Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript title: Investigation of the mechanisms of transition of gram-negative bacterial cells into induced anabiosis using computational methods of classical molecular dynamics

 

The presented study aimed to explain the effect of 4-hexylresorcinol on the cell membranes of gram-negative bacteria (Escherichia coli) by combination of experimental and computational methods. The topic fits well within the journal’s scope, and the manuscript is generally well-written, logically structured, and supported by an appropriate selection of literature. The Materials and Methods section is very detailed, especially the computational part. The results are presented comprehensively, following a logical flow, and the discussion gives appropriate answers to questions raised in the Introduction.

 

The following minor points should be addressed:

1) Check the proper way to write the phrase “gram-negative”, and uniform it throughout the manuscript.

2) In lines 128-132, there is mention of 4-GR. Check if it is a typo or give full name before using the abbreviation.

3) Why use the concentration of 1000 μM, when in the literature 200 μM was the highest. Isn’t this cutoff too high?

4) Line 275 – what is meant by “data not shown”?

5) If I understood correctly, mummified cells cannot be perceived as dead, even though they are non-viable? Maybe it would be beneficial to wider audience to insert a sentence or two explaining the difference in the Introduction.

Author Response

Dear reviewer, thank you very much for your attention to our article. All your valuable comments have been taken into account.

 

1) Check the proper way to write the phrase “gram-negative”, and uniform it throughout the manuscript.

Thank you very much for your comment. We've checked and fixed everything.

 

2) In lines 128-132, there is mention of 4-GR. Check if it is a typo or give full name before using the abbreviation.

Yes, that was a typo. It's been fixed. Thank you.

 

3) Why use the concentration of 1000 μM, when in the literature 200 μM was the highest. Isn’t this cutoff too high?

Thank you for your question. A concentration of 200 μM is used to obtain anabiotic cells that can return to a viable state. A concentration of 1000 μM 4-hexylresorcinol is used to ensure rapid production of nonviable mummified cells. We use a fivefold higher concentration to ensure that all cells have lost viability, and at this concentration, the process occurs very quickly. We have used this high concentration to obtain mummified cells in numerous other previous studies. Therefore, this concentration was also used in this study.

4) Line 275 – what is meant by “data not shown”?

We meant that these data were not presented in this study. We have corrected the phrase to read: "These data are not provided in this study." Thank you.

 

5) If I understood correctly, mummified cells cannot be perceived as dead, even though they are non-viable? Maybe it would be beneficial to wider audience to insert a sentence or two explaining the difference in the Introduction.

Thank you for your comment. Mummified cells are dead cells that can no longer be revived; they are nonviable. They differ from other dead cells in that they do not undergo lysis, as occurs in most cases when a bacterial cell dies. After a bacterial cell dies, its enzymes begin to destroy all structures. In this case, 4-hexylresorcinol, rapidly penetrating all cellular structures, mummifies them, so the cell does not decompose, just like the Egyptian mummies of the pharaohs. We've added a new section to the introduction to make this information clearer.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

        I have reviewed the manuscript entitled "Investigation of the mechanisms of transition of gram-negative bacterial cells into induced anabiosis using computational methods of classical molecular dynamics"  and found it to be well written and of interest for the scientific community as it provides valuable insight into the mechanisms of transition of E. coli into induced anabiosis, using 4-hexylresorcinol as an anabiosis autoinducer. Therefore, there are only a few minor observations to be taken into account to improve the quality of the manuscript. They are mentioned below:

1. Please write all Latin names of bacteria and Latin expressions using italic letters (line 42 - Escherichia coli, line 104 - in vivo, line 105 - in vitro, line 274 - E. coli).
2. Please choose the preferred form: "Gram-negative" or " gram-negative" and "Gram-positive" or "gram-positive"  and use throughout the entire manuscript to ensure uniformity.
3. Line 76: Please correct "bio-technology" to "biotechnology".
4. Line 90: Please correct "al-most"  to "almost". 
5. Lines 129-131: What does 4-GR stand for? Did you mean 4HR? Please modify accordingly or detail the abbreviation.
6. Line 147: I recommend using "ethanolic solutions"  instead of "ethyl solutions".
7. Line 183: I recommend adding the appropriate reference for Reynolds procedure.
8. Table 2, line 5, last column: There is a dot after the multiplication sign and before 10⁷ that should be eliminated.
9. Line 190: Please correct " pepidoglycan"  to "peptidoglycan".
10. Please make sure that all abreviations used are detailed the first time they appear in the manuscript (e.g. POPE, POPG, SPC/E water etc.)
11. Line 192: The preposition "in" is doubled.

Author Response

Dear reviewer, thank you very much for reading our manuscript so carefully. We have taken all your comments into account and made the necessary revisions to the article.

1. Please write all Latin names of bacteria and Latin expressions using italic letters (line 42 - Escherichia coli, line 104 - in vivo, line 105 - in vitro, line 274 - E. coli).

Thanks for the comment, everything has been fixed.

2. Please choose the preferred form: "Gram-negative" or " gram-negative" and "Gram-positive" or "gram-positive"  and use throughout the entire manuscript to ensure uniformity.

