Dear Phoebe,

Thank you for submitting your Review Commons manuscript entitled "Magnesium depletion unleashes two unusual modes of colistin resistance with different fitness costs" for consideration as a Research Article by PLOS Biology.

Your manuscript, and the reviews and your Revision Plan, have now been evaluated by the PLOS Biology editorial staff, as well as by an academic editor with relevant expertise, and I'm writing to let you know that we would like to consider your manuscript further.

However, before we can send a Major Revision decision inviting you to revise the manuscript, 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. After your manuscript has passed the checks I'll send the Major Revision decision. To provide the metadata for your submission, please Login to Editorial Manager (https://www.editorialmanager.com/pbiology) within two working days, i.e. by Sep 15 2025 11:59PM.

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

Kind regards,

Roli

Roland Roberts, PhD

Senior Editor

PLOS Biology

rroberts@plos.org

Dear Phoebe,

Thank you for your patience while we assessed your manuscript "Magnesium depletion unleashes two unusual modes of colistin resistance with different fitness costs" and read the Review Commons reviews and your revision plan. These have now been evaluated by the PLOS Biology editors and an Academic Editor with relevant expertise

In light of the Review Commons reviews, we would like to invite you to revise the work to thoroughly address the reviewers' reports, broadly along the lines of your revision plan.

IMPORTANT: In case it's helpful, the Academic Editor said the following:

"I think this is an interesting manuscript. The topic is certainly timely and relevant. The work is technically strong, and the manuscript is well-written. One could probably argue that large parts are better suited to a specialized audience, and that some aspects seem anecdotal. However, I feel that the work is strong enough overall to be considered for PLOS Biology. Revision Plan: Some of reviewer #1's requests (e.g., additional bacterial species and in vivo models) are excessive, and I agree that the authors do not need to perform these experiments. Furthermore, some of reviewer #3's more serious concerns seem to be misunderstandings. In any case, I am optimistic that the reviewers will be satisfied with the planned revisions."

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,

Roli

Roland Roberts, PhD

Senior Editor

PLOS Biology

rroberts@plos.org

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REVIEWS: see REVIEW COMMONS.

Dear Phoebe,

Thank you for your patience while we considered your revised manuscript "Magnesium depletion unleashes two unusual modes of colistin resistance with different fitness costs" for publication as a Research Article at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor, and the original Review Commons reviewers.

Based on the reviews and our Academic Editor's assessment of your revision, we are likely to accept this manuscript for publication, provided you satisfactorily address the remaining points raised by the reviewers, and the following data and other policy-related requests.

IMPORTANT - please attend to the following:

a) Please include the species names in the Title, i.e. "Magnesium depletion by Candida albicans unleashes two unusual modes of colistin resistance in Pseudomonas aeruginosa with different fitness costs"

b) Please address the remaining requests from reviewers #2 and #3.

c) Please address my Data Policy requests below; specifically, we need you to supply the numerical values underlying Figs 1DEF, 2ABCDEF, 3ABCDEG, 4ABCD, 5BCDE, 6AB, 7ABCD, S2, S3ABC, S5ABCDEFGH, S6ABCD, S7ABCDEFGHI, S8, S9ABC, S10, S11AB, S13AB, S14, S15ABC, S16ABCDEF, S17ABCDEF, S18, either as a supplementary data file or as a permanent DOI’d deposition.

d) 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://zenodo.org/records/XXXXXXXX

e) Please make any custom code available, either as a supplementary file or as part of your data deposition.

f) Please include the URLs of your funders in the Financial Disclosure statement.

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,

Roli

Roland Roberts, PhD

Senior Editor

rroberts@plos.org

PLOS Biology

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DATA POLICY:

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

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

1) 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).

2) Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication.

Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are essential for readers to assess your analysis and to reproduce it: Figs 1DEF, 2ABCDEF, 3ABCDEG, 4ABCD, 5BCDE, 6AB, 7ABCD, S2, S3ABC, S5ABCDEFGH, S6ABCD, S7ABCDEFGHI, S8, S9ABC, S10, S11AB, S13AB, S14, S15ABC, S16ABCDEF, S17ABCDEF, S18. 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).

IMPORTANT: Please also ensure that figure legends in your manuscript include information on where the underlying data can be found, and ensure your supplemental data file/s has a legend.

Please ensure that your Data Statement in the submission system accurately describes where your data can be found.

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CODE POLICY

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

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DATA NOT SHOWN?

- Please note that per journal policy, we do not allow the mention of "data not shown", "personal communication", "manuscript in preparation" or other references to data that is not publicly available or contained within this manuscript. Please either remove mention of these data or provide figures presenting the results and the data underlying the figure(s).

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REVIEWERS' COMMENTS:

Reviewer #1:

[identifies himself as Samir Giri]

This revised version of the manuscript shows a significant improvement in clarity. The authors have provided thoughtful and satisfactory responses to all my previous comments. I have no further remarks and would like to thank the authors for their excellent work on this study.

Reviewer #2:

I reviewed this manuscript a while a go through the Review Commons platform. Now, I see that it is under consideration with PLOS Biology. I was already very positive during the initial round of review and I'm still positive. The authors have adequately addressed my comments. This is strong and very detailed work on an important topic.

I have only one minor comment. The sentence on lines 427+428 starting with "As expected …" is awkward. First, the end-point resistance of P2, P5, P8 was already known from the previous work by the same authors. Moreover, the reference to Fig. 5C makes little sense as this figure shows membrane permeability and not resistance patterns. Please check these inconsistencies.

Reviewer #3:

This is a revised version of a previously assessed manuscript on how Mg levels can alter the evolution of colistin resistance in Pseudomonas aeruginosa. The authors have largely addressed all the previous concerns, and I have only a few minor comments:

1. It would be useful if the authors could list the specific mutations that are present in the chosen endpoint clones (shown in e.g. Fig.2 D-F). This will make it easier to interpret the reversion mutants.

2. Some of the figure call-outs appear to be wrong. E.g.

Lines 190-194 refer to Figures 2D-F, and mention survival, but both Figures 2D-F and Fig S3 show MICs, so it is not clear where the reversion data is shown.

Line 316: It should be Figure S10 instead of S9A-B.

3. On lines 454-455, the authors state "However, the same mutations incur high fitness costs under high Mg2+ conditions, explaining why they are unlikely to occur in Mg2+-replete conditions."

However, the authors show in Fig. 6A that the fitness costs are seen in both high and low Mg conditions, and that the likely reason these mutations are not seen in Mg-replete conditions is that they don't confer resistance under those conditions (as the authors mention and show on lines 391-393 and in Fig S15). Thus, that sentence needs to be revised.

4. On lines 264-266 the authors conclude that the htrB2 evolved mutations were partial loss-of-function alleles. But in the Discussion, on line 459, they are called "null mutations in htrB2" - this should be fixed.

5. Line 486: At what position in the gene/protein, is the 35bp deletion?

Dear Phoebe,

Thank you for the submission of your revised Research Article "Magnesium depletion by Candida albicans unleashes two unusual modes of colistin resistance in Pseudomonas aeruginosa with different fitness costs" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Tobias Bollenbach, 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

Roland G Roberts, PhD, PhD

Senior Editor

PLOS Biology

rroberts@plos.org