-->PONE-D-25-38532-->-->The allelopathic vitamin B1 antagonist bacimethrin impacts microbial gene expression in a hypereutrophic watershed dominated by cyanobacterial blooms-->-->PLOS ONE

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PLOS ONE

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2. Thank you for stating the following financial disclosure: [This work was funded by United States Fish and Wildlife Service grant F22AC01810-01 and California Department of Fish and Wildlife grant Q2196012, both to Christopher P. Suffridge. Additional personnel funding for Christopher P. Suffridge was provided by National Science Foundation grant DEB-1639033. Mass spectrometry instrumentation at the OSU Mass Spectrometry Center was supported by National Institutes of Health grant 1S10RR022589-1.]

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3. Thank you for stating the following in the Acknowledgments Section of your manuscript: [We thank the USFWS Klamath Falls Office including Rodger Gwiazdowski, Charlee Cramer, Ronald Twibell, and Christinia Kruse and U.S. Bureau of Reclamation staff Brock Phillips for their assistance with field sampling and project development. We thank Stephen Giovannoni for his support, mentorship, and use of his laboratory space. We thank Jeff Morré and Jaewoo Choi for assistance with mass spectrometry. Finally, we thank the broader community of Salmonid Thiamin Deficiency Complex scientists for helpful and inspirational conversations that have shaped the direction of this research (workshops funded by NCEAS Morpho, USGS John Wesley Powell Center for Analysis and Synthesis, and NSF). We thank Beth Ahner for her advice, support, and collaboration surrounding bacimethrin. This work was funded by United States Fish and Wildlife Service grant F22AC01810-01 and California Department of Fish and Wildlife grant Q2196012, both to Christopher P. Suffridge. Additional personnel funding for Christopher P. Suffridge was provided by National Science Foundation grant DEB-1639033 Mass spectrometry instrumentation at the OSU Mass Spectrometry Center was supported by National Institutes of Health grant 1S10RR022589-1. The authors declare no conflict of interest.]

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: [This work was funded by United States Fish and Wildlife Service grant F22AC01810-01 and California Department of Fish and Wildlife grant Q2196012, both to Christopher P. Suffridge. Additional personnel funding for Christopher P. Suffridge was provided by National Science Foundation grant DEB-1639033. Mass spectrometry instrumentation at the OSU Mass Spectrometry Center was supported by National Institutes of Health grant 1S10RR022589-1.]

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. [A small subset of the dissolved bacimethrin data (2 stations) was recently published in Yazadani et al. In that manuscript these data were used to provide evidence that bacimethrin is present in the environment. No further analysis of the data was included in that paper.

The complete bacimethrin dataset is included here to allow for complete ecological analysis. We have explained htis overlap in the methods and have cited Yazdani et al.

Mohammad Yazdani, Christopher P. Suffridge, Fangchen Liu, Cait M. Costello, Zhiyao Zhou, Gillian St. John, et al. Harmful algal bloom species Microcystis aeruginosa releases thiamin antivitamins to suppress competitors. mbio 2025. doi: https://doi.org/10.1128/mbio.01608-25.] Please clarify whether this [conference proceeding or publication] was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript.

5. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 

Additional Editor Comments:

Reviewer #1:

n the manuscript “ The allelopathic vitamin B1 antagonist bacimethrin impacts microbial gene expression in a hypereutrophic watershed dominated by cyanobacterial blooms” it is clearly indicated that increased cyanobacterial bloom abundance is associated with increased concentrations of the thiamine and its antagonist, bacimethrin, and increased expression of thiamine biosynthesis genes (thiC, thiF, and dxs). However, some minor errors and obscurities need to be clarified before publishing.

1. The year "2025" in the Figure 1B, C caption seems to be a typographical error. Because the Materials and Methods section states that “ samples were collected May 1-3 and August 28-30, 2023, during daylight”.

2. Although linear regressions show that cyanoHAB abundance significantly predicts HMP and bacimethrin concentrations. But the low adjusted R² values (0.18 for HMP and 0.38 for bacimethrin) indicate that there are also other factors controlling the levels of these two chemicals. And authors were unable to identify potential genes responsible for bacimethrin synthesis. However, previous study in M. aeruginosa has suggested that genes encoding glycosyltransferase, methyltransferase, and thiaminase I may be involved in bacimethrin production, like their homologs in the bcm gene cluster found in Clostridium botulinum.

