The Vibrio vulnificus stressosome is dispensable in nutrient-rich media
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The stressosome is a protein complex that senses environmental stresses and mediates the stress response in several Gram-positive bacteria through the activation of the alternative sigma factor SigB. The stressosome locus is found in 44 % of Gram-negative Vibrio vulnificus isolates. However, V. vulnificus does not possess SigB. Nonetheless, in nutrient-limited media, the stressosome modulates gene transcription and bacterial behaviour. In this work, the expression of the stressosome genes was proven during stationary phase in nutrient-rich media and co-transcription as one operonic unit of the stressosome locus and its putative downstream regulatory locus was demonstrated. The construction of a stressosome mutant lacking the genes encoding the four proteins constituting the stressosome complex (VvRsbR, VvRsbS, VvRsbT, VvRsbX) allowed us to examine the role of this complex in vivo . Extensive phenotypic characterization of the ΔRSTX mutant in nutrient-rich media showed that the stressosome does not contribute to growth of V. vulnificus . Moreover, the stressosome did not modulate the tolerance or survival response of V. vulnificus to the range of stresses tested, which included ethanol, hyperosmolarity, hypoxia, high temperature, acidity and oxidative stress. Furthermore, the stressosome was dispensable for motility and exoenzyme production of V. vulnificus in nutrient-rich media. Therefore, in conclusion, although stressosome gene transcription occurs in nutrient-rich media, the stressosome neither has an essential role in stress responses of V. vulnificus nor does it seem to modulate these activities in these conditions. We hypothesise that the stressosome is expressed in nutrient-rich conditions as a sensor complex, but that activation of the complex does not occur in this environment.
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Thank you very much for diligently implementing all the suggested amendments on the manuscript. This is now suitable for publication. Congratulations!
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Thank you very much for implementing some of the suggestions and comments made by the reviewers in this revised manuscript. However, some amendments and clarifications are still needed. Please, address the following points: • Include dispersion measurements (i.e. error bars or shadowed zones, as suggested by Reviewer 2) in all graphs. • Start data series over time at t=0 and intersect all axis pairs at (0,0) (e.g. Figure 3, Figure S2, Figure S3). • Include the statistical analysis performed for each result to assess significance, as well as its result in terms of p-value, either in a ‘Statistical analysis’ subsection in Materials and Methods or in each figure legend. • Make clear in the main text that the results shown in Figure 4 are from one biological replicate only.
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In summary, the manuscript is well written and the experimental strategy seems appropriate. However, several concerns have emerged after the peer-review process. Please, consider all the reviewers’ suggestions and comments thoroughly, especially those concerning: • The methodology and experiments under anaerobic conditions and the conclusions obtained from gene expression quantification experiments. • Absence of statistical analyses and error bars, graph formatting with axis intersections different from (0,0). • If edited for presentation, original images must be provided as supplementary material if the manipulation could affect the interpretation of the results. • Please consider a re-writing of the methodology section describing the construction of the ΔRSTX mutant. As this mutant strain and its construction have …
In summary, the manuscript is well written and the experimental strategy seems appropriate. However, several concerns have emerged after the peer-review process. Please, consider all the reviewers’ suggestions and comments thoroughly, especially those concerning: • The methodology and experiments under anaerobic conditions and the conclusions obtained from gene expression quantification experiments. • Absence of statistical analyses and error bars, graph formatting with axis intersections different from (0,0). • If edited for presentation, original images must be provided as supplementary material if the manipulation could affect the interpretation of the results. • Please consider a re-writing of the methodology section describing the construction of the ΔRSTX mutant. As this mutant strain and its construction have already been published by the authors, they should refer to it providing a citation rather than describing it again. Please also consider a revision of the title and conclusions of manuscript. As raised by the reviewers, some of the final conclusions of this work do not accord entirely to the results and the conditions tested, or may need further experimental development to be fully conclusive. Additionally, according to the Access Microbiology open data policy, I strongly recommend that the authors make available all data repeats of their experiments to the community. Please, consider attaching them as supplementary information or uploading them to a public repository (for example Figshare), which will generate a citable DOI that can be added to the data availability section of the manuscript. More information about the Access Microbiology open data policy can be found in https://www.microbiologyresearch.org/open-data Please provide a revised manuscript containing all changes and a point-by-point response to all the reviewers’ comments within 2 months.
