Dear Dr. Choi,

Thank you very much for submitting your manuscript "Two novel genes identified by large-scale transcriptomic analysis are essential for biofilm and rugose colony development of Vibrio vulnificus" 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.

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,

Matthew Parsek, PhD

Associate Editor

PLOS Pathogens

Karla Satchell

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):

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: In this manuscript, Lee and colleagues use a machine learning algorithm to identify groups of co-regulated genes, termed iModulons, in the Vibrio vulnificus genome linked to biofilm formation. The bioinformatic deep-dive was executed by using independent component analysis (ICA) to re-analyze hundreds of transcriptomic datasets publicly available through NCBI. This approach was validated by identifying 2 additional genes, cabH and bprN, that are part of the BpsT regulon, which regulates extracellular polysaccharide production and biofilm formation in V. vulnificus.

The manuscript is technically sound. The approach to genetic analysis for cabH and bprN is thorough – including phenotyping of mutants, complementation analysis, and demonstration of BpsT-dependent gene expression using EMSAs, which are not trivial. Taken together, this comprehensive approach provides confidence in the genetic linkages and provides proof-of-principle for the bioinformatic approach. I enjoyed reading this article.

Reviewer #2: The ability to form multicellular bacterial communities, or biofilms, is predominant among bacterial species. Biofilm formation provides protection to the bacterial community and allows opportunities for nutrient acquisition/concentration and horizontal gene transfer. This protected communal lifestyle allows for the survival and persistence of bacteria in nature. Among biofilm-forming bacteria, are naturally occurring aquatic Vibrio species. Vibrionaceae species such as V. cholerae, V. parahaemolyticus, and V. vulnificus are facultative human pathogens, with significant and devastating impacts to public health. Biofilm formation is key to the environmental survival and persistence of Vibrio species and can impart a substantial role in host pathogenesis and disease. In this work, Lee et al. performed an in-depth analysis of transcriptome data comparing planktonic and biofilm modalities within the seafood-borne pathogen V. vulnificus. The objective was to use an unbiased machine learning algorithm, Independent Component Analysis (ICA), to identify genetic regulons (iModulons) related to biofilm and rugose colony formation in V. vulnificus.

The authors identified several biofilm-associated iModulons; one of which contained genes modulated by the known V. vulnificus biofilm transcriptional regulator, BrpT. Along with genes known to be regulated by BrpT, the authors identify two novel BrpT-regulated genes of unknown function: VV1_3061(cabH) and VV2_1694 (brpN). The authors next validate these two genes as BrpT-regulated through qRT-PCR, and predict their function based on structural homology to known V. vulnificus genes. The authors then beautifully demonstrate that CabH appears to be involved in surface-attachment, and BrpN appears to be involved in production or secretion of exopolysaccharide. Overall, I feel that the quality of work and data analysis is strong, and that the results match the interpretation developed by the authors.

I feel that the work presented here is novel and represents a very significant advancement for the study of biofilm regulation in bacteria. The authors present a very well written and detailed step-by-step procedure for the computational identification of previously unknown genes involved in V. vulnificus biofilm formation, and for molecular verification of results predicted by computational analysis. Not only is this work significant for the study of both V. vulnificus and biofilm biology, but it is also a significant step forward in development and utilization of unbiased data analysis methods for the interpretation of large transcriptome datasets. I do feel that this work is acceptable for publication in PLoS Pathogens and would be of key interest to its audience. Therefore, with consideration of the minor revisions outlined below, I would recommend publication of this work.

**********

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. Perhaps the most interesting thing in the paper is the analysis of transcriptional regulatory networks in V. vulnificus using ICA to identify iModulons. However, relatively little information about this machine learning approach is presented in the manuscript. The descriptions leave me wanting to know more. I suspect it will be the same for other readers. Could the authors provide an expanded description of these methods and the “Modulome” workflow? This is exciting, new technology.

2. There is a lot of information yielded by the Modulome workflow that is glossed over in the manuscript. Could the authors consider providing a display item that summarizes some of the insights into V. vulnificus regulatory networks provided by this analysis? I realize that this is not a small ask – but it comes across as an omission. As a microbiologist, I want to know more about what machine learning can reveal about gene expression in this bacterium.

3. Can the authors add their datasets to iModulonDB? See (https://academic.oup.com/nar/article/49/D1/D112/5921295).

Reviewer #2: (No Response)

**********

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: 1. In all bar graphs, could the authors please show their individual datum points so that the readers can evaluate data distributions for themselves.

2. While it is possible that rendering in Editorial Manager may have affected figure quality, it is very difficult to see rugose colony morphology in the figures. Could the authors double check the image quality, or alternatively, take better photos so that rugosity is easily visible to the reader.

Reviewer #2: Minor Modifications:

- Figure 3A-B: How many biological replicates were used for qRT-PCR analysis? Please state in the Figure Legend or Materials and Methods.

- Figure 3C-D: How many independent times were the EMSA analyses performed? Provided gel images are representative of how many assays? Please state in the Figure Legend or Materials and Methods.

- Figure 4: How many biological replicates were used for the crystal violet staining biofilm assay? Please state in the Figure Legend or Materials and Methods.

- Figure 6: How many biological replicates were used for the crystal violet staining biofilm assay? Please state in the Figure Legend or Materials and Methods.

- Figure 6: While I agree with the conclusions of the authors that CabH appears to be involved in surface attachment, and BrpN impacts biofilm formation independent of attachment, I do feel that this assertion could be strengthened by quantification of the crystal violet used for staining here after washing of the biofilm. This would not only allow for quantification of the differences between the isogenic parent strain and each mutant tested but would also allow for determination of more subtle differences between the strains.

This assertion could also be strengthened by the microscopic analysis of the ability of the cabH mutant to attach to surfaces. Is the mutant impaired in initial attachment, or can it initially attach to a surface and the impairment is in the long-term ability to stay on a surface (given that the biofilms are analyzed at the mature state od 24H)? While this analysis may not be required for this publication, I do believe it is something that the authors should consider.

**********

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: Yes: Joe J. Harrison

Reviewer #2: Yes: Kyle A. Floyd

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, we recommend that 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 to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References:

Please 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 rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Dear Dr. Choi,

We are pleased to inform you that your manuscript 'Two novel genes identified by large-scale transcriptomic analysis are essential for biofilm and rugose colony development of Vibrio vulnificus' 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.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

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

Best regards,

Matthew Parsek, PhD

Academic Editor

PLOS Pathogens

Karla Satchell

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):