PONE-D-22-09616Contribution of Stenotrophomonas maltophilia MfsC transporter to protection against diamide and the regulation of its expression by the diamide responsive repressor DitRPLOS 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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: No

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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 presented characterization of MfsC transporter to protection against diamide and the regulation of its expression by the diamide responsive repressor DitR in Stenotrophomonas maltophilia. Before the paper could be accepted for publication in PLOS ONE, the authors need make revisions as follow:

1. What’s the function of MfsB? What’s the relationship between MfsB and MfsC? Please add discussion.

Reviewer #2: This is a very nice characterization of a transporter operon and its regulator that mediated resistance to diamide in Stenotrophomonas maltophilia (Steno). The authors show deletion of mfsC leads to increased sensitivity to diamide and that the regulator smlt0547 (named ditR here) is a repressor (based on deletion, complementation, and EMSA behavior) that binds the mfsC operon promoter (shown by EMSA and DNase footprinting) and is released by the presence of diamide (shown by EMSA) resulting in operon expression specific to diamide presence (qRT-PCR and reporter assays). Release by diamide is reversible by DTT treatment and loss of conserved reactive cysteines in DitR result in a diamide-insensitive and thus constitutively repressive protein (shown by reporter expression). These findings are related to the field via appropriate discussion and phylogenetic analyses. I think this is a really nice story and really enjoyed reading it. I have a few minor things to suggest or to be clarified, followed by some suggested text edits to make the manuscript more clear.

1. For the various compounds, particularly the oxidants, why were these concentrations chosen? It seems as if diamide is used at the highest concentration and one could argue that the repressor could be responsive and/or transporter active if the other compounds were present at higher concentrations. I do not think this is the case, but an explanation of why these concentrations were chosen would be very helpful.

2. Statistics: For all of the comparisons in which there are more than two groups (all of them) any t-tests need to be corrected for multiple comparisons by an appropriate post-test. For Fig 3 it would be a Dunnett’s post-test with WT as the comparator tested within each compound. For Fig 4 it would be a Dunnett’s post-test with untreated as the comparator. I don’t think this increased statistical rigor will change the conclusions but use of multiple-comparison corrected stats is important.

3. Figure suggestions: Fig 1 is unnecessary, the description in the text is sufficient. For the phylogenetic trees, I would prefer plotting the trees with quantitative branch lengths (actual branch lengths with an amino-acid difference scale) so that relative similarity is visually represented.

4. Methods: Line 97: I find it really hard to believe that a bunch of molecular biology was conducted according to a 21 year old method manual without the use of any kits or any deviations from those published methods. Please expand detail as needed. Line 110: Which plasmid was the cre recombinase expressed from and how was the strain cured of that plasmid?

5. This is really just a comment to improve future work. Having myself tried lacZ in Steno many times, it really is not a great reporter and seems to be due to poor protein folding. The authors have appropriately used a Unit based measure, as these numbers would look ridiculously low if the common Miller unit were used (1 Bgal unit in this manuscript would be 0.002 Miller Units, if my calculations are correct). I don’t think anything needs to change for this manuscript, but I would suggest using a better reporter for Steno in the future. XylE works very well as an enzymatic reporter and mScarlet works really well as a fluorescent reporter.

Text edit suggestions:

Line 25: change ‘transports’ to ‘transporters’

Line 44: edit first sentence to- “Stenotrophomonas maltophilia is a Gram-negative, rod-shaped, aerobic bacterium, which is ubiquitous in the aqueous environment and soil.”

Line 50: edit start of sentence to- “Efflux transporters are recognized…”

Line 52: Since the assumption of kingdom number keeps changing, I suggest using “all domains of life” instead of numbering the kingdoms.

Line 57: change ‘several’ to ‘many’ and replace ‘however’ with ‘and’

Line 82: suggest “Here, we are interested in the MFS transporters because of their…”

Line 83: change ‘gene’ to ‘transcriptional’

Line 86: Suggest putting the smlt gene number for DitR and stating ‘… which we have named DitR’

Line 351: change ‘direction’ to ‘orientation’

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Reviewer #1: No

Reviewer #2: No

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Contribution of Stenotrophomonas maltophilia MfsC transporter to protection against diamide and the regulation of its expression by the diamide responsive repressor DitR

PONE-D-22-09616R1

Dear Dr. Vattanaviboon,

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.

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Kind regards,

Yixin Shi

Academic Editor

PLOS ONE

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: (No Response)

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 #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

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 #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

**********

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: The responses to reviewer’s comments were clear, and the revised manuscript was satisfied. The authors know the importance of elucidation of MfsB function and the relationship between MfsB and MfsC. I recommend the paper could be accepted for publication in PLOS ONE.

Reviewer #2: (No Response)

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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 #1: No

Reviewer #2: No

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