Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

Dear Dr Beeby,

Thank you very much for submitting your manuscript "Campylobacter jejuni motility integrates specialized cell shape, flagellar filament, and motor, to coordinate action of its opposed flagella" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

All three reviewers feel enthusiastic about the study and think that it provides valuable input to the biophysical aspect of bipolar flagella-mediated motility. They have some questions and suggestions that the authors should respond to and reflect on the manuscript.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. 

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. 

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

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

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

Sincerely,

Tomoko Kubori, Ph.D.

Associate Editor

PLOS Pathogens

Nina Salama

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

All three reviewers feel enthusiastic about the study and think that it provides valuable input to the biophysical aspect of bipolar flagella-mediated motility. They have some questions and suggestions that the authors should respond to and reflect on the manuscript.

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: A complicated movement of bipolar flagella of C.jejuni was analysed using Total Internal Reflection Fluorescence Microscopy.

Visualysing each flagellum and the cell body by fluorescent labelling clearly showed the shape and handedness of flagella on the cell body.

Flagellum on the head wrapped the cell body. Handedness of flagella and the cellbody explained how they could produce a strong force in viscous media without tangling each other.

Results were well written to describe the phenomenon. An excellent work worth publishing in this jouranal. However I have several questions before publication.

Reviewer #2: This is a really interesting manuscript that attempts to solve the old problem of how bacteria with bipolar flagella coordinate these rotary motors to achieve synchronous motion. The authors discover that in high viscosity environments the leading flagellum wraps up around the cell body, thereby flipping itself to be coordinated with the lagging flagellum. The authors argue that the leading flagellum and the helical geometry of the cell body itself both contribute to the propulsion. This is a new model for bipolar helical motility and therefore exciting.

Reviewer #3: Campylobacter jejuni is amphitrichously flagellated and swims using two flagella located at each cell pole. Remarkably, these bacteria reach swimming speeds of up to 100 microns per second. How C. jejuni coordinates rotation of two opposed flagellar motors and how two flagella contribute to the motility behavior of the bacteria remains poorly understood. In the present manuscript, Cohen and colleagues used high-speed fluorescent microscopy to visualize the flagellar filament and analyze the swimming behavior of C. jejuni. Using a clever combination of various C. jejuni mutants and sophisticated microscopy approaches, the authors found that C. jejuni wraps the leading flagellar filament around the cell body, while both leading and lagging filaments rotate counter-clockwise. These results allowed the authors to propose a novel model for C. jejuni motility, where the wrapped-mode swimming behavior enables C. jejuni to efficiently move through high-viscosity environments, such as the mucus environment of the gastrointestinal tract.

The manuscript has been a pleasure to read, the data is well-presented and supports the author’s conclusions. I especially appreciated the use of a DcheY mutant to unequivocally demonstrate that both filaments of C. jejuni rotate counter-clockwise by default.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: 1. Although I have completely understood usage of "wrapped" and "unwrapped" in the text, I felt uneasy with usage in some sentences.

For example, 163: filament is wrapping the cell body but not wrapped.160: wrapped filament.

2. 172. It is better to use the pitch distance and the diameter of a helix than pitch angle, which is often used in engineering and particle physics.

Reviewer #2: 1. A key question is whether the leading flagellum provides propulsive force. I do not understand the authors' line of reasoning for why they conclude that it does. Cells with wrapped flagella move faster, but couldn't this be an inverse causation: what if high speed causes wrapping?

2. The authors use several mutants to try to address questions like flagellar number of cell shape, but they assume that these mutants do not disrupt other properties of the cell. For example, the straight mutant affects cell wall - what if it affects cell stiffness, which would also affect speed? Or if the flagella of fliI mutants are weaker?

Reviewer #3: None.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: 126: what does "stored" mean?

386: E.coli and Salmonella should be in Italic.

423: "yank" may be "pull".

448: typo, facilate.

Reviewer #2: I really enjoyed this paper and think it would be a valuable contribution to understanding the biophysical basis of bacterial motility. However, I wonder if its best home is really PLoS Pathogens. If the editor and authors think yes, then I am highly supportive. I just hope it will reach the right audience, as opposed to something like Biophysical Journal.

