Dear Dr Bassler,

Thank you very much for submitting your Research Article entitled 'Natural Silencing of Quorum-Sensing Activity Protects Vibrio parahaemolyticus from Lysis by an Autoinducer-Detecting Phage' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. All three reviewers appreciated the attention to an important topic but suggested some minor suggestions that we ask you address in a revised manuscript.

In addition we ask that you:

1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

2) Upload a Striking Image with a corresponding caption to accompany your manuscript if one is available (either a new image or an existing one from within your manuscript). If this image is judged to be suitable, it may be featured on our website. Images should ideally be high resolution, eye-catching, single panel square images. For examples, please browse our archive. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License. Note: we cannot publish copyrighted images.

We hope to receive your revised manuscript within the next 30 days. If you anticipate any delay in its return, we would ask you to let us know the expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments should be included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool. 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.

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

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

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.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

Please let us know if you have any questions while making these revisions.

Yours sincerely,

Ivan Matic

Academic Editor

PLOS Genetics

Lotte Søgaard-Andersen

Section Editor

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: In recent years, there have been exciting new studies (particularly from the Bassler lab) focused on connections between quorum-sensing and symbioses of phage and their prokaryotic hosts. The Bassler lab recently reported a vibriophage VP882, capable of sensing host-produced QS signals that trigger prophage induction. Presumably host QS molecules act as a signal of high prey availability to a prophage. Here, Duddy et al. sequence the genome of the natural host of the VP882 prophage, and find that two host QS systems are disabled. They demonstrate that, if functionality of these systems is restored, virion production and host cell lysis by the phage are enhanced, suggesting that inactivation of host QS confers a fitness benefit to VP882 lysogens.

The data in this paper are beyond reproach and strongly support the conclusions. The paper is also very well-written and was a pleasure to read. I do not have any experimental requests, but have a few points of interest that could be clarified via additional discussion:

1. Is there any correlation between Vibrio isolates that have VqmR-VqmA deletions and those that contain a resident prophage encoding a VqmA homolog?

2. It seems that strain 882 produces DPO at high cell density, as restoration of VqmA-R results in cell density-dependent phenotypes. Is the DPO synthase known? It might be worth pointing out whether it’s present in strain 882.

3. Could the mysterious signal activating transcription of VqmA phage be a direct target of Qrr-mediated translational silencing? Or conversely, is OpaR required for the observed QS-dependent phage phenotype?

4. Once VP882 is established in a host, triggering prophage induction in response to high cell density might be disadvantageous if the producers are lysogenic kin, as these cells would presumably not constitute susceptible prey (since lysogens are typically resistant to superinfection by the phage they already harbor). Therefore it might benefit both the host and phage if the host QS systems are disabled, but phage VqmA remains competent to respond to QS signals from neighboring un-lysogenized cells.

5. Very minor, but it might be worth emphasizing multiple times that prophage VqmA is silent until the unknown signal is produced. I found myself wondering why phage VqmA wasn’t responding to HCD, and this took a while to realize.

Reviewer #2: This is a well conducted and written study characterising loss of function mutations in Vibrio parahaemolyticus that prevent lysis by prophage that detect Quorum Sensing signals. The study is of general interest for understanding how phages regulate their behaviour in response to host ecology, and of more specific interest because the focus is on the natural host of the archetypal “QS eavesdropping” phage that has been extensively studied.

I only have minor comments.

1. End of introduction. Phage could also be beneficial (acting as biowarfare agents), so suggest refrain from use of parasite.

2. Any information linking mutations in vqmA, vqmQ or lux genes and prophage incidence in general across the approx. 5000 genomes?

3. Interesting to carry outs test of associations between presence of defective Lux and VqmA systems across the genomes

4. Page 13, first paragraph. Explain more clearly interactions between Lux and VqmA on phage gene expression. They cant be both additive and epistatic at the same time.

5. The implication throughout is that preventing lysis confers a fitness advantage to the host. But you don’t show this: bacterial growth isn’t affected by QS-induced lysis. Please clarify this pint in the discussion, linking back to point 1.

Reviewer #3: Duddy et al examine the sequence and signalling characteristics of a strain of Vibrio parahaemolyticus of interest because it is the source of a bacteriophage, VP882, encoding VqmAPhage, a protein homolog of the receptor for DPO, a quorum sensing (QS) molecule for Vibrios. In previous work, the Bassler lab first demonstrated that DPO activates a host-encoded protein, VqmA, which leads to group behaviors, including biofilm formation. In work on the phage homolog, they found that VqmAPhage could also bind DPO, and expression of this protein initiated a signalling cascade leading to induction of the phage lysogen. Here, they find that the normal VqmA host signalling is defective in the natural host of VP882, and explore the consequences of this. By “repairing” the mutations, they show that the variants found in this host help to keep the expression of phage genes low, presumably avoiding inappropriate induction of phage lytic growth. Comparative genomics on the components highlighted here provide some novel insights into evolution of the QS pathways among Vibrio strains, although the implications of these have not been fully explored.

1. P. 2, introduction: A rationale provided here for the induction of the VqmAphage pathway and phage lysis at high cell density is that this is a situation where “the probability of infecting the next host cell is maximized.” That would not work well if the other host cells in the vicinity are also lysogens. Given that it is not known what the inducing signal for VqmAphage is, might there be a signal that transmits information about sensitive, non-lysogenic hosts? While this is not a focus of this paper, the work here does suggest that lysogenizing Vibrios that have fully competent QS systems may create problems.

