PONE-D-22-09403The regulon of Brucella abortus two-component system BvrR/BvrS reveals the coordination of metabolic pathways required for intracellular lifePLOS ONE

Dear Dr. Guzman-Verri,

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.

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Sorry for the delay in getting your paper reviewed!  As you can see from their comments, both reviewers feel that the manuscript presents information that will be important to the field. But both also have concerns about the presentation that will need to be corrected/clarified before the paper will be considered suitable for publication in PLOS ONE. A particular concern that was raised was that in some cases genes are being identified as targets of  BvrR regulation by ChIPseq analysis without independent verification that BvrR actually regulates these genes.

Considering the reviewers' evaluations, I am going to ask that you submit a revised version of  the manuscript that adequately and appropriately addresses all of  the concerns raised by both reviewers. 

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Please submit your revised manuscript by Jun 06 2022 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.

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We look forward to receiving your revised manuscript.

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Marty Roop

Academic Editor

PLOS ONE

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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: Partly

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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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

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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

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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: The manuscript by Rivas-Solano and colleagues describes the regulatory link between the BvrR/BvrS two-component system and metabolism in Brucella abortus. The authors have a long history with this system, including the identification of BvrR/BvrS and the genetic and biochemical characterization of the system, and this manuscript expands on that work to demonstrate the role that BvrR/BvrS plays in controlling the expression of genes related to metabolism. The bvrR/bvrS genes are encoded as part of a 16-gene operon, which includes may genes that putatively encode proteins involved in nitrogen metabolism, DNA repair, stress responses, and cell cycle processes. ChIP-seq analysis demonstrated that BvrR binds to more than 300 sites in the genome of B. abortus, and EMSAs confirmed direct binding to several of the identified regions. Further bioinformatic and biochemical (i.e., DNase footprinting) analyses defined a consensus DNA-binding sequence for BvrR, and the authors have developed a model for BvrR/BvrS-mediated control of metabolic systems (as well as other important virulence-related systems) in B. abortus.

Overall, the authors have performed a robust analysis of BvrR binding to DNA elements in B. abortus, and while many of the conclusions are supported by the data, there are some concerns about the data and conclusions. The specific concerns are:

-The authors have previously demonstrated that the BvrR/BvrS system is a transcriptional activator of virB. Here, the authors suggest that BvrR binds to the virB promoter at approximately -12 from the transcriptional start site. Mechanistically, this is difficult to understand. How does binding to that site promote transcription? In the same vein, in lines 558-568 the authors discuss the different potential binding sites for BvrR, but the data in Fig. 5 using limited DNA segments show that only one region is bound by BvrR. Is it possible that multiple binding sites are needed for optimal binding?

-Fig. 1 - This is a very minor point, but it is difficult to determine which gels/lanes correspond to the map and primer sets. Moreover, the authors have included a control to demonstrate that transcription stops that the 3' end of the message (i.e., the primer set represented by the black bar). Why is a similar control not included on the 5'-end of the message?

-Fig. 4 - Overall, the EMSAs are convincing, but there are some issues with some of them. For example, the binding to the virB promoter is highly variable between panels A and B. Why is this? It is understandable that differences exists between experiments, but in this case, the data are very difficult to interpret in terms of the competition controls when the control for those experiments looks nothing like the results in the panel A.

-Line 68 - Brucella replicates in a vacuole composed of (or associated with) the ER, and thus it may be incorrect to say that the bacteria replicate "within the ER."

Reviewer #2: This study aims to expand our knowledge of the genes controlled by the Brucella BvrR/S two component system.

While the experiments are well performed and the data presented solid, the conclusions are not fully supported by the data, more experimental work is needed.

Major Concerns

1 The authors write (L114) ‘We expand our knowledge of the BvrR/BvrS regulon, describing the genes controlled directly by this TCS and under conditions that mimic the intracellular environment confronted by B. abortus while trafficking to its replicative niche.’

This is not correct, the data show binding of Bvr-P to DNA, not regulation of gene expression. While there is some evidence that BvrR/S controls expression some genes, including virB and omp25, yo support this claim, it is essential that the authors provide data for the new set of genes that they claim to be controlled by BvrR/S.

2 The authors say that most BvrR-P binding sites are in regions upstream of the target genes. They also find binding sites in the virB4 and virB5 genes, several thousand bases into the operon. This is not at all discussed or commented on in the manuscript. How does this work? Are there internal promotors? This should be clarified.

