The influence of Pseudomonas syringae on water freezing and ice melting

Maria A. Majorina; Victoria R. Veselova; Bogdan S. Melnik

doi:10.1371/journal.pone.0265683

Peer Review History

Original SubmissionNovember 17, 2021
20 Dec 2021 Decision Letter - Lei Li, Editor PONE-D-21-36407The influence of Pseudomonas syringae on water freezing and ice melting.PLOS ONE Dear Dr. Melnik, 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 Feb 03 2022 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. 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Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. [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: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: I Don't Know ****** 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: No Reviewer #2: No ****** 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 language level of the manuscript must be increased. There is also a need to explain the nature of the phenomenon under study much better. Why and how the P. syringae affect the freezing point of water and the temperature of the coexistence of ice and water should be answered on more detailed level. The conditions of the liquid/solid phase transition must be more precisely discussed in the context of physical background of the complex freezing-thawing processes: I. Kratochvilova et al. Theoretical and experimental study of antifreeze protein AFP752, trehalose and dimethyl sulfoxide cryoprotection mechanism: correlation with cryopreserved cell viability, RSC Advances 7 (2017) 352-360. Reviewer #2: These authors have built a simple apparatus to measure three temperatures in the presence of a bacterium (Pseudomonas syringae) that has surface displayed ice nucleation proteins (INPs). These temperatures are i) the freezing point of water; ii) the temperature at which ice and water co-exist; and iii) the melting point of ice. They have measured these parameters for a wide range of bacterial optical densities in 0.5-mL volumes in a home-made apparatus. However, it is not clear from the paper what has been discovered. For example, the brief (8-line) Abstract states: “P. syringae appears to affect both the freezing point of water and the temperature of the co-existence of ice and water.” But it does tell us the direction and magnitude of the effects and the scientific basis for them. The last sentence of the Abstract states: “…we propose a novel research technique that is simple and requires no complex equipment.” but it does not say what the technique is for, or what it can accomplish towards solving a scientific problem. These points are also not answered in the paper. The technique described seems to be a crude version of a manually operated differential scanning calorimeter. This is not a useful instrument for measuring the activity of ice-nucleating bacteria on the freezing point of water because ice nucleation is stochastic event. This parameter is typically measured on small sample volumes (nL to uL) in a multiplexed apparatus where 102 droplets are simultaneously studied and the mid-point where 50% of the droplets are frozen is taken as the nucleation temperature. The authors say: “The methods of visual droplet monitoring are clear but very labor-consuming and insufficiently accurate.” This is simply not true because the appearance of the 102 droplets can be simultaneously recorded through a polarizing filter during the freezing process, and the data can be tabulated in minutes on replay of the video. Another rapid method is to record the exotherms of 102 individual droplets and tabulate the freezing process this way. The relatively large volume (0.5 mL) of the sample is a problem when measuring the melting process as described in the last paragraph prior to the conclusions. Ice remains around the thermometer probe while the ice melts at the sample edges. This prevented measuring the dependence of the melting temperature on the amount of ice nucleating bacteria present, which was one of the objectives of the study. This seems like a cumbersome technique and it effectiveness has not been demonstrated here. One way to assess the potential of this system is to do a parallel analysis with ice-binding proteins (IBPs). These proteins have well-documented effects on the temperature at which ice and water co-exist, and the melting point of ice. The former is a temperature range for IBPs that is called the thermal hysteresis value and can be as high as 5-6 C for some insect IBPs. The latter is elevated by a fraction of a degree for most IBPs but would be again a good test of the sensitivity of the apparatus. This manuscript has been inadequately edited and would have benefitted from a peer review by a colleague. There are many issues to address. It talks about the ‘concentration’ of bacteria. This term applies to something in solution, whereas the bacteria are in a suspension. Cell density is a more appropriate term. E. coli needs a space between E. and coli, as does 20 mM. Elsewhere mM is written as mm. ****** 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.0265683.r001
Revision 1
24 Feb 2022 Author Response Reviewer #1: The language level of the manuscript must be increased. There is also a need to explain the nature of the phenomenon under study much better. Why and how the P. syringae affect the freezing point of water and the temperature of the coexistence of ice and water should be answered on more detailed level. The conditions of the liquid/solid phase transition must be more precisely discussed in the context of physical background of the complex freezing-thawing processes: I. Kratochvilova et al. Theoretical and experimental study of antifreeze protein AFP752, trehalose and dimethyl sulfoxide cryoprotection mechanism: correlation with cryopreserved cell viability, RSC Advances 7 (2017) 352-360. We have made a lot of corrections, which, in our opinion, have significantly improved the English. The main changes are marked in the text. You can see that we have tried to work on almost every sentence. As for the detailed discussion of the transition of water from the liquid phase to the solid phase. Unfortunately, this is a very complicated process. Just mentioning the process of water crystallization will not help the understanding of the article, and detailed explanations require some mathematical calculations that are not relevant in this article. We have inserted a reference to article AV. Finkelstein “Some particularities of water freezing at small sub-zero temperatures”, which discusses the process of freezing water and the role of nuclei in ice formation. Reviewer #2: These authors have built a simple apparatus to measure three temperatures in the presence of a bacterium (Pseudomonas syringae) that has surface displayed ice nucleation proteins (INPs). These temperatures are i) the freezing point of water; ii) the temperature at which ice and water co-exist; and iii) the melting point of ice. They have measured these parameters for a wide range of bacterial optical densities in 0.5-mL volumes in a home-made apparatus. However, it is not clear from the paper what has been discovered. For example, the brief (8-line) Abstract states: “P. syringae appears to affect both the freezing point of water and the temperature of the co-existence of ice and water.” But it does tell us the