Protein profile of the Escherichia coli strain, BW25113, exposed to two novel iron-halide compounds: Fe(Hampy)2Cl4 and Fe(Hampy)2Br4
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Characterizing the proteomic profile of the Escherichia coli strain, BW2513, exposed to two novel iron-halide complexes: iron tetrachloride and iron tetrabromide.
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Thank you very much for submitting your revised manuscript to Access Microbiology and your efforts in applying the suggested correction to the manuscript. I am pleased to inform you that the manuscript has now been accepted for publication. Congratulations to all authors!
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Thank you very much for submitting your revised manuscript to Access Microbiology and for implementing the suggested changes. It has now been reviewed by one of the reviewers that provided comments on the original submission, whose comments are attached at the bottom of this email. We both agree that the quality of the manuscript has increased considerably, so thanks again for your efforts in improvement. However, there are some new amendments and suggestions that would need to be applied. One important aspect that would need to be amended is that, as the reviewer highlights, there is no biological validation of the proteomics and in silico findings. We appreciate that the experiments performed and their interpretation are comprehensive and correct, but this is a major limitation of the study that needs to be acknowledged in the …
Thank you very much for submitting your revised manuscript to Access Microbiology and for implementing the suggested changes. It has now been reviewed by one of the reviewers that provided comments on the original submission, whose comments are attached at the bottom of this email. We both agree that the quality of the manuscript has increased considerably, so thanks again for your efforts in improvement. However, there are some new amendments and suggestions that would need to be applied. One important aspect that would need to be amended is that, as the reviewer highlights, there is no biological validation of the proteomics and in silico findings. We appreciate that the experiments performed and their interpretation are comprehensive and correct, but this is a major limitation of the study that needs to be acknowledged in the discussion. Another important aspect that can be improved is the discussion of a potential mechanism of action of these iron complexes. It is true that a potential mechanism of action is mentioned based on previous literature in L664-667, however I believe a more elaborated paragraph on this aspect in the Discussion section would be greatly beneficial for the reader’s understanding of the study. Apart from the reviewer’s minor points to amend, please find here some additional comment that would need correction: • L247-248, L391, L682: here are some mentions to “protein expression”. However, only genes are expressed, whilst proteins are produced. Please correct this terminology throughout the text. • L254-255: “These were chosen as concentrations that inhibited but did not prevent bacterial cell growth”. Inhibiting and preventing can actually be interchangeable here. I assume the meaning of the sentence is that these concentrations decreased growth rate but did not completely inhibit it. If that is the case, please correct it accordingly. • L255-256: “The iron halide complexes caused a decrease in cell density over a 24 h period”. Decrease may mean that there was a certain OD that went down, whereas I interpret from the Methods section that the treated cultures just reached a lower OD after 24 h. Please correct this accordingly. • Fig. 4 is cited before Fig. 3 in the text, and Fig. 7 before Fig. 6 • L372 Figure legend 7 and the main text are in the same paragraph • L375 and L558 and L563, L565: correct “LoN” protease to “Lon” protease • The Discussion section is very comprehensive and extensive, I would suggest that it is shortened a bit highlighting the most important aspects of the study and contextualising them with the literature. Furthermore, the Conclusion section is a bit redundant with the Discussion, I would suggest that it is made more direct and concise. There are also further comparisons with previous literature in the Conclusion section that would be better moved to the Discussion section itself. Please provide a revised version of the manuscript (including a tracked changes version) along with point-by-point responses to the reviewer’s comments and the ones above within 2 months.