2. Presumably, previously published work on VqmAphage and its ability to activate synthesis of VqmR was not done in this host. It would be useful to spell that out someplace.

3. In reporting the sequence changes in the 882 strain, the focus is on expressionf VqmA882. However, if VqmR is also absent, it would seem that signalling from VqmAphage to VqmR and thus to host behaviors would also be absent in this strain. In swaps into other Vibrios, just the intergenic region was traded, but it was unclear in the text exactly what was “repaired” in some of the experiments. For instance, in Fig. 2b, is it only the vqmA and upstream region that is restored? Is this then different from the strains used later in the paper (Fig. 4).

4. Is the statement “Of the >5000 sequenced strains, strain 882 is the only sequenced Vibrio that lacks vqmR but retains vqmA” referring to the phage version of vqmA? Is it also the only phage VqmA? Maybe it would be worth returning vqmR but not vqmA to the 882 genome, to see the effect of the lysogen in that context.

5. Fig. 2C , 2D, and S1C: These two clades of vqmR/vqmA are interesting. In Clade I, VqmA is expressed constitutively, and a reasonable -10 region seems to be present, although the lack of function for a swap of this sequence into RIMD suggests either something further upstream or possibly another problem. If the transcription start site is known for that, I might be mentioned. In Clade II, no decent conserved -10 is obvious. Is there any evidence where transcription actually starts? Or that transcription (and not translation) is responding to cell density? If the swap to look at what is needed for expression includes the upstream vqmR gene, does this work better? What if it includes first part of the vqmA genes?

6. Fig. 3B, Fig. S3B, Fig. 5B, Fig. 6C: It is not very easy to see what colors are what in many of these graphs (luxO882 and luxOD61A are particularly difficult to tell apart in 3B, for instance). It was also not entirely clear that luxOD61E is full-length luxO, while luxO882D61E is the one with the internal deletion. Maybe spell out more in the legend.

7. Fig. 3F: Since these are all strains with the deletion (other than RIMD2210633), it is difficult to really follow how many times the authors think this deletion arose. It might be more useful to look at trees and close relatives for a few other Vibrios not closely related. If this happened multiple times, aside from some expectation of selection for LCD state, does the DNA sequence around this deletion reveal a likely recombination hot-spot?

8. If LuxO is constitutively active in these strains, locked into an LCD state, is there any loss/change in upstream signalling machinery or downstream output (Qrr sRNAs or OpaR?)? This is an interesting observation but does suggest a very different lifestyle (or maybe a more active VqmA system?).

9. Fig. 4 and epistasis of luxO+. A bit more discussion of why luxO+ is needed for the vqmR/A genes to have any effect would be useful. In Fig. 1A, the implication is that there are parallel but independent inputs into “group behaviors”. Here, the data focuses on phage genes; do group behavior genes show a similar pattern of luxO epistasis? Has that been looked at before in other Vibrios, or does the observation here suggest the evolution of different wiring, maybe due to the LuxO constitutive derivative?

10. Fig. S3B: Again, hard to see color differences for V. cholerae and parahaemolyticus. Since only derivatives with this deletion are shown, other changes are not particularly informative (the species differ). Are there other differences relative to luxO+ strains?

11. Discussion: Is it really true that the same phage was found in Salmonella and Shewanella strains? How clear is it that this is real? Does the phage infect Salmonella? Shewanella? Is it possible these sequences were contaminants?

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #3: No

Dear Dr Bassler,

We are pleased to inform you that your manuscript entitled "Natural Silencing of Quorum-Sensing Activity Protects Vibrio parahaemolyticus from Lysis by an Autoinducer-Detecting Phage" has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional acceptance, but your manuscript will not be scheduled for publication until the required changes have been made.

Once your paper is formally accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you’ve already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosgenetics@plos.org.

In the meantime, please log into Editorial Manager at https://www.editorialmanager.com/pgenetics/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production and billing process. Note that PLOS requires an ORCID iD for all corresponding authors. Therefore, please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field.  This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

If you have a press-related query, or would like to know about making your underlying data available (as you will be aware, this is required for publication), please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Ivan Matic

Academic Editor

PLOS Genetics

Lotte Søgaard-Andersen

Section Editor

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

----------------------------------------------------

Comments from the reviewers (if applicable):

----------------------------------------------------

Data Deposition

If you have submitted a Research Article or Front Matter that has associated data that are not suitable for deposition in a subject-specific public repository (such as GenBank or ArrayExpress), one way to make that data available is to deposit it in the Dryad Digital Repository. As you may recall, we ask all authors to agree to make data available; this is one way to achieve that. A full list of recommended repositories can be found on our website.

The following link will take you to the Dryad record for your article, so you won't have to re‐enter its bibliographic information, and can upload your files directly: 

http://datadryad.org/submit?journalID=pgenetics&manu=PGENETICS-D-23-00618R1

More information about depositing data in Dryad is available at http://www.datadryad.org/depositing. If you experience any difficulties in submitting your data, please contact help@datadryad.org for support.

Additionally, please be aware that our data availability policy requires that all numerical data underlying display items are included with the submission, and you will need to provide this before we can formally accept your manuscript, if not already present.

----------------------------------------------------

Press Queries

If you or your institution will be preparing press materials for this manuscript, or if you need to know your paper's publication date for media purposes, please inform the journal staff as soon as possible so that your submission can be scheduled accordingly. Your manuscript will remain under a strict press embargo until the publication date and time. This means an early version of your manuscript will not be published ahead of your final version. PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via plosgenetics@plos.org.