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This Pre Peer Review is an exercise performed by Master 2 students from Paul Sabatier University, Toulouse, France (Master Microbiologie Moléculaire). Writing a pre-review from a bioRxiv article is a part of their module "Scientific analysis".

It looks us 3 sessions to finalise the peer-review :

Session 1: presentation of the scientific edition landscape + article reading

Session 2: critical reading of each figure

Session 3: pooling students peer-reviews

The final text was slightly edited for grammar and syntax but the peer-review reflects the work of the students. 

The article discusses the role of a small RNA (sRNA) named OueS in the evolution and virulence of Vibrio cholerae, the bacterium responsible for cholera. OueS is a sRNA with a unique bimodular structure, and it is partially located within the ompU gene. The authors elucidated the major role of a toxigenic OueS allele in the emergence of virulence traits by using a set of genetic constructs. The article shows that OueS variants from toxigenic strains repress biofilm formation, which is essential for effective colonization, by inhibiting the RyhB RNA. Moreover, with plaque assays, they demonstrated that this allele activates the phage defense system (CBASS), thereby promoting resistance to intestinal bacteriophages. It was also shown that OueS is expressed from the OmpU promoter and is regulated by ToxR, a major virulence regulator. Interestingly, the transcriptional studies showed that the toxigenic OueS allele controls a large portion of the ToxR regulon, acting as a "substitute" for ToxR in regulating many genes. Finally, using a mice infection model, the study's results suggest that variations in OueS alleles greatly influence the ability of V. cholerae to colonize various hosts and environmental reservoirs, indicating a trade-off in fitness.

Major issues 

(1) In figure 2 (D and H), the predicted interaction regions between OueS/RyhB and OueS/PCBASS are located within a region of OueS which is already involved in duplex formation (last stem loop). Would you please comment how this pairing region could be involved in interactions with mRNA targets? 

Furthermore, the OueS regions which are predicted to interact with RahB and PCBASS are in the invariable part of OueS (in other words, these regions are identical in C6706 and GBE1114). The autors should comment on this fact and discuss possible reasons why OueS from GBE1114 does not give the same effect, even though the predicted interaction-sequence is the same.

(2) Statistical analysis with indicative pvalues are in general included along the whole article, however we noticed they were issing, and would be appreicated, for Fig 1I, 1J, 2B and 2J

Minor issues 

(1) Figure 1 :

        (1.1)  We agree figure 1C is quite convincing in showing that the 0.2kb sRNA half-life is affected by the absence of the RNA/RNA mediating interaction protein Hfq, however, a complementation assay would be welcome, specially because we are wondering why the band in the hfq mutant would be higher than in the wt. Could you please discuss this particular point?

        (1.2) For panels E and F, showing the OueS secondary structure of each allele:

            - Was the OueS C6706 secondry structure the only structure predicted, or the one with the highest energy level? 

            - To be able to compare the 2 secondary structures and sequences, the reader would appreciate  if the nucleotides that differ between the two versions of OueS could be clearly indicated.

        (1.3) Perhaps panels I and J would fit better in supplementary data, since Figure 1 is already heavy and these panels account mostly for control verification of ompU deletion effect.

(2) Figure 2 : 

(2.1) We suggest splitting the ten panels into two separate figures by thematics: one with panels A to E focused on biofilm formation through iron metabolism, and another with panels F to J centered on defense systems against bacteriophages

(2.2) For panel B, the strain names don't seem logically organized to us. We suggest aligning the strain names on the same line and placing the plasmid complementation on a line below the strain names. 

(2.3) On all the panels, it would be wise to give the same name for OueS complementation. All conditions needs to be named OueS or all named pOueS.

(2.4) A few writing errors : there is probably a typo on fig 2.E, it may be "nts", not "ntd". In the legend for Fig. 2D, "C" is written instead of "D". 

(2.5) In the panel E legend, it may no be relevant to talk about the role of phage resistance system because it is not what the experiment is about. It would be preferable to link, if there one, the expression modulation of RhyB, the phage resistance and its impact on the intestine survival, host colonization and seasonality of cholera outbreaks in the main text, where that link is not made . In other words, a little introduction of the phage part that connects it to the previous one would be appreciated.