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Comments to Author
The authors provide a solid set of experiments investigating the role of the stressosome in Vibrio vulnificus. The development of a clean deletion strain is very useful and can provide better information about the role of the stressosome. However, the authors do not compare the effects of the RSTX operon on most of the phenotypes both under aerobic and anaerobic conditions. Since the stressosome is suggested to sense and response to O2 levels, the effects of the stressosome will likely only be observed by either comparing the full transcriptome of the WT and deletion strains grown under aerobic and anaerobic conditions, or by performing all of their phenotypic assays (stress responses, motility, etc) in the presence and absence of O2. The current data supports the fact that the stressosome doesn't …
Comments to Author
The authors provide a solid set of experiments investigating the role of the stressosome in Vibrio vulnificus. The development of a clean deletion strain is very useful and can provide better information about the role of the stressosome. However, the authors do not compare the effects of the RSTX operon on most of the phenotypes both under aerobic and anaerobic conditions. Since the stressosome is suggested to sense and response to O2 levels, the effects of the stressosome will likely only be observed by either comparing the full transcriptome of the WT and deletion strains grown under aerobic and anaerobic conditions, or by performing all of their phenotypic assays (stress responses, motility, etc) in the presence and absence of O2. The current data supports the fact that the stressosome doesn't affect growth in rich media in the presence or absence of O2, but further studies investigating the effects +/- O2 are required to support the statements that the stressosome isn't utilized in rich media. - The stressosome in Vibrio species has previously been shown to be regulated by oxygen levels. Does the presence/absence of the stressosome modulate responses to O2 in the WT and deletion? - It would be very useful to measure the O2 concentration in the O2 depleted conditions at different time points during the growth. Or if that is not possible, to use the sachets that generate an anaerobic atmosphere to deplete O2 from the media before starting the growth curves. That would help to better control the O2 levels and compare the strains. - What are the errors associated with the growth curve data? It would be very useful to show the error bars on the plots (as either bars or shadowed zone) or list the % in the caption. - Please label the figure panels in each of the figures to make it easier to refer to the captions. - For the stress response assays, the presence/absence of O2 should be combined with the H2O2 and NaCl stresses. By eye, it looks like the deletion strain is not able to grow as well on solid media as WT, despite the equivalent growth in liquid culture. The differences in growth may be further exacerbated by the addition of the stressors, especially since the stressosome may be regulating stress responses in different O2 environments. - Motility and exoenzyme experiments should also be performed anaerobically to determine if there is an effect.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very 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
In general, the work is well designed, methods are adequate and are well described and the conclusions are consistent with the results obtained. Reading is easy and results are properly presented. However, there are certain aspects that must be reconsidered or improved. *One of the objectives proposed in this work is to study the expression of the stressosome genes in nutrient-replete conditions. However, RT-PCR expression analysis have been performed with cells growing in LBN to stationary phase when conditions are nutrient limited. Therefore, these results, which demonstrate transcriptional organization, do not serve to conclude that the stressosome genes are expressed in nutrient-replete conditions as the authors state in the lines 30 and 269. To demonstrate it, RT-PCR experiments should be …
Comments to Author
In general, the work is well designed, methods are adequate and are well described and the conclusions are consistent with the results obtained. Reading is easy and results are properly presented. However, there are certain aspects that must be reconsidered or improved. *One of the objectives proposed in this work is to study the expression of the stressosome genes in nutrient-replete conditions. However, RT-PCR expression analysis have been performed with cells growing in LBN to stationary phase when conditions are nutrient limited. Therefore, these results, which demonstrate transcriptional organization, do not serve to conclude that the stressosome genes are expressed in nutrient-replete conditions as the authors state in the lines 30 and 269. To demonstrate it, RT-PCR experiments should be performed in exponential phase, similar to the approach used by the same authors in Heinz et al, 2022 in minimal medium. In fact, they observe low expression in the exponential phase and induction due to nutrient limitation in the stationary phase. In addition, the documentation of these results is inadequate, please provide the raw files for review as supplementary material in future versions of this manuscript. *In my opinion, the authors need to improve their use of the statistical tools to address the findings they present in this paper: - The authors do not properly use the term 'significant differences' (lines 329 and 416). The terms significant/insignificant differences should only be used when the appropriate statistical test has been applied to the results. Recommended re-write. - Dispersion measures of the data in Figures 4, S2A (16h) and S3 are missing. This statistical analysis will allow to the reader to visualize how reliable the results are. *Panel letters in Fig.5 should be added to improve comprehension.
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?
Yes: Fig1. Has been edited for presentation and this does not allow adequate determination of the size of the transcription products. The authors have provided the raw image files to the editorial office and reviewer, and these will be included in future versions of the manuscript.
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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