Morphological peculiarities of the DNA-protein complexes in starved Escherichia coli cells

Natalia Loiko; Yana Danilova; Andrey Moiseenko; Vladislav Kovalenko; Ksenia Tereshkina; Maria Tutukina; Galina El-Registan; Olga Sokolova; Yurii Krupyanskii

doi:10.1371/journal.pone.0231562

Peer Review History

Original SubmissionMarch 24, 2020
7 Jul 2020 Decision Letter - Maria Gasset, Editor PONE-D-20-08434 Morphological peculiarities of DNA-protein complexes in dormant Escherichia coli cells, subjected to prolonged starvation PLOS ONE Dear Dr. Yurii Krupyanskii, Thank you for submitting your manuscript to PLOS ONE, and my personal apologies for getting back to you with a propper review. 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 convincingly addresses all the points raised during the review process and that both reviewers have constructively indicated. Please submit your revised manuscript by september 6th. 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. 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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.PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. 3. 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. [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: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No ****** 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: The paper by Loiko et al reports an electron microscopy study of the complexes formed by the nucleoid-associated protein Dps of E.coli and DNA in cells that survive a prolonged starvation. To this end, Authors prepare strains that overproduce Dps upon induction of alternate expression systems (either arabinose-induced or T7-based) and test their viability along time in different growth media. The long-lasting cells are then inspected with various types of EM and found to contain a range of structures which in one case resemble nucleosomes. The paper is interesting and visually appealing, but there is a number of issues that should be tackled: 1. A clarification of the biological momenclature would be very welcome. I do not understand the pecise meaning of dormant. Is it that they keep some membrane potential—but can they generate viable colonies? What is vegetative in this context (eg Table 1)? How do we know the cells observed under the microscope are alive or dead? 2. The Achilles's heel of this work is that the interesting structures observed under EM come from cells overexpressing Dps. The wt is mentioned in Table 2, but not analyzed further. It is worrisome that the distribution of the various DNA-protein complexes depends on the overexpression system. BTW, the Table lacks various controls eg strains carrying insert-less plasmids (not just the wt) with and without inducers. 3. Is Dps protein essential? It would be highly informative to inspect starved cells lacking the protein. The nucleoid has many other associated proteins which could contribute to the structures shown in the figures. Reviewer #2: Bacterial cells have a variety of mechanisms of molecular adaptation for survival under stress conditions. One of them relates to the protein Dps (DNA-binding protein of starved cells), which acts as a protective element against starvation by interacting with bacterial DNA and promoting its condensation in supramolecular structures. This work describes the analysis of the structural organization of the condensed DNA-Dps assemblies in E. coli cells starved for extended periods (months) by electron microscopy and electron tomography. The authors have found three types of condensed DNA-Dps structures in dormant cells, two previously reported (nanocrystalline and liquid crystalline structures). In contrast, the third one - termed folded nucleosome-like structures - is novel being more abundant in cells grown in synthetic medium. They conclude that the morphological heterogeneity of DNA condensates in dormant cells found in this study here is an additional factor contributing to the response of bacterial cells to stress conditions and environmental changes. COMMENTS Title: please try to shorten it. Introduction: it supplies sufficient background information to allow the reader to understand and evaluate the results of the present study and to provide the rationale of this work. The experimental procedures used are appropriate to ask the proposed questions. I encourage the improvement of the statistical analysis of the observations summarized in Table 2, including some statistical parameters (i.e., the standard deviation of the mean values, or equivalent) to allow the reader to evaluate if the abundance of the difference morphologies found is significant within experimental uncertainty or not. The description of the results is again concise and clear. The figures complement well the main text. The manuscript will improve if the authors further elaborate on the significance of the novel structures found here (namely the ones denoted as folded nucleosome-like structures). In this regard, Figure 6 would need further improvements. How these results and the interpretations of them agree (or contrast) with previously published work? In the conclusions, could the authors define more precisely the unsettled points for future explorations? What are the principles, relationships, and generalizations shown by the results? ******** 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.0231562.r001