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Comments to Author
After the first revision, the article has improved considerably. The authors have included all suggested changes and explained those points that were unclear. The article is very complete in terms of the results obtained by proteomic analysis, showing a great work of bioinformatic analysis and a very good discussion of the data using an extensive literature review. This article is interesting and is the beginning of a line of work, although it lacks the biological validation to be able to validate the hypotheses put forward. Mutant generation, molecular interaction assays, enzyme activity assays, among others, would be necessary to verify the predictions made using the different bioinformatics tools. Some small formatting errors remain, such as: - Line 384: There are two semicolons, delete one. - Line …
Comments to Author
After the first revision, the article has improved considerably. The authors have included all suggested changes and explained those points that were unclear. The article is very complete in terms of the results obtained by proteomic analysis, showing a great work of bioinformatic analysis and a very good discussion of the data using an extensive literature review. This article is interesting and is the beginning of a line of work, although it lacks the biological validation to be able to validate the hypotheses put forward. Mutant generation, molecular interaction assays, enzyme activity assays, among others, would be necessary to verify the predictions made using the different bioinformatics tools. Some small formatting errors remain, such as: - Line 384: There are two semicolons, delete one. - Line 388: Put E. coli in italics. - Line 481: Figure 9 A is incomplete in the lower part. - Line 564: References 74 and 75 are in different format, make them bold and italic, same as the rest.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Thank you very much for submitting your manuscript to Access Microbiology. It has now been reviewed by two experts in the field, whose comment are attached at the bottom of this email. I agree with them that this work is timely and has potential, however he reviewers have flagged a number of concerns about it. In particular, the comments on lack of replicates of the proteomics experiments need to be addresses. As there is no individualised validation of the protein variation with/without treatment, I am afraid the conclusions of the study cannot be entirely supported if there are not 2-3 biological replicates of this experiment. Additionally, I would like to enquire you to disclose the full proteomic dataset of each sample, not just those proteins whose quantities change significantly, according to the Open Research nature of Access …
Thank you very much for submitting your manuscript to Access Microbiology. It has now been reviewed by two experts in the field, whose comment are attached at the bottom of this email. I agree with them that this work is timely and has potential, however he reviewers have flagged a number of concerns about it. In particular, the comments on lack of replicates of the proteomics experiments need to be addresses. As there is no individualised validation of the protein variation with/without treatment, I am afraid the conclusions of the study cannot be entirely supported if there are not 2-3 biological replicates of this experiment. Additionally, I would like to enquire you to disclose the full proteomic dataset of each sample, not just those proteins whose quantities change significantly, according to the Open Research nature of Access Microbiology. This can be done in the supplementary material or uploading it to a public repository such as ProteomeXchange and indicating the accession number in a Data Summary section within the manuscript. There are also several reviewer suggestions on the figure style and format and the literature presentation that would greatly benefit the quality of the manuscript. Please provide a revised version of this manuscript (including a tracked changes document) and a point-by-point response to all the reviewer comment within 4 months.
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Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data The methods are largely well-described and a clear rationale is made for the choice of methods. Explanation of the purpose, parameters and data used and the outputs generated by the different bioinformatics analyses is good. Minor comments: Line 94-96 Previous study is cited [14] where bactericidal activity was discovered. What bacteria were investigated in this study? Was E. coli, and specifically E. coli BW2513, investigated and did it show sensitivity to the halide compounds? What is the rationale for choosing this bacterial species and strain for the submitted manuscript? Line 100 - the proteome of BW2513 is well-characterised, a reference is needed here. NOTE ; this reference to Swiss-Prot is included in the Results but …
Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data The methods are largely well-described and a clear rationale is made for the choice of methods. Explanation of the purpose, parameters and data used and the outputs generated by the different bioinformatics analyses is good. Minor comments: Line 94-96 Previous study is cited [14] where bactericidal activity was discovered. What bacteria were investigated in this study? Was E. coli, and specifically E. coli BW2513, investigated and did it show sensitivity to the halide compounds? What is the rationale for choosing this bacterial species and strain for the submitted manuscript? Line 100 - the proteome of BW2513 is well-characterised, a reference is needed here. NOTE ; this reference to Swiss-Prot is included in the Results but should also be included in the introduction) Line 120 - Was the 5 ml incubation at 16h or 18h? Line 133-134 - What level of sonication was used? You state that concentration of the supernatant was determined, what centrifugation conditions were used to generate supernatants? 2. Presentation of results * Results are presented with detailed figure legends and are described well in the text. * The graphical abstract has some bits of the halide complex names that are not clear - this may be an issue with the way this figure has been assembled. In the Proteomics Workflow section of the abstract the following corrections are needed: Bacterial growth vs Bacterial cell grow and DAVID vs David. I also think that the blue highlighted text6 does not fit with the colour scheme and is not really necessary. * The GO terms and KEGG analysis would be more easily interpreted and summarised in a table or other type of figure, rather than stated in the text as percentages. This would also allow the reader to be able to more clearly see how differentially regulated proteins have been identified and classified using these systems. For example, there seems to be a merging of the different types of GO analysis in figures 3 and 4, with the highlighted GO terms including both biological function and cellular location in 3A, 4A and 4C. The distribution of cellular location, biologicals processes and molecular function is clear in the supplementary data figures S1-S3 and I suggest incorporating this data into the main part of the manuscript. * Fig 3C - It would help interpretation of Fig 3C if the key with GO terms was listed in the same order top to bottom as they appear on the heat map from left to right. The rationale for the further investigation of these 96 proteins could also be explained better as it is not clear why these proteins were focused on out of the 244 shared dysregulated proteins. * Fig 5 - the legend says that red arrows indicate stimulatory signals, but no red arrows are visible. Is this an error in the legend or are there no stimulatory signals in these networks? * Fig 6 - the resolution of this figure was very low and when I zoomed in I could not decipher the gene names so the quality of this interactome figure needs to be improved. NOTE: quality of other interactome figures were better than Fig 6 but could still benefit from greater resolution. 3. How the style and organization of the paper communicates and represents key findings * Overall, there are a large number of typographical errors and, while this sort of large data set is very difficult to draw firm conclusions from, the trends and overall findings could be more clearly described to demonstrate what the key outcomes are from this work. * Abstract - the abstract uses "suggests" vs "hints at" when describing the effects of tetrachloride and tetrabromide compounds respectively. Do these mean the same or different things? Does this word choice imply differences in the size of effects or confidence in these conclusions? * There appears to be a very large space preceding reference numbers. Not sure what this is but it looks a bit strange so check the formatting instructions for references for this journal. * Line 220 - not sure what this phrase means - "such sub-total growth inhibitory concentrations". Suggest changing it to "such sub-inhibitory concentrations" if the intention is to describe how lower concentrations of antimicrobials can still have growth effects even if they are not bacteriostatic or bacteriocidal. * Line 222- see above * Line 225-228 and Fig 2 - this data shows that the iron halide complexes do inhibit growth at the concentrations used in this study. Therefore the section above discussing sub-inhibitory concentrations should be revised. Were lower concentrations than 0.5 and 1M investigated at all? * lines 431-433 - the results text states that "most" of the key regulators had differences in expression, but in fact Fig7A shows that 3/6 had changes in expression with 3/6 not showing significantly different expression. This is certainly a contrast to the results in Fig 7B, but a more nuanced interpretation of this result would improve it, including whether increased/decreased expression levels of regulators correspond to their inhibitory/stimulatory effects on the pathways analysed. * The interpretation of the results lacks any sort of insight into the mechanism of action of these two antimicrobial compounds. What is already known about their likely mechanisms and do the data in this paper add any further evidence for this? The data clearly show that there are differences in the response to the different ligands, so what is likely to be some compound-specific and/or common mechanisms for these antimicrobials? 