, provide full forms of DTT and DHLA.

Reviewer's Comments 2:

MS # PONE-D-24-26458 intends to identify major site of ROS generation, which is ubiquitously loosely attributed to FADH2 oxidation during aerobic respiration. However, there are many oxidative pathways wherein FADH2 generates and enters the oxidative electron transport system for ATP production and electron leakage responsible for ROS generation. With the help of E. coli fadE (Acyl-CoA dehydrogenase) mutant, the generation of hydrogen peroxide has been monitored during the in vitro βoxidation of dodecanoic acid, a fatty acid, for the mutant was incapable of scavenging the generated hydrogen peroxide. The authors are inclined to believe that βoxidation generates the major source of ROS within the limits of their experimental setup. Of course, the findings are significant and prompts the further advanced work.

Experimental design and data generation is sound and appears to be reproducible. Results and Discussion are posited logically. However, the MS needs through scrutiny for standard usage of English language e.g., use “must arise” in place of “must be arise” in Line 45-46 “However, the investigators quickly inferred that these oxygen species must be arise inside cells and be capable of damaging the organism.”

[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: Review comment on “Evidence for endogenous hydrogen peroxide production by E. coli fatty acyl-CoA dehydrogenase”

General comment: This is a nice piece of work on detail understanding on how superoxides and H2O2 are produced continuously in bacterial cells. Results presented in figures but not described elaborately, indeed results are given partially as in typical “results & Discussion” section, while this article has again a separate discussion section. So, it is actually better combine both sections as “Results and Discussion”, and describe a little of the Figures as a result component of the section.

Specific comment:

Lines22-23: “To avoid this problem cells maintain high levels of scavenging enzymes” provide names of a few important scavenging enzymes relevant to this study.

Lines115-116: “We observed that when the dehydrogenase, encoded by FadE, is strongly expressed, it elevates the rate of cellular H2O2 production.” Is there any information or did you try the rate of cellular H2O2 production in absence of FadE?

Line130-131: What is the source of dodecanoic acid? What concentration of ethanol was used to prepare dodecanoic acid/ and at what ratio to the LB medium ethanol was used (what would be final ethanol concentration in LB growth medium)? For H2O2 production studies in various genetic backgrounds, why you did not use WT cells along with media as controls?

Table1: write the full form of NEB. NO need to write “REF” just numbering is enough. Check for accurateness of the “Reference” column, which should have appropriate references/sources only.

Figure 6 legend, provide full forms of DTT and DHLA.

Reviewer #2: MS # PONE-D-24-26458 intends to identify major site of ROS generation, which is ubiquitously loosely attributed to FADH2 oxidation during aerobic respiration. However, there are many oxidative pathways wherein FADH2 generates and enters the oxidative electron transport system for ATP production and electron leakage responsible for ROS generation. With the help of E. coli fadE (Acyl-CoA dehydrogenase) mutant, the generation of hydrogen peroxide has been monitored during the in vitro βoxidation of dodecanoic acid, a fatty acid, for the mutant was incapable of scavenging the generated hydrogen peroxide. The authors are inclined to believe that βoxidation generates the major source of ROS within the limits of their experimental setup. Of course, the findings are significant and prompts the further advanced work.

Experimental design and data generation is sound and appears to be reproducible. Results and Discussion are posited logically. However, the MS needs through scrutiny for standard usage of English language e.g., use “must arise” in place of “must be arise” in Line 45-46 “However, the investigators quickly inferred that these oxygen species must be arise inside cells and be capable of damaging the organism.”

**********

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: No

Reviewer #2: Yes: Shamim A Ansari

**********

[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.

Evidence for endogenous hydrogen peroxide production by  E. coli  fatty acyl-CoA dehydrogenase

PONE-D-24-26458R1

Dear Dr. Sirithanakorn,

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,

Vipin Chandra Kalia, FNASc, FAMI

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

There is a minor suggestion which may be rectified at the Galley Proof stage.

Specific comment:

The following comment was answered in the response letter but the information included in the materials and methods section was incomplete. Please re-write providing the detail rationale of not using WT cells. It is also not true that any living organism with Respiratory Electron Transport (RET) system wouldn’t generate H2O2, it may be undetectable levels.

“For H2O2 production studies in various genetic backgrounds, why you did not use WT cells along with media as controls?”

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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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 #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: N/A

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: Specific comment:

The following comment was answered in the response letter but the information included in the materials and methods section was incomplete. Please re-write providing the detail rationale of not using WT cells. It is also not true that any living organism with Respiratory Electron Transport (RET) system wouldn’t generate H2O2, it may be undetectable levels.

“For H2O2 production studies in various genetic backgrounds, why you did not use WT cells along with media as controls?”

Reviewer #2: The revised text incorporates suggestions/queries raised by both reviewers and has improved significantly. I trust the revised MS deserves to be accepted.

**********

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: Yes: Shamim A Ansari

**********