Pyruvate decarboxylase and thiamine biosynthetic genes are regulated differently by Pdc2 in S. cerevisiae and C. glabrata

Christine L. Iosue; Julia M. Ugras; Yakendra Bajgain; Cory A. Dottor; Peyton L. Stauffer; Rachael A. Hopkins; Emma C. Lang; Dennis D. Wykoff

doi:10.1371/journal.pone.0286744

Peer Review History

Original SubmissionMarch 2, 2023
4 Apr 2023 Decision Letter - Hari S. Misra, Editor PONE-D-23-06178Pyruvate decarboxylase and thiamine biosynthetic genes are regulated differently by Pdc2 in S. cerevisiae and C. glabrata.PLOS ONE Dear Dr. Wykoff, 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. Please submit your revised manuscript by May 19 2023 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. We look forward to receiving your revised manuscript. Kind regards, Hari S. Misra Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. 3. 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. [Note: HTML markup is below. Please do not edit.] 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 ******** 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: No 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: This is a nice article about differences between S. cerevisiae and C. glabrata thiamine biosynthesis genes, their promoters and regulation. It is written well and the data is shown in a clear and simple manner. The results show that S. cerevisiae and C. glabrata differ in their ability to respond to thiamine deprivation, including the promoter responses of key thiamine-inducible genes. There are differences in the genomes of the two organisms, and the Thi2 transcriptional regulator is missing in C. glabrata. While these results are perhaps not surprising, the authors have carried out a detailed analysis of the transcriptional responses of different promoters in different knock-out strains of S. cerevisiae and C. glabrata and show that regulation is different on the two organisms. Major comments: 1) The major concern is that the analysis of the promoters is done uniformly with 1 kb of sequence upstream of the start codon. Is this sequence inclusive of all promoter elements? Has it been characterized? Is the sequence similar in S. cerevisiae and C. glabrata? If the sequence is very different in the two organisms, then the comparisons are not meaningful. It would be really helpful to get information about the 1kb sequences of the promoters from both S. cerevisiae and C. glabrata, in terms of their sequence identity. 2) The authors mention that the CgPMU3 promoter is different. It is not clear how. This needs to be explained and the points made above are relevant to this as well. 3) In Figure 4, S. cerevisiae promoters are being tested in C. glabrata and and vice versa. Again, if the sequences are very different, these results simply show that the promoters are not the same, rather than differences in regulation. The bottom line is that it would be very important to show some data about the sequences of the 1 kb regions and compare them. Minor comments: 1) At the end of the paper, the reader is left with the main finding that the thiamine-inducible gene promoters are different in S. cerevisiae and C. glabrata. It would be very helpful to have a schematic to bring all the information together. 2) The authors talk about “complexity” of regulation (e.g. Abstract, lines 35,254, 279, 281, 336, 339, 364). What do they mean by this word? The results show differences but it is not clear why the words “complexity/complex” is used to describe these differences Reviewer #2: In this manuscript, the authors have addressed the differential regulation of Pyruvate decarboxylase and thiamine biosynthetic genes by the regulator Pdc2 in two yeast species S. cerevisiae and C. glabrata. The differences are addressed at the level of differences in the cis-acting components in the promoters and the structural organization of the trans-acting components of the regulator protein Pdc2 in the two species. Although the two species differ in that deletion of PDC2 is lethal in S. cerevisiae but not in C. glabrata, the cross-complementation studies reveal presence of cryptic cis elements in the C. glabrata promoters that are not apparently regulated by CgPdc2 but can be regulated by the ScPdc2. Among other difference could be ascribed to absence of Thi2 in C. glabrata, with its inclusion in S. cerevisiae possibly playing a role in imparting greater complexity of regulation of THI and PDC genes through the formation of a compendium of transcription regulatory Pdc2-containing complexes S. cerevisiae but a simpler regulatory machinery independent of Pdc2 in C. glabrata. The authors also demonstrate the role of Intrinsically disordered regions in the activation function of the C-terminal region of CgPdc2. The authors opine that a deeper analysis of the different regulation among the two species could help identify potential druggable targets that are unique to C. glabrata. I find this study well-structured and conclusions consistent with the results. Accordingly, I recommend it for publication in PlosOne. However, I think that the manuscript could improve in clarity and continuity of logic, especially in the discussion section, where some sentences are lacking in clarity. For example: lines 425-430 , 434-436. ****** 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 ******** [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. https://doi.org/10.1371/journal.pone.0286744.r001