(3) Figure 3:

(3.1) We proposed the legend to be more detail for better understanding, as a suggestion pelase find an example for the panel A : "Ectopic expression of OueS represses biofilm formation in a ΔtoxR mutant. The OD600nm were measured after crystal violet essay on strains that harbor either the empty vector or vectors encoding OmpU or OueS mutants and allelic variants. n > 6. Two-tailed T-test p-value **** < 0.0001.

(3.2) For panel 3A, we suggest aligning the strain names on the same line and placing the plasmid complementation on a line below the strain names. 

(3.3) For panel B, the strains genotypes are not clear (all strains are delta toxR, which should be specified).

(3.4) For panel 3C, the graphical representation choice is not easy to understand. It would be better to use a graph that overlays the two strains  like the Venn diagram or 100% stacked histogram

(3.5) For panel F, we ask to see the northern blot in its uncut version

(4) Figure 4 

(4.1) Legend : The legend is not very detailed, in our opinion, the genotype descriptions of the strains used are not very informative. It would be more appropriate to include the precise genotype in the legend for the different panels, to improve the comprehension of the figures (genetic background of the strain)

(4.2) Panel A and B : The x-axis lacks clarity regarding the genotype of the strains used. It would be advisable to specify the deletions/mutations they carry in more detail, particularly mentioning the ompU gene deletion for the strains (instead of just “OueS”)

(4.1) For all panels, in both legends and axis on the figures: the precise genotype is not clear to us, is it delta OmpU + pOues?

(4.3) Panel D : It would be relevant to replace the current label above the fluorescence images (“DC and DC+GFP”) with more specific information about the strain used for the infection, that is, the wild-type strain labeled with GFP. For example, it could be labeled as  "DC Without infection and DC Infection with V. cholerae C6706 expressing GFP”.

Also, the panel presents the absolute fluorescence measurement correlated with infection by the wild-type strain. It would be interesting to perform the same infection test using both the C6706 strain labeled with GFP and a strain deleted for ompU or mutated in oueS, labeled with a different fluorophore. This would allow for a direct comparison of host colonization efficiency according to the strain’s genotype and provide a relative quantification of the infection capacity of the deleted strain.

(5) Regarding Supplementary Figures : 

(5.1) Supplementary 2, F : logo plot irrelevant since it's a visual representation of base frequency and that it was done on 2 sequences, the basic alignment shows it already.

(5.2) Supplementary S5 : The last column show that the ompU promoter is the one that transcribes OueS so we do not really understand the relevance of the 10 mutants done beforehand. We do not understand how the mutants strains were constructed (to show where the sequences were inserted, one could add a scheme just like the Fig 1D) and for what purpose, this might be better to remove those data from the pannel since they do not provide clear and usefull information (since the last column does it already). 

Positive feedbacks

Overall, the study's results are robust and include necessary controls. We believe that the conclusions of this paper will provide a solid base for future research on Vibrio cholerae.

(1) Regarding Figure 1 : 

Every necessary control experiments were done in panel A and H, making the panels more robust overall. The different gels are really clean and easy to read and the panel D make the location of the sRNA really clear as well. 

(2) Regarding Figure 2 : 

All the panels are clear and necessary for the author's conclusion and for the understanding of lecturers. The panels which represent GO enrichment results are intuitive and it's a good representation of what gene is expressed or not in the strain.  The legend is complete and easy to understand.

(3) Regarding Figure 3 :

The figure is very successful due to the use of appropriate controls and reliable statistics in figure A. These elements ensure an accurate assessment of the results, which enhances confidence in the conclusions. This methodological approach allows for a solid and convincing interpretation of the data.

(4) Regarding figure 4 : 

The study effectively demonstrates how different alleles of oueS lead to fitness trade-offs between virulence (intestinal colonization) and survival in environmental reservoirs. The use of appropriate host and environmental models strengthens the conclusions regarding the fitness costs associated with virulence. The use of statistical tests supports and adds value to the results.

Competing interests

The author declare that they have no competing interests

Competing interests

The authors declare that they have no competing interests.