3 Fig 3. Stress conditions increase binding of BvrR. Is this specific to BvrR. What happens with another TCS regulator, will it also bind to its targets more efficiently?

Other concerns

4 Fig 1 hard to follow with respect to text, there is a confusing mix of mix of gene names and gene numbers. It would be easier to follow if the figure showed the gene names referred to in the text. A more extensive color code could also help with calrity…so all pts genes in one color, unknown function in another etc.

5 The legend for Fig1B does not fit with the figure. First, the authors write that there are 31 primer pairs; this implied 31 PCR reactions, why are only 15 shown. If this is an English language problem and the authors meant 15 primer pairs (so 30 primers), where doses the number 31 come from?

6 The authors write ‘These co-transcription events happened at log and stationary growth phases…’ however they do not show data for different growth phases. What was the growth phase tested in Fig 1?

7 The introduction is rather confused.

Paragraph from L72-Are you talking about TCS in general or Brucella and BvrR/S? The refs suggest the latter the text the former.

L73 Define TCS

Paragraph from L80.

Here there are mixed references to TCS then PTS. It would be much clearer to introduce the Bvr family in the alphas and then talk about PTS.

8 L69 Bacteria then reach an autophagosome-like exit compartment where they are ready to egress from the host cell and start a new infection cycle [5]

Not an appropriate reference

9 L94 ‘Bacteria grown in a nutrient-rich medium at neutral pH (rich conditions), transiently activate BvrR through phosphorylation’

Modify to ‘When bacteria are grown in a nutrient-rich medium at neutral pH (rich conditions), BvrR is transiently activated through phosphorylation’

10 L392 typo rplI?

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Reviewer #1: No

Reviewer #2: No

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PONE-D-22-09403R1The regulon of Brucella abortus two-component system BvrR/BvrS reveals the coordination of metabolic pathways required for intracellular lifePLOS ONE

Dear Caterina,

Thanks for doing such a conscientious job with the revision!  Reviewer 2 has a couple of  minor suggestions and I think the one about mining the literature to see how many of  your BvrR targets have been identified in previous studies  is a good one. So, I am going to ask you to submit a revised manuscript that addresses this point. I agree with the reviewer that it will strengthen the paper.  Please submit your revised manuscript by Aug 27 2022 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.

Thanks for your patience with the review process. I look forward to seeing the revised manuscript!

Sincerely,

Marty Roop

Academic Editor

PLOS ONE

Journal Requirements:

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.

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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 #1: All comments have been addressed

Reviewer #2: (No Response)

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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 #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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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 #1: Yes

Reviewer #2: Yes

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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 #1: Yes

Reviewer #2: Yes

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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 #1: Overall, the authors have appropriately addressed the comments raised in the previous review, and I do not have any addition major concerns.

Reviewer #2: The revised manuscript is greatly improved and much easier to follow.

The authors misunderstood my request to define TCS, I meant to explain what the abbreviation means (even though is is defined in the abstract). I would add this to line 73, where they first introduce BvrRS . The additional text they added, to describe what a TCS is, is very good!

While the authors argue that confirming the BrvRS targets experimentally can wait for another study, they should mine the available transcriptomic and proteomic studies comparing WT and TCS mutants to see whether any of their predicted targets have been found. This will strengthen the manuscript.

While the manuscript is understandable, the authors should have the text proof read by a native english speaker to correct the numerous small errors in grammar and syntax.

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Reviewer #1: No

Reviewer #2: No

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[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.

The regulon of Brucella abortus two-component system BvrR/BvrS reveals the coordination of metabolic pathways required for intracellular life

PONE-D-22-09403R2

Dear Caterina,

Thanks for making the requested modifications to your paper and I'm recommending approval. The paper will be formally accepted for publication once it meets any outstanding technical requirements. Please note that a reviewer suggested modifying a sentence for clarity, but I will leave that up to you whether on not you want to make that modification. 

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.

Sincerely,

Marty Roop

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

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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

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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

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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

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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 manuscript is very much improved. I have one comment

The three studies converged on identifying four common target genes, while our study compared only to proteomics or transcriptomics presented respectively 20 and 15 additional common target genes (S4 Table).

I don't understand this sentence...there may just be punctuation problems (commas missing). Please revise.

Is this what you meant?

The three studies converged, identifying four common target genes, while our study, compared only to proteomics or transcriptomics, identified respectively 20 and 15 additional common target genes (S4 Table).

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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

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