direction and magnitude of the effects and the scientific basis for them. The last sentence of the Abstract states: “…we propose a novel research technique that is simple and requires no complex equipment.” but it does not say what the technique is for, or what it can accomplish towards solving a scientific problem. These points are also not answered in the paper. The whole article is devoted to explaining what technique we use can give. In particular, this method made it possible to continuously monitor the freezing/melting process and made it possible to reveal the influence of PSeringae cells on the temperature of the coexistence of ice and water. Our method allows us to study three temperature parameters (on one installation) and, accordingly, compare them with each other. Reviewer #2 The technique described seems to be a crude version of a manually operated differential scanning calorimeter. Our setup does not look like a bad calorimeter, but a good cryosmometer - an instrument that is usually used to study IBP proteins. Reviewer #2 This is not a useful instrument for measuring the activity of ice-nucleating bacteria on the freezing point of water because ice nucleation is stochastic event. This parameter is typically measured on small sample volumes (nL to uL) in a multiplexed apparatus where 102 droplets are simultaneously studied and the mid-point where 50% of the droplets are frozen is taken as the nucleation temperature. The authors say: “The methods of visual droplet monitoring are clear but very labor-consuming and insufficiently accurate.” This is simply not true because the appearance of the 102 droplets can be simultaneously recorded through a polarizing filter during the freezing process, and the data can be tabulated in minutes on replay of the video. Another rapid method is to record the exotherms of 102 individual droplets and tabulate the freezing process this way. We discuss all of the above in the introduction and refer to articles that explore droplets. But when examining droplets, it is impossible to investigate temperature parameters with an accuracy of 0.01 °C. In addition, droplets technique does not allow one to determine the temperature of the coexistence of ice and water. In our work, we measure temperature parameters with high accuracy, and this is what allows us to draw the main conclusions. Which technique is more difficult or easier is a moot point. Perhaps there are researchers who will decide that a polarizing microscope should be considered a simple technique, and a thermometer that continuously measures temperature is difficult. We think the opposite. Reviewer #2 The relatively large volume (0.5 mL) of the sample is a problem when measuring the melting process as described in the last paragraph prior to the conclusions. Ice remains around the thermometer probe while the ice melts at the sample edges. This prevented measuring the dependence of the melting temperature on the amount of ice nucleating bacteria present, which was one of the objectives of the study. This seems like a cumbersome technique and it effectiveness has not been demonstrated here. One way to assess the potential of this system is to do a parallel analysis with ice-binding proteins (IBPs). These proteins have well-documented effects on the temperature at which ice and water co-exist, and the melting point of ice. The former is a temperature range for IBPs that is called the thermal hysteresis value and can be as high as 5-6 C for some insect IBPs. The latter is elevated by a fraction of a degree for most IBPs but would be again a good test of the sensitivity of the apparatus. We are discussing the problem of measuring the melting of ice. And we are discussing the fact that the point is not in the installation and its accuracy, but in the fact that it is impossible to start the process of ice melting in the entire volume of ice. Melting always starts at the surface. Therefore, the large volume of the sample (0.5ml) allowed us to somehow measure this parameter. Small droplets melt quickly and it is not possible to fix the exit of the curve to a plateau. We have an idea to make these measurements more accurately, but for this it is necessary to modernize our installation or do joint work with a laboratory that has a “simple” technique for measuring solutions in droplets. In this case, their installation will also have to be changed. Simply put, these measurements cannot be taken quickly. Regarding the comparison of the results of our experiments with ice binding proteins. That most IBPs are examined using cryo-osmometers. The cryo-osmometer differs from our installation only in the presence of a vibrator, which "starts" the process of water crystallization at relatively high temperatures (-5 oC). We do not need it, because we have studied the influence of cells on the process of ice nucleation. That is, to put it simply, our work is carried out in exactly the same way as most IBP studies. And, therefore, the results of the study can be compared with works in which a cryo-osmometer is used. We used our facility to study IBP proteins The article in print, but, the results are available in BioRxiv (Glukhova K.A., Okulova J.D., Melnik B.S. (2020) Designing and studying a mutant form of the ice-binding protein from Choristoneura fumiferana. BioRxiv. https://doi.org/10.1101/2020.08.31.275651) Reviewer #2 This manuscript has been inadequately edited and would have benefitted from a peer review by a colleague. There are many issues to address. It talks about the ‘concentration’ of bacteria. This term applies to something in solution, whereas the bacteria are in a suspension. Cell density is a more appropriate term. E. coli needs a space between E. and coli, as does 20 mM. Elsewhere mM is written as mm. As for typos. You are absolutely right. Minor typos, such as mm instead of mM, greatly interfere with the understanding of the article. We tried to fix it. Attachments Attachment Submitted filename: Response_to_rev12e.docx https://doi.org/10.1371/journal.pone.0265683.r002
7 Mar 2022 Decision Letter - Lei Li, Editor The influence of Pseudomonas syringae on water freezing and ice melting. PONE-D-21-36407R1 Dear Dr. Melnik, 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, Lei Li 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 #3: 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 #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #3: 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 #3: 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 #3: 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 adapted to a form suitable for publication. The authors have sufficiently included all my requirements in the revised manuscript. Reviewer #3: Obviously, the authors have made great efforts to modify the manuscript. This manuscript is highly innovative and should be published. ****** 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 #3: No https://doi.org/10.1371/journal.pone.0265683.r003
Formally Accepted
2 May 2022 Acceptance Letter - Lei Li, Editor PONE-D-21-36407R1 The influence of Pseudomonas syringae on water freezing and ice melting. Dear Dr. Melnik: 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. Lei Li Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0265683.r004

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