3. Why did you set a threshold of >1,000 cells mL⁻¹ for CyanoHAB high and low?

Reviewer #2:

� The study establishes bacimethrin as an allelopathic factor influencing microbial gene expression, however, the discussion does not clearly differentiate how much of the observed gene expression is bacimethrin-specific versus broader Eutrophication /cyano HAB effects. Stronger evidence separating correlation from causation is needed.

� The chemical quantification of bacimethrin and thiamin should include detection limits, calibration methods, and reproducibility for LC-MS/MS measurements.

� The metatranscriptomics is briefly mentioned, add details on read mapping thresholds, normalization, and statistical treatment (e.g., correction for multiple testing in LEfSe analyses).

� The conclusion that bacimethrin confers a competitive advantage to prototrophs (e.g., cyanoHAB taxa) is plausible, but the manuscript does not present direct experimental evidence (e.g., controlled culture assays with bacimethrin). This should be acknowledged as a limitation.

� Alternative explanations such as nutrient limitation, redox dynamics, or co-occurring toxins could also structure microbial communities. The discussion should weigh these factors.

� The manuscript uses a large dataset (metabolites, community composition, transcripts), but integration sometimes feels descriptive rather than mechanistic. For example, correlations between bacimethrin and gene expression are presented, but no causal or predictive modeling is attempted.

� The claim that bacimethrin is “nearly equimolar” with HMP may be misleading given the high variability and large error ranges in Table 1. Statistical support for “equimolar” is weak.

� Redundancy analysis (RDA) is included, but effect sizes are low, raising questions about biological significance.

� The introduction is strong and well-referenced, but could more explicitly state the hypothesis: is the expectation that bacimethrin actively suppresses auxotrophs, or that it is merely a biomarker of bloom dominance?

� The discussion occasionally overstates certainty (e.g., “bacimethrin provides a competitive advantage”) without caveats.

� Some pathway schematics (e.g., Fig. 6) are visually complex and may be simplified for clarity.

� Supporting information tables (S1–S4) are extensive but not fully summarized in the main text.

� “TRCs” (thiamin and related compounds) is introduced but should be used more consistently throughout.

� The term “allelopathy” may imply direct inhibition; clarify that the evidence here is correlative.

� Only 16 of 24 samples yielded usable RNA. This introduces bias, but the limitation is not fully acknowledged.

� Metatranscriptome and 16S community analyses show inconsistent patterns (DNA shows environment/season as stronger drivers; RNA shows cyanoHAB-driven changes). This contradiction is under-discussed.

� The presence of biosynthesis/salvage genes in reference genomes is used to infer ecological function, but no expression or enzyme activity data confirm functionality.

� Some tests (e.g., Wilcoxon signed-rank, PERMANOVA) are mentioned, but exact test statistics, effect sizes, and corrections for multiple testing are not always reported.

� Revise to emphasize limitations and uncertainties (lack of experimental causation, possible confounders).

� Strengthen methodological transparency for chemical and transcriptomic analyses.

� Clarify the central hypothesis and refine the narrative to avoid overstated conclusions.

� Simplify and better integrate figures/tables with the text.

� Some citations like HMP auxotrophy studies are outdated, try to add recent ones.

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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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

-->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

**********

-->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

**********

-->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: In the manuscript “ The allelopathic vitamin B1 antagonist bacimethrin impacts microbial gene expression in a hypereutrophic watershed dominated by cyanobacterial blooms” it is clearly indicated that increased cyanobacterial bloom abundance is associated with increased concentrations of the thiamine and its antagonist, bacimethrin, and increased expression of thiamine biosynthesis genes (thiC, thiF, and dxs). However, some minor errors and obscurities need to be clarified before publishing.

1. The year "2025" in the Figure 1B, C caption seems to be a typographical error. Because the Materials and Methods section states that “ samples were collected May 1-3 and August 28-30, 2023, during daylight”.