Revision 1
16 May 2023 Author Response Response to reviewers Reviewer #1: This is a nice article about differences between S. cerevisiae and C. glabrata thiamine biosynthesis genes, their promoters and regulation. It is written well and the data is shown in a clear and simple manner. The results show that S. cerevisiae and C. glabrata differ in their ability to respond to thiamine deprivation, including the promoter responses of key thiamine-inducible genes. There are differences in the genomes of the two organisms, and the Thi2 transcriptional regulator is missing in C. glabrata. While these results are perhaps not surprising, the authors have carried out a detailed analysis of the transcriptional responses of different promoters in different knock-out strains of S. cerevisiae and C. glabrata and show that regulation is different on the two organisms. Major comments: 1) The major concern is that the analysis of the promoters is done uniformly with 1 kb of sequence upstream of the start codon. Is this sequence inclusive of all promoter elements? Has it been characterized? Is the sequence similar in S. cerevisiae and C. glabrata? If the sequence is very different in the two organisms, then the comparisons are not meaningful. It would be really helpful to get information about the 1kb sequences of the promoters from both S. cerevisiae and C. glabrata, in terms of their sequence identity. We have now included an additional supplementary figure (S4 Fig) indicating that whether we choose 1kb or 2 kb upstream of the start codon, we observe similar expression for six of the promoters that we examine. It is possible that indeed we are missing some subtleties of expression by only examining 1 kb promoters, but we do believe we are capturing most of the expression regulation in the 1 kb promoter. In our experience in analyzing promoters, we often find that 500 bp recapitulates much of a promoter response, and core promoter elements (TATA boxes, etc.) are often 100-150 bp upstream of the start codon. There are clearly exceptions, but we believe our supplementary figure should assuage these concerns. Alignment of orthologous promoters from the two species do not result in meaningful alignments. Using relaxed BLASTn algorithms can sometimes find significant similarity in the promoters, but overall there is too much sequence divergence. At the protein level, the two species share 60-80% identity, but at the DNA level, similarity drops dramatically. These two species diverged from one another too long ago for promoters to be able to be aligned. However, we would argue that comparing the promoters is useful. We observe similar cis elements in both species’ promoters, and the ScPDC5 promoter contains cis elements that are recognized by Pdc2 in each species. Our study was designed to begin to understand how similar the promoter architecture is between these two yeast species that likely diverged from one another millions of generations before present. We believe it underlies that these promoter behaviors have been strongly selected for given little sequence similarity, except at likely Pdc2 cis elements. 2) The authors mention that the CgPMU3 promoter is different. It is not clear how. This needs to be explained and the points made above are relevant to this as well. We agree with the reviewer that we were not clear about why we think this promoter is interesting. We have inserted some clarification and background. We find it exciting that C. glabrata has evolved a brand-new phosphatase gene (CgPMU3) that cleaves thiamine pyrophosphate (TPP), is required for C. glabrata to survive when TPP is the only thiamine source, and that this promoter acquired the ability to be regulated by the same transcription factors as other THI genes. Because CgPMU3 promoter does not use a cis element that is common to other THI promoters, it begins to give us clues as to how a new promoter can evolve to be regulated simply by promoter substitutions. We think in the long-term, this promoter will give us clues as to how the transcriptional complexes function at THI promoters, but we do not have enough information yet. 3) In Figure 4, S. cerevisiae promoters are being tested in C. glabrata and and vice versa. Again, if the sequences are very different, these results simply show that the promoters are not the same, rather than differences in regulation. The bottom line is that it would be very important to show some data about the sequences of the 1 kb regions and compare them. We addressed this important concern in the answer above. We disagree