Revision 1
7 Sep 2020 Author Response 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.PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. 3. 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. [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: Partly Reviewer #2: Yes 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No 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: The paper by Loiko et al reports an electron microscopy study of the complexes formed by the nucleoid-associated protein Dps of E.coli and DNA in cells that survive a prolonged starvation. To this end, Authors prepare strains that overproduce Dps upon induction of alternate expression systems (either arabinose-induced or T7-based) and test their viability along time in different growth media. The long-lasting cells are then inspected with various types of EM and found to contain a range of structures which in one case resemble nucleosomes. The paper is interesting and visually appealing, but there is a number of issues that should be tackled: 1. A clarification of the biological momenclature would be very welcome. I do not understand the pecise meaning of dormant. Is it that they keep some membrane potential—but can they generate viable colonies? What is vegetative in this context (eg Table 1)? How do we know the cells observed under the microscope are alive or dead? 2. The Achilles's heel of this work is that the interesting structures observed under EM come from cells overexpressing Dps. The wt is mentioned in Table 2, but not analyzed further. It is worrisome that the distribution of the various DNA-protein complexes depends on the overexpression system. BTW, the Table lacks various controls eg strains carrying insert-less plasmids (not just the wt) with and without inducers. 3. Is Dps protein essential? It would be highly informative to inspect starved cells lacking the protein. The nucleoid has many other associated proteins which could contribute to the structures shown in the figures. Reviewer #2: Bacterial cells have a variety of mechanisms of molecular adaptation for survival under stress conditions. One of them relates to the protein Dps (DNA-binding protein of starved cells), which acts as a protective element against starvation by interacting with bacterial DNA and promoting its condensation in supramolecular structures. This work describes the analysis of the structural organization of the condensed DNA-Dps assemblies in E. coli cells starved for extended periods (months) by electron microscopy and electron tomography. The authors have found three types of condensed DNA-Dps structures in dormant cells, two previously reported (nanocrystalline and liquid crystalline structures). In contrast, the third one - termed folded nucleosome-like structures - is novel being more abundant in cells grown in synthetic medium. They conclude that the morphological heterogeneity of DNA condensates in dormant cells found in this study here is an additional factor contributing to the response of bacterial cells to stress conditions and environmental changes. COMMENTS Title: please try to shorten it. Introduction: it supplies sufficient background information to allow the reader to understand and evaluate the results of the present study and to provide the rationale of this work. The experimental procedures used are appropriate to ask the proposed questions. I encourage the improvement of the statistical analysis of the observations summarized in Table 2, including some statistical parameters (i.e., the standard deviation of the mean values, or equivalent) to allow the reader to evaluate if the abundance of the difference morphologies found is significant within experimental uncertainty or not. The description of the results is again concise and clear. The figures complement well the main text. The manuscript will improve if the authors further elaborate on the significance of the novel structures found here (namely the ones denoted as folded nucleosome-like structures). In this regard, Figure 6 would need further improvements. How these results and the interpretations of them agree (or contrast) with previously published work? In the conclusions, could the authors define more precisely the unsettled points for future explorations? What are the principles, relationships, and generalizations shown by the results? 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. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0231562.r002
16 Sep 2020 Decision Letter - Maria Gasset, Editor Morphological peculiarities of DNA-protein complexes in starved Escherichia coli cells PONE-D-20-08434R1 Dear Dr. Yuri Krupyanskii, 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, Maria Gasset, Ph.D. 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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: (No Response) ****** 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 https://doi.org/10.1371/journal.pone.0231562.r003
Formally Accepted
24 Sep 2020 Acceptance Letter - Maria Gasset, Editor PONE-D-20-08434R1 Morphological peculiarities of DNA-protein complexes in starved Escherichia coli cells Dear Dr. Krupyanskii: 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 Dr. Maria Gasset Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0231562.r004

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