Reviewer #3: 1) Abstract, line 19: Enterobacteriaceael? Perhaps rephrase to ‘the norm of Enterobacteriaceae’?

2) Single flagellated cells. The authors used a DfliI mutant, which assembles flagella less frequently and thus most cells have none or only one flagellum. While Fig. S2 shows that assembled flagella have approximately the same length, can the authors exclude that the ratio of the two flagellins (FlaA/FlaB) is not changed, which would result in different properties of the filament? This might also account for the observed decreased motility?

Are there any cells in the WT, which only produce one flagellum? Even if there are only a few, it might be useful to compare motility of these uniflagellated cells with uniflagellated cells of the DfliI mutant.

3) The DcheY mutant experiment to demonstrate that the default rotational direction of the motor is CCW is a beautiful example of how simple genetics can answer a fundamental problem.

4) Does flagellin glycosylation contribute to the observed cell body wrapping of the filament? Is flagellin in the flaA_S397C mutant still glycosylated?

5) Concerning the question of how unwrapped cells are able to swim in low-viscosity media (i.e. both flagella at the opposing cell poles are unwrapped and would cancel each other’s propulsion). One possibility is that – contrary to the proposed model – the motors at the opposing cell poles rotate in different directions. Did the authors analyze the swimming behavior of their DcheY mutant in low-viscosity media? This would at least ensure that both flagella are rotating CCW and might support alternative hypotheses, e.g. that different filament lengths or helical pitches enable swimming of unwrapped cells.

6) Line 384 + 396: define the abbreviations ‘LH’ and ‘RH’ in the manuscript text.

7) Line 386: E. coli and Salmonella should be italics

8) Fig. 1B y-axis should be labeled (e.g. ‘proportion of cells’)

9) Fig. 2: The rotational direction of flagella and the cell body is indicated by arrows. Consider to use different colors for the arrows indicating rotational direction of flagella and the cell body. Also, for the first schematic, both the top flagellum and cell body are indicated to rotate CCW. This is wrong for the cell body, which should rotate CW?

10) Fig. 3: Bad quality of the schematic and figure labels

11) Fig. 4A: Scale bar is missing.

12) Fig. 4B, D, E: Should read ‘∆pgp1’

13) Fig. 5: Bad quality of the schematic and figure labels

14) Fig. 1B, 4E, 5C, S1B, S2B, S7B, S8B: Information concerning the number of replicates or samples, as well as error bars are missing and should be indicated in the figure legend.

15) Fig. S2A+B: Should read ‘∆fliI’.

16) Fig. S2B + S2: y-axis label should read ‘µm’

17) Fig. S7: Bad quality of the figure labels

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all 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.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit 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. For instructions see http://journals.plos.org/plospathogens/s/submission-guidelines#loc-materials-and-methods

Dear Dr Beeby,

Thank you very much for submitting your manuscript "Campylobacter jejuni motility integrates specialized cell shape, flagellar filament, and motor, to coordinate action of its opposed flagella" for consideration at PLOS Pathogens. 

I am grateful that the authors comprehensively addressed the comments raised by all the reviewers. Before I will officially accept the manuscript, I would like to request the authors to supply the video of the delta cheY in low viscosity media according to the Reviewer 3's Minor comment 5), as I think that the information is important for supporting the model. Please give the minor change of the text accordingly.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. 

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. 

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

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

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

Sincerely,

Tomoko Kubori, Ph.D.

Associate Editor

PLOS Pathogens

Nina Salama

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

I am grateful that the authors comprehensively addressed the comments raised by all the reviewers. Before I will officially accept the manuscript, I would like to request the authors to supply the video of the delta cheY in low viscosity media according to the Reviewer 3's Minor comment 5), as I think that the information is important for supporting the model. Please give the minor change of the text accordingly.

Reviewer Comments (if any, and for reference):

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all 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.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit 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. For instructions see http://journals.plos.org/plospathogens/s/submission-guidelines#loc-materials-and-methods

Dear Dr Beeby,

We are pleased to inform you that your manuscript 'Campylobacter jejuni motility integrates specialized cell shape, flagellar filament, and motor, to coordinate action of its opposed flagella' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted 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.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Tomoko Kubori, Ph.D.

Associate Editor

PLOS Pathogens

Nina Salama

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):