Dear Dr Walhout,

Thank you for submitting your manuscript entitled “Bioplastic producing bacteria kill the nematode C. elegans” for consideration as a Research Article by PLOS Biology under our Portable Peer Review process.

Your manuscript has now been evaluated by the PLOS Biology editorial staff and discussed with an Academic Editor with relevant expertise. Based on this initial assessment, we would be interested in inviting you to proceed further with your submission. However, I would like to clarify how we would proceed should you wish to continue.

At this stage, no changes to the manuscript are required for submission. We ask that you complete your submission by providing the required metadata only. Once this step is completed, we would issue a Major Revision decision, outlining a set of substantial textual and interpretive revisions that we consider necessary for the manuscript to be suitable for publication at PLOS Biology. The revised manuscript would then be assessed by the Academic Editor after the revision period, without a further round of external peer review.

In particular, while the study presents compelling evidence that bacterial production of poly-β-hydroxybutyrate (PHB) is necessary and sufficient to induce lethality and physiological disruption in C. elegans, we have concerns about how this finding is currently framed and interpreted throughout the manuscript. As written, the title, abstract, Results section headings, and parts of the Discussion give the impression that the study addresses the toxicity of “bioplastics” or PHB itself. However, the data support a more specific conclusion: that C. elegans dies when fed bacteria that produce PHB (rather than PHB by itself), under the experimental conditions tested. We consider this to be an important finding in its own right, without the need to overstate the implications.

The forthcoming Major Revision decision would therefore request revisions that more closely align the claims with the data. This would include, for example, revising the title to focus on PHB-producing bacteria rather than bioplastics more broadly, and adjusting the abstract and Results language to remove or modify statements that imply direct toxicity of PHB or bioplastics per se, among other related changes.

We recognize that similar concerns were raised during prior peer review at Cell, and we appreciate the additional experiments and clarifications you have already provided. While we do not anticipate requesting further experimental work, we would require these textual and interpretive changes for the manuscript to be suitable for publication at PLOS Biology.

If you are willing to proceed under these conditions, we would be happy to invite completion of the submission. If you feel that these changes would not be compatible with your intended presentation of the work, you may of course choose not to proceed. Should you decide to continue, we would also appreciate it if you could provide the formal rejection letter from Cell, in addition to the response to the reviewers.

However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Once your full submission is complete, your paper will undergo a series of checks. To provide the metadata for your submission, please Login to Editorial Manager (https://www.editorialmanager.com/pbiology) within two working days, i.e. by Feb 07 2026 11:59PM.

During the process of completing your manuscript submission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF.

Feel free to email us at plosbiology@plos.org if you have any queries relating to your submission.

Kind regards,

Melissa

Melissa Vazquez Hernandez, Ph.D.

Associate Editor

PLOS Biology

mvazquezhernandez@plos.org

Dear Marian,

Thank you for your patience while your manuscript "Bioplastic producing bacteria kill the nematode C. elegans" was peer-reviewed at PLOS Biology.

As I mentioned before, your manuscript has now been evaluated as Portable Peer Review from Cell by the editorial team in consultation with the Academic Editor. We find the experimental work strong and the evidence convincing that bacterial production of poly-β-hydroxybutyrate (PHB) is necessary and sufficient to induce lethality and physiological disruption in C. elegans.

However, as I mentioned in the invitation letter, we have concerns about how the findings are framed throughout the manuscript. As written, the title, abstract, Results headings, and parts of the Discussion give the impression that the study demonstrates toxicity of “bioplastics” or PHB itself. In contrast, the data support a more specific conclusion: that C. elegans dies when fed bacteria that produce PHB under the experimental conditions tested.

While no additional experiments are required; we do require substantial textual revisions to align the claims with the data.

In particular, we request:

- revision of the title to focus on PHB-producing bacteria rather than “bioplastics” broadly.

- removal or revision of the Abstract statement (lines 23–24) referring to “non-toxic” biodegradable bioplastics.

- systematic adjustment of language throughout the Results and Discussion (e.g., line 170; lines 510–512) to avoid implying direct toxicity of purified PHB or commercial bioplastics, unless it is experimentally proven

- clear framing of broader environmental or biomedical implications as speculative rather than demonstrated

More specific *examples* and comments from the Academic Editor can be found at the end of this e-mail.

Given the extent of revision needed, we cannot make a decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is likely to be sent for further evaluation by all or a subset of the reviewers.

In addition to these revisions, you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests shortly.

We expect to receive your revised manuscript within 3 months. Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension.

At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may withdraw it.

**IMPORTANT - SUBMITTING YOUR REVISION**

Your revisions should address the specific points made by each reviewer. Please submit the following files along with your revised manuscript:

1. A 'Response to Reviewers' file - this should detail your responses to the editorial requests, present a point-by-point response to all of the reviewers' comments, and indicate the changes made to the manuscript.

*NOTE: In your point-by-point response to the reviewers, please provide the full context of each review. Do not selectively quote paragraphs or sentences to reply to. The entire set of reviewer comments should be present in full and each specific point should be responded to individually, point by point.

You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

2. In addition to a clean copy of the manuscript, please also upload a 'track-changes' version of your manuscript that specifies the edits made. This should be uploaded as a "Revised Article with Changes Highlighted" file type.

*Re-submission Checklist*

When you are ready to resubmit your revised manuscript, please refer to this re-submission checklist: https://plos.io/Biology_Checklist

To submit a revised version of your manuscript, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' where you will find your submission record.

