Understanding the impact of the transcription-supercoiling coupling on bacterial genome evolution

DNA supercoiling, the under or overwinding of DNA

are locally organized to leverage the transcription-supercoiling coupling for activation or inhibition, but largerscale networks of genes are required to strongly inhibit genes (sometimes up to networks of 20 genes).Thus, supercoiling-mediated interactions between genes can implicate more than just local genes.Finally, they construct an "effective interaction graph" between genes by successively simulating gene knock-outs for all of the genes of an individual and observing the effect on the expression level of other genes.They observe a densely connected interaction network, implying that supercoiling-based regulation could evolve concurrently with genome organization in bacterial genomes.

Authors' reply, 27 May 2024
Dear Editor and Reviewers, The main file containing our response to the reviewers is the reply round 2.pdf file found below.
As the edits to the main manuscript in response to the reviewers are minor, we have noted precisely in the reply file where they can be found in the main text for easy readability.

Reviewed by anonymous reviewer 1, 02 April 2024
After carefully reading the modifications provided by the authors, the manuscript has been extended by incorporating the impact of gene triplets into the analysis.Following these revisions, the manuscript now presents a clearer communication of the main findings and the methods employed.
Therefore, I have no further requirements for the manuscript and am pleased to proceed with its publication.
Regarding the additional questions: The results of this study highlights the importance of gene organization and its direct impact on gene expression levels, driven by DNA supercoiling.These interactions extend beyond pairwise interactions between neighboring genes, revealing a complex web of connections.
Overall, the article is well-written and effectively communicates the main findings and methods.
Here are some minor suggestions to enhance the quality and clarity of the paper: 1.In the abstract, "two different environments" are mentioned without immediate context.After reading the paper, now I know what it means, but it would be beneficial to clarify that these two environments refer to settings with different global superhelical levels.This clarification will help readers better understand the specific conditions being discussed.
2. While the introduction effectively presents the research question, which investigates the impact of transcriptionsupercoiling coupling on genome evolution in bacteria, consider adding a more explicit discussion of the potential broader implications and significance of answering this research question.Mentioning how this research could advance the field, benefit experimentalists, contribute to synthetic biology, inform future models, etc., would provide a clearer picture of the research's potential impact.
3. Explicitly mention the mathematical objective of the model in the methods section (I didn't see it there).Specifically, state that the model aims to maximize the fitness function 'f' based on gene type assignments (A, B, AB).Including this clarification will provide a clearer understanding of the primary mathematical goal and purpose of the model within the methods section.
4. I found it somewhat unclear from the text whether, for each generation, there were N=100 copies of individuals with the same mutation or if random mutations were introduced for each of the 100 individuals.
While the latter interpretation seems more reasonable to me, explicitly mentioning this in the text would greatly enhance clarity and help readers better understand the experimental setup.6. Mention the threshold used to define the minimal subnetwork size (around line 325).Additionally, it would be helpful to have figures depicting the expression levels of both the minimal subnetworks and the complete genome.Perhaps these figures could be included as supplementary material to provide a more comprehensive view of the data.7. Revise the captions of Figure 10 to explicitly explain and mention the box plots, ensuring that each figure is understandable on its own.8.In line 603, where it states "n= 1 if the genes are on the same strand, and n=-1 otherwise" shouldn't it be the other strand?9.In Section 5.3 (Experimental Setup), include a reminder that each individual is tested in the two environments.
Varoquaux , posted 07 May 2024, validated 07 May 2024 Minor changes Dear Théotime, Sam, and Guillaume, The article you have re-submitted have been reviewed a second time (see below for the two reviews).A reviewer suggests additional minor changes.We would be delighted if you would propose a new version of this preprint addressing the reviewer's comments.I look forward to receiving your revised preprint, upon which I will submit my recommendation.

•--
Title and abstract -Does the title clearly reflect the content of the article?[X ] Yes, [ ] No (please explain), [ ] I don't know -Does the abstract present the main findings of the study?[X] Yes, [ ] No (please explain), [ ] I don't know • Introduction -Are the research questions/hypotheses/predictions clearly presented?[X] Yes, [ ] No (please explain), [ ] I don't know -Does the introduction build on relevant research in the field?[X] Yes, [ ] No (please explain), [ ] I don't know • Materials and methods -Are the methods and analyses sufficiently detailed to allow replication by other researchers?[X] Yes, [ ] No (please explain), [ ] I don't know -Are the methods and statistical analyses appropriate and well described?[X] Yes, [ ] No (please explain), [ ] I don't know • Results In the case of negative results, is there a statistical power analysis (or an adequate Bayesian analysis or equivalence testing)?[X] Yes, [ ] No (please explain), [ ] I don't know -Are the results described and interpreted correctly?[ X] Yes, [ ] No (please explain), [ ] I don't know • Discussion Have the authors appropriately emphasized the strengths and limitations of their study/theory/methods/argument?[ X] Yes, [ ] No (please explain), [ ] I don't know -Are the conclusions adequately supported by the results (without overstating the implications of the findings)?[X ] Yes, [ ] No (please explain), [ ] I don't know Evaluation round #1 DOI or URL of the preprint: https://doi.org/10.1101/2022.09.23.509185Version of the preprint: 2 5. ForFigures 5, 7, 9 & 10, explicitly indicate the significance of the circular markers 'o' in the captions.' It might not be immediately clear to readers what these markers represent, and including this information in the captions would facilitate a better understanding of the data and visuals.