with the reviewer that the data simply show the promoters are not the same. We believe what is key here is that many of the promoters between species have similar patterns of expression (e.g. regulation by thiamine and different regulation of the promoter when THI3 is deleted). It is a “feature, rather than a bug” of our study that the promoter sequence between the two species is very different. Thus, when we know where transcription factors bind, we can identify the key cis elements that dictate this behavior. We have promising preliminary results that suggest that CgPdc2 binds to specific elements using ChIP-seq, but we believe that data is best in another publication. Minor comments: 1) At the end of the paper, the reader is left with the main finding that the thiamine-inducible gene promoters are different in S. cerevisiae and C. glabrata. It would be very helpful to have a schematic to bring all the information together. We agree with the reviewer. We spent a lot of time trying to figure out how to condense the data that we have generated into a schematic, and we found all of them wanting. We believe that the reviewer may be indicating the same issues in the next concern and mirroring the concerns of reviewer #2 that there is a weakness in the “clarity and logic” of the discussion and we have worked to make the discussion clearer such that a schematic is not needed. Unfortunately, we are still trying to figure out how everything works together and so there is going to be some lack of clarity in our discussion. We have endeavored to make the writing clear and to highlight the outstanding questions that we still have. 2) The authors talk about “complexity” of regulation (e.g. Abstract, lines 35,254, 279, 281, 336, 339, 364). What do they mean by this word? The results show differences but it is not clear why the words “complexity/complex” is used to describe these differences We agree with the reviewer that we were not clear about what we mean. We have edited the manuscript to avoid these vague statements and appreciate both reviewers concerns that there is a vagueness in parts of the manuscript. Reviewer #2: In this manuscript, the authors have addressed the differential regulation of Pyruvate decarboxylase and thiamine biosynthetic genes by the regulator Pdc2 in two yeast species S. cerevisiae and C. glabrata. The differences are addressed at the level of differences in the cis-acting components in the promoters and the structural organization of the trans-acting components of the regulator protein Pdc2 in the two species. Although the two species differ in that deletion of PDC2 is lethal in S. cerevisiae but not in C. glabrata, the cross-complementation studies reveal presence of cryptic cis elements in the C. glabrata promoters that are not apparently regulated by CgPdc2 but can be regulated by the ScPdc2. Among other difference could be ascribed to absence of Thi2 in C. glabrata, with its inclusion in S. cerevisiae possibly playing a role in imparting greater complexity of regulation of THI and PDC genes through the formation of a compendium of transcription regulatory Pdc2-containing complexes S. cerevisiae but a simpler regulatory machinery independent of Pdc2 in C. glabrata. The authors also demonstrate the role of Intrinsically disordered regions in the activation function of the C-terminal region of CgPdc2. The authors opine that a deeper analysis of the different regulation among the two species could help identify potential druggable targets that are unique to C. glabrata. I find this study well-structured and conclusions consistent with the results. Accordingly, I recommend it for publication in PlosOne. However, I think that the manuscript could improve in clarity and continuity of logic, especially in the discussion section, where some sentences are lacking in clarity. For example: lines 425-430 , 434-436. We appreciate the reviewer’s comments, and agree that there are some lapses in our logic. We have reworked the discussion and clarified our use of “complexity” throughout the manuscript. Attachments Attachment Submitted filename: Final response to reviewers.docx https://doi.org/10.1371/journal.pone.0286744.r002
23 May 2023 Decision Letter - Hari S. Misra, Editor Pyruvate decarboxylase and thiamine biosynthetic genes are regulated differently by Pdc2 in S. cerevisiae and C. glabrata. PONE-D-23-06178R1 Dear Dr. Wykoff, 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. 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. Kind regards, Hari S. Misra, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0286744.r003
Formally Accepted
29 May 2023 Acceptance Letter - Hari S. Misra, Editor PONE-D-23-06178R1 Pyruvate decarboxylase and thiamine biosynthetic genes are regulated differently by Pdc2 in S. cerevisiae and C. glabrata Dear Dr. Wykoff: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Hari S. Misra Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0286744.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .