Dear Tom,

Happy new year! Thank you for submitting your manuscript entitled "Membrane permeability differentiation at the lipid divide" for consideration as a Research Article by PLOS Biology. Many thanks for your patience while I sought advice over the busy holiday period.

Your manuscript has now been evaluated by the PLOS Biology editorial staff, and an academic editor with relevant expertise has agreed to assess your revisions and responses to the authors (in an ideal world, they'll be able to make a decision themselves. There's a chance that they might ask me to obtain further advice from a specialist, but we'll cross that bridge when we come to it...)

However, whichever route we end up taking, 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 in preparation for the next step. To provide the metadata for your submission, please Login to Editorial Manager (https://www.editorialmanager.com/pbiology) within two working days, i.e. by Jan 11 2023 11:59PM.

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

Best wishes,

Roli

Roland Roberts, PhD

Senior Editor

PLOS Biology

rroberts@plos.org

Dear Tom,

Thank you for your patience while your manuscript "Membrane permeability differentiation at the lipid divide" was assessed at PLOS Biology. It has now been evaluated by the PLOS Biology editors, and an Academic Editor with relevant expertise.

Based on our Academic Editor's assessment of your revisions and your responses to the prior PNAS reviews, we are likely to accept this manuscript for publication, provided you satisfactorily address the remaining points raised by the Academic Editor (see foot of this email), and the following data and other policy-related requests.

IMPORTANT. Please attend to the following:

a) Please attend to the comments from the Academic Editor that can be found at the foot of this email. The Academic Editor (whose identity will be fairly self-evident) made it clear that while these are not mandatory, they might be advisable.

b) The current Title will be very opaque to anyone outside the immediate field, and we ask that you change it to something much more explicit and informative. We suggest "Systematic comparison of unilamellar vesicles reveals that archaeal lipid membranes are more permeable than bacterial ones."

c) Please address my Data Policy requests below; specifically, we need you to supply the numerical values underlying Figs 2ABCDEFGHIJKLMNOPQR, 3ABCDEFG, 4AB, S1ABCDEFGHIJKLMNOPQR, S2ABC, S3AB, S4ABCDEF, S5ABCDEF, S6ABCDEF, S7ABC, 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://doi.org/XXXX”

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.

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)

- 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*

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

*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 2ABCDEFGHIJKLMNOPQR, 3ABCDEFG, 4AB, S1ABCDEFGHIJKLMNOPQR, S2ABC, S3AB, S4ABCDEF, S5ABCDEF, S6ABCDEF, S7ABC. 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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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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ACADEMIC EDITOR'S COMMENTS:

1. On page 2 line 70 Introduction) the authors write:

“An additional study has shown that, in the absence of counter-ions, liposomes made of diether lipids extracted from archaea (e.g. from Halobacterium salinarum) display lower permeability to protons compared to liposomes made of lipids extracted from bacteria”.

I do not think it is possible to conduct a study without counter-ions: H+ has a charge and that charge must be balanced by something. That might be a Cl- ion. If an H+ crosses a membrane a positive charge moves into the cell, and that positive charge will oppose the influx of other H+ ions into the cell. If a Cl- also crosses into the cell, that positive charge will be partially dispersed by the negative charge. The rate at which H+ moves in therefore ultimately depends on the permeability of the membrane to Cl-, not H+. A counter-ion does not mean a positive charge moving the other way, but an ion that counters the build-up of charge, and that would include Cl-. It is therefore a counter-ion. If you used SO42- instead of Cl- and it crossed the membrane more slowly (which it probably would) then you would measure a lower permeability to protons. I have not read the papers cited but either they did not say what is imputed to them, or if they did then the authors did not know what they were talking about. Either way, this should be clarified for the authors not to look foolish to any bioenergeticist.

2. At the bottom of page 4 the authors write:

“These data suggest that the metabolic selectivity of these membrane mimics is complex and does not rely solely on basic molecular properties, such as molecular weight or hydrophobicity.”

As an aside, we have also found some strange patterns between hydrophobicity and binding interactions in Harrison et al (A biophysical basis for the emergence of the genetic code in protocells. BBA Bioenerg. 2022 1863: 148597)… It seems that a more subtle measure such as partition energy, discussed by in Caldararo & di Giulio (The genetic code is very close to a global optimum in a model of its origin taking into account both the partition energy of amino acids and their biosynthetic relationships, Biosystems 214 (2022), 104613). Some brief discussion of partition energy in terms of properties of metabolites crossing the membrane might be helpful.

3. Page 9 bottom of page:

The authors write:

“Regardless, transporter reduction may have been facilitated, in part, by the increased metabolite permeability of archaeal lipid membranes which, in turn, reduces the requirement for, or utility of, transporter proteins.”

This argument is interesting and may be true. But another possible explanation is simply differential loss of membrane proteins since then, as they are especially labile. See for example Sojo et al (Membrane Proteins Are Dramatically Less Conserved than Water-Soluble Proteins across the Tree of Life. MBE 33 (2016) 2874–2884). If, for example, archaea were more likely to be autotrophic than bacteria, then there may have been a greater tendency to lose membrane transporters as there was less need for them. Such a possibility might at least be mentioned.

4. Conclusions (page 10):

These are now admirably concise and give very little perspective. That might be a virtue, though a few extra words of context might not go amiss. I had previously specifically raised questions about the likelihood of the common ancestor of archaea being a heterotroph, and there are good recent papers from Williams et al and others on LACA and LBCA (which also imply autotrophic ancestry. Does this paper imply that archaea were heterotrophs, lived by recycling in biofilms, were autotrophs, or what? A few words that tentatively point to the significance of the paper in relation to what we think we might know from phylogenetics might not go amiss. Some of this was raised in response to the referees and could possibly be incorporated into the paper itself. I don’t need to agree; I would like them to offer something, even if couched tentatively.

Dear Tom,

Thank you for the submission of your revised Research Article "Systematic comparison of unilamellar vesicles reveals that archaeal core lipid membranes are more permeable than bacterial ones" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Nick Lane, 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.

IMPORTANT: Many thanks for making the requested changes to the manuscript and supplying data underlying the main Figs. However, I couldn't find the data underlying the Supplementary Figs (specifically, S1ABCDEFGHIJKLMNOPQR, S2ABC, S3AB, S4ABCDEF, S5ABCDEF, S6ABCDEF, S7ABC), so I'll be asking my colleagues to pass on a request to you (alongside their additional requests) to supply these and to cite the location of the data in the respective legends.

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