PONE-D-23-33472

Overexpression of the flagellar motor protein MotB sensitizes Bacillus subtilis to aminoglycosides in a motility-independent manner

PLOS ONE

Dear Dr. Kaito,

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.

The reviewers highlight major concerns about potential physiological complications arising from MotA/MotB protein overexpression and question the relevance of findings in a biological context. Differential expression levels of different stator types and the lack of systematic testing of different antibiotic concentrations hinder drawing reliable conclusions. Reviewer 2 raises a key point about protein misfolding potentially influencing observations. The revised manuscript should address these concerns and alternate possibilities through additional experiments and textual modifications.    

Please submit your revised manuscript by Jan 04 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,

Pushkar P Lele

Academic Editor

PLOS ONE

Journal Requirements:

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

1. 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. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.  

Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services.  If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free.

Upon resubmission, please provide the following: 

● The name of the colleague or the details of the professional service that edited your manuscript

● A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)

● A clean copy of the edited manuscript (uploaded as the new *manuscript* file)

3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. 

  

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

[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: No

**********

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

Reviewer #1: Yes

Reviewer #2: N/A

**********

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: This manuscript is admirable for its clarity and conciseness. I have absolutely no problems with it except as noted below with regard to Figure 5 and its interpretation and some suggestions for improving the figures. I also have some suggestions for further work, but I do not think they need to be incorporated into this preliminary report.

1) Questions about Figure 5A.

a) In the middle panel, the photographs do not seem consistent with the statistical evaluation. Overexpressed

MotB(D24E) looks no worse than EV, and overexpressed MotB(D24A) looks 10X better than either EV or MotB(D24E).

b) In the bottom panel. MotB(D24A) looks much worse than EV.

c) In the bottom panel, there is something wirtten that is illegible for the comparison of EV and MotB(D24E). This is

also true in Figure 1B for gentamycin.

d) Any idea why MotBD24A seems to be more abundant than WT MotB or MotBD24E?

2) General observation about the figures. The asterisks used to indicate P values are so small and blurred that they look

the same as the data points. Make them bigger/clearer.

3) The identity of SigP and AtpB should be made clear in the description of Figure 5B.

4) It is not clear from Figure 5A how important it is to have a D or an E at residue 24. I think the conclusion should be

tentative. It is also not clear why MotS does not have the same effect as MotB. Doesn't it also have an Asp residue at

the same relative position as D24 of MotB? I think the difference between proton-driven and sodium-ion-driven motility

relies on the MotA/MotP component. Was MotS expressed at the same level as MotB? It would seem that ion conduction

is not critical, so why would MotB and MotS be different?

5) On line 65, "played" should be "plays."

6) I think it would be useful to show the structures of kanamycin and gentamycin, perhaps in the Discussion, to highlight

why they might behave differently than the other antibiotics.

7) Suggestions for further work. I think they can be addressed as future investigations in the Discussion.

a) Does MotB have to associate with the peptidoglycan to have this effect? My guess would be no, as until MotB

associates with the flagellar motor, it probably does not extend its periplasmic domain to interact with the PG. However,

it should be easy enough to make MotB constructions that cannot interact with the PG.

b) Assuming that B. subtilis MotB also has a plug region, does deleting it affect the kanamycin/gentamycin

susceptibility either in the presence or absence of MotA.

c) To what extent does MotB overexpression disrupt the pmf? The diameter of a colony spreading in soft agar is a very

qualitative assay for motility, and even substantial reductions in the pmf might not affect the colony diameter very

much. This is a critical point, as the authors postulate that it is a decrease in the pmf that causes the increased

susceptibility to kanamycin and gentamycin. Therefore, better quantitative measures of flagellar performance and pmf

will be necessary to draw definitive conclusions. I think this question should be addressed a bit more fully in the

Discussion and alternative explanations, if any suggest themselves, should be considered.

Reviewer #2: Uneme and authors propose to test the effects of flagellar proteins on susceptibility to aminoglycosides. To test this, they overexpress flagellar proteins MotA and MotB individually and together in B. subtilis and examine the effects on motility and sensitivity to aminoglycosides. Overexpression of MotB leads to an increase in aminoglycoside sensitivity but does not affect sensitivity to the other classes of antibiotics tested. This sensitivity does not require overexpression of MotA, its usual binding partner. Over-expression of MotA does not alter sensitivity to aminoglycosides. Mutations in the D24A proton binding site of MotB eliminates the sensitivity observed when overexpressed. Overexpression of MotB doesn’t affect motility-dependent spreading on a soft-agar plate. The authors conclude that MotB has a novel function by increasing the susceptibility to aminoglycosides.

My primary concern is that the authors are overexpressing MotB at levels that are far an above what would normally be observed in the cell, and in doing so, are measuring phenotypes that aren’t relevant in a biological context. Therefore, the conclusion that MotB has a novel function is not supported.

Instead of a novel function, it seems more likely that MotB is misfolding because it doesn’t have its binding partner, MotA, or because protein expression is too high for the membrane to adequately support. This could lead to slightly leaky membranes that are sufficient to alter the membrane potential but still support cell division and growth. The D24A mutation in the proton binding site could be support for MotB folding properly, but it’s also likely that D24A mutation leads to a different misfolding/mis-localization since you’re eliminating a charged amino acid in the hydrophobic/transmembrane portion of the protein.

Is the doubling time altered upon overexpression of MotB?

A p-value of 0.0549 isn’t significant so the authors cannot conclude that there is a “tendency of decreased growth compared to the vector-transformed strain.” (line 76)

The statement that overexpression of MotB is motility-independent needs to be qualified. The soft-agar assay is a single time point assay that requires motility but is not sufficient to say that there is no difference in motility. The rate of spreading depends on the motility of single cells but also the rate of nutrient depletion in the agar and rate of cell division. If nutrient depletion is rate limiting, then differences in motility may not be observed. Single-cell motility assays are needed to conclude that motility isn’t impacted by overexpression of MotB.

How are single cells counted in the spot assays in Figure 2 when no individual colonies are observed, e.g., wt motB-FLAG(OE) with kanamycin or gentamicin? Where does the data come from in the bottom half of the figure? The authors state that there is “decreased growth” (line 109), but isn’t it a decreased survival since you’re reducing the number of colonies?

**********

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: 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. Please note that Supporting Information files do not need this step.

Overexpression of the flagellar motor protein MotB sensitizes Bacillus subtilis to aminoglycosides in a motility-independent manner

PONE-D-23-33472R1

Dear Dr. Kaito,

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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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,

Pushkar P Lele

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #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 #2: Yes

**********

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

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 #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 #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 #2: The authors' revised manuscript addressed all of my concerns. The growth curve and single-cell motility assays together demonstrate that the cells aren't significantly stressed with an altered PMF. The native gel is a valuable addition the manuscript because it reveals the distribution of motB in different complexes (at least under the conditions of the gel, which has detergent present). The authors hypothesize that some motB are in homo-dimers, some might be in MotAB complexes, and some are in higher molecular weight complexes, perhaps with other flagellar proteins or as very large homo-aggregates.

It's still not clear how an increased motB leads to an increased kanamycin susceptibility, which is the primary limitation of the study. Is motB acting by itself because there is not enough motA to form a complex? Is motB acting with another protein that it natively associates with but only when there is excess motB? Or is motB associating with another protein that it never binds but can at these high expression levels? In other words, is overexpressing motB revealing a new, perhaps rare, function for motB or is it an overexpression artifact?

**********

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 #2: No

**********