Please make sure to read the following important policies and guidelines while preparing your revision:

*Published Peer Review*

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*PLOS Data Policy*

Please note that as a condition of publication PLOS' data policy (http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information (N.B. this includes any numerical values that were used to generate graphs, histograms etc.). For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5

*Blot and Gel Data Policy*

We require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare them now, if you have not already uploaded them. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. 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

Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Melissa

Melissa Vazquez Hernandez, Ph.D.

Associate Editor

PLOS Biology

mvazquezhernandez@plos.org

------------------------------------

From the Academic Editor:

Title: To me, the most important thing to change would be the title. I'd be OK with "bacterial production of poly-β-hydroxybutyrate kills the nematode C. elegans" or "poly-β-hydroxybutyrate-producing bacteria kill the nematode C. elegans" or along the lines what you had suggested as well.

Abstract: In the abstract, ideally, the sentence on bioplastics would be removed ("One such polymer, poly-β-hydroxybutyrate (PHB), is used commercially to produce ‘non-toxic’, biodegradable bioplastics."). At bare a minimum, to avoid the appearance that this study is about bioplastic toxicity, the word "non-toxic" should be removed.

Throughout the results: Replace "bioplastics", "bioplastic-producing bacteria", etc. with the specific compound name, i.e. "PHB" and "PHB-producing bacteria", etc.

→ Other examples that can be used to consider the modifications:

Chaning names of subsections in the Results and Figures similar to "PHB kills C-elegans" for something similar to "C. elegans dies when bacteria it produces PHB"

Dear Marian,

Thank you for your patience while we considered your revised manuscript "Bacteria producing the bioplastic polyhydroxybutyrate kill the nematode C. elegans" for publication as a Research Article at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors, and the Academic Editor.

Based on our Academic Editor's assessment of your revision, we are likely to accept this manuscript for publication. Please make sure to address the following data and other policy-related requests.

1) Thank you for the change in the title. We had further discussion within the team and we would like to suggest the following title to bring up also the host-microbe angle:

"Bacteria producing the biodegradable bioplastic polyhydroxybutyrate can kill their host nematode C. elegans"

2) Please add the weblink of the funding agencies in the Financial Disclosure statement in the manuscript details.

3) Please add to your Competing Interests the following statement “MA is a member of PLOS Biology’s Editorial Board. The other authors declare that no competing interests exist."

4) You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

Please supply the numerical values either in the a supplementary file or as a permanent DOI’d deposition for the following figures:

Figure 1A, 2DE, 3CFDHIE, 4A, 5CDEFIKLM, 6DEG, S1A, S2ABD, S3D

NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived (it should not present only the mean/average values).

5) Please cite the location of the data clearly in all relevant main and supplementary Figure legends, e.g. “The data underlying this Figure can be found in S1 Data” or “The data underlying this Figure can be found in https://doi.org/10.5281/zenodo.XXXXX”

6) Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

7) Please ensure that your Data Statement in the submission system accurately describes where your data can be found and is in final format, as it will be published as written there

8) Per journal policy, if you have generated any custom code during the course of this investigation, please make it available without restrictions. Please ensure that the code is sufficiently well documented and reusable, and that your Data Statement in the Editorial Manager submission system accurately describes where your code can be found. More information on our Code Policy, what and how to share can be found here: https://journals.plos.org/plosbiology/s/code-availability

Please note that we cannot accept sole deposition of code in GitHub, as this could be changed after publication. However, you can archive this version of your publicly available GitHub code to Zenodo. Once you do this, it will generate a DOI number, which you will need to provide in the Data Accessibility Statement (you are welcome to also provide the GitHub access information). See the process for doing this here: https://docs.github.com/en/repositories/archiving-a-github-repository/referencing-and-citing-content

***Insert bulleted list of Editorial Requirements HERE, including REPORTING, ETHICS & DATA REQUIREMENTS AS NECESSARY (BRING THEM UP FROM BELOW THE SIGNATURE, AS THEY ARE MUCH LESS LIKELY TO BE IGNORED)***

As you address these items, please take this last chance to 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 cover letter that accompanies your revised manuscript.

In addition to these revisions, you may need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests shortly. If you do not receive a separate email within a few days, please assume that checks have been completed, and no additional changes are required.

We expect to receive your revised manuscript within two weeks.

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include the following:

- a cover letter that should detail your responses to any editorial requests, if applicable, and whether changes have been made to the reference list

- a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable, if not applicable please do not delete your existing 'Response to Reviewers' file.)

- a track-changes file indicating any changes that you have made to the manuscript.

NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

https://journals.plos.org/plosbiology/s/supporting-information

*Published Peer Review History*

Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://plos.org/published-peer-review-history/

*Press*

Should you, your institution's press office or the journal office choose to press release your paper, please ensure you have opted out of Early Article Posting on the submission form. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. 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

Please do not hesitate to contact me should you have any questions.

Sincerely,

Melissa

Melissa Vazquez Hernandez, Ph.D.

Associate Editor

mvazquezhernandez@plos.org

PLOS Biology

Dear Dr Walhout,

Thank you for the submission of your revised Research Article "Bacteria producing the bioplastic polyhydroxybutyrate kill the nematode C. elegans" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Sebastian Winter, I'm pleased to say that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.

Please take a minute to log into Editorial Manager at http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.

PRESS: We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have previously opted in to the early version process, we ask that you notify us immediately of any press plans so that we may opt out on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study.

Sincerely,

Roli Roberts

Roland G Roberts, PhD

Senior Editor

PLOS Biology

rroberts@plos.org

on behalf of

Melissa Vazquez Hernandez, Ph.D., Ph.D.

Associate Editor

PLOS Biology

mvazquezhernandez@plos.org