Thanks for the comment. We've corrected all the terms in the article so they're spelled the same: "gram-positive" and "gram-negative."

3. Line 76: Please correct "bio-technology" to "biotechnology".

Thanks for the comment, everything has been fixed.

4. Line 90: Please correct "al-most"  to "almost". 

Thanks for the comment, everything has been fixed.

5. Lines 129-131: What does 4-GR stand for? Did you mean 4HR? Please modify accordingly or detail the abbreviation.

It was a typo which we have corrected.

6. Line 147: I recommend using "ethanolic solutions"  instead of "ethyl solutions".

Thank you, we have corrected this term.

7. Line 183: I recommend adding the appropriate reference for Reynolds procedure.

Thank you, we've included a reference to this method.

8. Table 2, line 5, last column: There is a dot after the multiplication sign and before 10⁷ that should be eliminated.

Thanks for the comment, everything has been fixed.

9. Line 190: Please correct " pepidoglycan"  to "peptidoglycan".

Thank you, we have corrected this term.

10. Please make sure that all abreviations used are detailed the first time they appear in the manuscript (e.g. POPE, POPG, SPC/E water etc.)

We thank the reviewer for this comment, the abbreviations have been detailed.

11. Line 192: The preposition "in" is doubled.

Thanks for the comment, everything has been fixed.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript present a combined experimental and simulation data which indicate that 4-hexylresorcinol is able to penetrate into phospholipid membranes, and at increasing concentrations can lead to lipid reorganization, membrane stress, and accumulation of amphiphile aggregates. Authors wish to suggest that membrane perturbations correlate with ultrastructural changes observed experimentally at high 4HR concentrations.

The manuscript is original and the topic is timely but authors are requested to clarify the following point before publishing:

1. The membrane model consists of a symmetric POPE/POPG bilayer, which is generally considered representative of the Gram-negative inner Since the Gram-negative outer membrane LPS-rich outer leaflet is not included, how do the authors expect this simplification to affect the adsorption, and penetration of 4-hexylresorcinol? In particular, to what extent might the absence of the LPS barrier lead to an overestimation of membrane accessibility and disruption in vivo?
2. Based on the slow relaxation times of lipid membranes and amphiphile partitioning, how can the authors be confident that simulations at the shorter end of this range (∼50–100 ns) are sufficiently converged?
3. In Sec 3.4 the observed adsorption, aggregation, and penetration behavior of 4HR may depend on the initial placement and orientation of molecules relative to the membrane. Could the authors comment on whether alternative initial conditions were tested, or whether a preliminary diffusion/equilibration stage (e.g., Brownian dynamics or restrained MD above the membrane surface) might reduce potential bias from the initial configuration?
4. The manuscript proposes a mechanistic link between membrane perturbation by 4HR and bacterial dormancy/mummification. Please clarify to which extent these biological states are directly demonstrated from membrane structural changes.

Author Response

Dear reviewer, thank you very much for your review. We have provided all responses to your questions and comments in a separate file.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript addresses an important biological problem and contains a substantial body of experimental and computational work. The topic is clearly relevant to fields of broad interest, including bacterial persistence, antimicrobial resistance, and membrane biophysics.

A central claim is that 4HR-induced membrane reorganization drives the transition from stress resistance to dormancy and ultimately to mummification. However, the evidence presented is largely correlative. TEM images demonstrate altered membrane ultrastructure. MD simulations show that 4HR can integrate into lipid bilayers and form aggregates.
What is missing is direct experimental validation that membrane restructuring is causative rather than a downstream consequence. For example: Are similar membrane changes observed during starvation-induced dormancy in the absence of 4HR? Can genetic or chemical perturbations of membrane composition modulate the 4HR effect?

The manuscript relies heavily on relatively high concentrations of 4HR (200–1000 µM). Although the authors argue that these concentrations correspond to membrane-level ratios derived from adsorption assumptions, the physiological plausibility remains unclear. The authors should explicitly discuss whether endogenous anabiosis autoinducers reach comparable local concentrations in natural or clinical settings.

How ethanol-mediated delivery may influence membrane permeability independently of 4HR.

The MD simulations are technically sound and carefully described, but their biological interpretation requires caution. The membrane model lacks lipopolysaccharides (LPS), which dominate the outer membrane of Gram-negative bacteria and strongly affect permeability and mechanics. Also, timescales (≤1 µs) remain short relative to biological membrane remodeling processes. These limitations should be more explicitly acknowledged, and conclusions should be correspondingly tempered.

Although the authors briefly mention similar effects in other Gram-negative bacteria, the manuscript is overwhelmingly centered on E. coli Top10. Claims regarding “Gram-negative bacteria” in general should be narrowed or supported by additional data.

Comments on the Quality of English Language

The manuscript is generally readable but would benefit from professional language editing to reduce redundancy and improve narrative flow, particularly in the Introduction and Results sections.

Author Response

Dear reviewer, thank you very much for your thoughtful review of our publication and valuable comments. We have provided answers to your questions in the attached file. Thank you.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have addressed all questions satisfactorily. I support the publication of this paper from now on.

Reviewer 4 Report

Comments and Suggestions for Authors

All issues were responded.