2. Although linear regressions show that cyanoHAB abundance significantly predicts HMP and bacimethrin concentrations. But the low adjusted R² values (0.18 for HMP and 0.38 for bacimethrin) indicate that there are also other factors controlling the levels of these two chemicals. And authors were unable to identify potential genes responsible for bacimethrin synthesis. However, previous study in M. aeruginosa has suggested that genes encoding glycosyltransferase, methyltransferase, and thiaminase I may be involved in bacimethrin production, like their homologs in the bcm gene cluster found in Clostridium botulinum.

3. Why did you set a threshold of >1,000 cells mL⁻¹ for CyanoHAB high and low?

Reviewer #2: � The study establishes bacimethrin as an allelopathic factor influencing microbial gene expression, however, the discussion does not clearly differentiate how much of the observed gene expression is bacimethrin-specific versus broader Eutrophication /cyano HAB effects. Stronger evidence separating correlation from causation is needed.

� The chemical quantification of bacimethrin and thiamin should include detection limits, calibration methods, and reproducibility for LC-MS/MS measurements.

� The metatranscriptomics is briefly mentioned, add details on read mapping thresholds, normalization, and statistical treatment (e.g., correction for multiple testing in LEfSe analyses).

� The conclusion that bacimethrin confers a competitive advantage to prototrophs (e.g., cyanoHAB taxa) is plausible, but the manuscript does not present direct experimental evidence (e.g., controlled culture assays with bacimethrin). This should be acknowledged as a limitation.

� Alternative explanations such as nutrient limitation, redox dynamics, or co-occurring toxins could also structure microbial communities. The discussion should weigh these factors.

� The manuscript uses a large dataset (metabolites, community composition, transcripts), but integration sometimes feels descriptive rather than mechanistic. For example, correlations between bacimethrin and gene expression are presented, but no causal or predictive modeling is attempted.

� The claim that bacimethrin is “nearly equimolar” with HMP may be misleading given the high variability and large error ranges in Table 1. Statistical support for “equimolar” is weak.

� Redundancy analysis (RDA) is included, but effect sizes are low, raising questions about biological significance.

� The introduction is strong and well-referenced, but could more explicitly state the hypothesis: is the expectation that bacimethrin actively suppresses auxotrophs, or that it is merely a biomarker of bloom dominance?

� The discussion occasionally overstates certainty (e.g., “bacimethrin provides a competitive advantage”) without caveats.

� Some pathway schematics (e.g., Fig. 6) are visually complex and may be simplified for clarity.

� Supporting information tables (S1–S4) are extensive but not fully summarized in the main text.

� “TRCs” (thiamin and related compounds) is introduced but should be used more consistently throughout.

� The term “allelopathy” may imply direct inhibition; clarify that the evidence here is correlative.

� Only 16 of 24 samples yielded usable RNA. This introduces bias, but the limitation is not fully acknowledged.

� Metatranscriptome and 16S community analyses show inconsistent patterns (DNA shows environment/season as stronger drivers; RNA shows cyanoHAB-driven changes). This contradiction is under-discussed.

� The presence of biosynthesis/salvage genes in reference genomes is used to infer ecological function, but no expression or enzyme activity data confirm functionality.

� Some tests (e.g., Wilcoxon signed-rank, PERMANOVA) are mentioned, but exact test statistics, effect sizes, and corrections for multiple testing are not always reported.

� Revise to emphasize limitations and uncertainties (lack of experimental causation, possible confounders).

� Strengthen methodological transparency for chemical and transcriptomic analyses.

� Clarify the central hypothesis and refine the narrative to avoid overstated conclusions.

� Simplify and better integrate figures/tables with the text.

� Some citations like HMP auxotrophy studies are outdated, try to add recent ones.

**********

-->6. 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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Bacimethrin, an allelopathic vitamin B1 antagonist, is linked with microbial gene expression patterns in a hypereutrophic watershed

PONE-D-25-38532R1

Dear Dr. Christopher,

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,

Barathan Balaji Prasath

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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

-->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

**********

-->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 manuscript has been properly revised and all comments have been addressed, and the manuscript is ready for acceptance and I would like to thank the respected editor for trusting me and assigning me the review of this manuscript.

Reviewer #2: Authors have successfully addressed my previous comments, resulting in a thoroughly improved and well-revised manuscript that demonstrates significant enhancement in clarity and quality.

**********

-->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

**********