4. Literature analysis or discussion * There is very little introductory material to give context to the study. A more thorough review of the relevant literature, including the authors' previous work, is needed. * Some key antibiotic resistance-associated genes are identified in the results section (LoN and FtsH line 336-7, AccD and acetyl CoA carboxylase line 343-4) but some further analyses of these in the discussion section would improve it. * The discussion identifies a trend in the upregulation of oxidative stress proteins (Lines 468-479) and compares this to other similar studies (Line 480-6), but this would be improved by more detailed comparison of these studies eg. of the bacteria/strains studied, the degree of chemical similarity of the antimicrobials and the methods used to detect responses. * What is the relevance to responses to tetrabromide/tetrachloride is the role of FliC in virulence? (Lines 494-503). The link between pathogenicity and antimicrobial effects could be made clearer, as well as some more detailed discussion of the mechanism of action of cranberry compounds for comparison. * The suggestion that MetK upregulation demonstrates a return to homeostasis following exposure to tetrachloride is not really explained (lines 513-522). What evidence is there for this idea from this study or others? Does MetK have a direct role in iron-ion homeostasis, TCA cycle, oxidative phosphorylation or other processes that were upregulated? 5. Any other relevant comments Typographical errors: Lines 2-3 - Subscript numbers are not used for compound names. Line 58 - remove "the" after profile. Line 70 - remove "a" before bacteria Line 79 - change actions to action Line 81 - change later to latter Line 85 - remove the before novel Line 96 - reference is in bold here, unlike others, this should be removed Line 129-130 - change bacteria to bacterial and state centrifugation conditions in g rather than rpm Line 131-133 - Put the components of cell lysis solution in square brackets [] Line 151 - change were to was as this is a singular sample Line 164 - remove repeated E. coli Line 165 - reference is in bold here, unlike others, this should be removed Line 214 - change bacteria to bacterial Line 353 - change mas-spectrometry data quantified to mass-spectrometry data are quantified Line 370 - component to components Line 385 - delete S13? Linr 413 - remove the "proteins" after (fig, 6A) Line 424-427, Fig 6, Fig 7 and their figure legends -several discrepancies on the name and nomenclature of SLP/SIP/S1P protein. This should be checked and the correct nomenclature standardised across the manuscript Line 452 - replace unpair t-test with unpaired t-test
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
The work is very interesting and has a lot of potential. The initial design is correct but has shortcomings that make the conclusions difficult to support. The first thing is that English must be improved. Second thing, I cannot find detailed in the text how many biological replicates are used for the proteomics study. This is a very important aspect because if there is no replication, data are not statistically significant and nothing can be concluded. Aditionally, your data with the fold change of the differentially expressed proteins are not complete. It can be very useful to create Tables with the MS data as a Supplementary table so people interested can check the quality of protein identification and quantitation. Figure 4 is difficult to analyse, letters are very small. And please, avoid the use …
Comments to Author
The work is very interesting and has a lot of potential. The initial design is correct but has shortcomings that make the conclusions difficult to support. The first thing is that English must be improved. Second thing, I cannot find detailed in the text how many biological replicates are used for the proteomics study. This is a very important aspect because if there is no replication, data are not statistically significant and nothing can be concluded. Aditionally, your data with the fold change of the differentially expressed proteins are not complete. It can be very useful to create Tables with the MS data as a Supplementary table so people interested can check the quality of protein identification and quantitation. Figure 4 is difficult to analyse, letters are very small. And please, avoid the use of colors in the names of the GO categories in panels B and C because is difficult to read Finally, below I list some errors in the text that need to be corrected: - Line 58: In the sentence protein-expression profile the of a bacteria..." Delete "the" - Line 70: Change "for comparing" with "to compare" - Line: 120: There is an inconsistency with the incubation time, 16 or 18 h? - Line 217: Change "permits" for "allows" - Line 540: Write E. coli in Italics - Line 540: Change the sentence "The central role of this protein has been shown in an E. coli study that demonstrated deletion of this gene decreased the bacterial cell growth and stress tolerance" with "The central role of this protein has been demonstrated in a study in which deletion of the gene decreased bacterial growth and stress tolerance in E coli" - Line 544 Delete the "s" in suggests - Line 544: Change "ligands do play" with "ligands play"
Please rate the manuscript for methodological rigour
Very poor
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Not at all
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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