DNA/Cell Mass Homeostasis: Coordinating DNA Replication and Cell Size with Central Carbon Metabolism During Bacterial Growth

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

The author, in his article entitled "DNA/cell mass Homeostasis: Coordinating Cell Size and DNA Replication during Bacterial Growth," has taken into consideration the important problem of the origin of life, i.e., prokaryotic replication. Moreover, the interesting point is the relationship between cellular homeostasis and DNA mass. The author discusses in additional detail the mutual relationship between DanA/RNR. However, at this point, the deep discussion on the role of these two proteins separately should be discussed. Moreover, the role of ribonucleosides in DNA must be explained, as well as the process of their removal. Moreover, the main point of bacteria and also eukaryotes' survival in the homeostasis condition is the effectiveness of DNA repair. Therefore, the author should discuss the role of DNA damage in bacteria and also the mechanism of their repair.

From the Editorial point of view, the article is well written and readable with correctly cited references. The figures are correct in their context, but I recommend making them more reader-friendly. Also, the captions for figures and tables should be useful for their correct understanding.

I have som ecritical relmarcke:

The article is partially hypothetical, which requires its separation from experimental (previous data)  with references.

Please check the text in the linguistic and stylistic correction – the sentences are too long, which makes it difficult to follow.

Author Response

Reviewer 1: The author, in his article entitled "DNA/cell mass Homeostasis: Coordinating Cell Size and DNA Replication during Bacterial Growth," has taken into consideration the important problem of the origin of life, i.e., prokaryotic replication. Moreover, the interesting point is the relationship between cellular homeostasis and DNA mass. The author discusses in additional detail the mutual relationship between DnaA/RNR. However, at this point, the deep discussion on the role of these two proteins separately should be discussed.

 

Author: The manuscript, as rewritten, has emphasized that DnaA, which is the prokaryote analog of the Origin Recognition Complex in eukaryotes, licenses the replication origin and loads the orisome onto DNA. RNR, which is cell cycle regulated both in terms of transcription and activity, feeds the orisome (DNA pol) with the necessary dNTPs to fire DNA synthesis and start replication elongation. Absent the dNTPs, the orisome/DNA pol cannot proceed or advance duplication of the chromosome. This is a crucial point the manuscript has tried to emphasize: stalled/blocked replication forks down stream of oriC will in the presence of ongoing dNTP synthesis result in de novo initiation of new forks at oriC. This is a hypothesis, however, as suggested in the rewrite, although substantial experimental evidence supports it as being scientifically plausible.

 

 

Moreover, the role of ribonucleosides in DNA must be explained, as well as the process of their removal. Moreover, the main point of bacteria and also eukaryotes' survival in the homeostasis condition is the effectiveness of DNA repair. Therefore, the author should discuss the role of DNA damage in bacteria and also the mechanism of their repair.

 

Author: this is an excellent point meriting attention in its own right. Indeed, The reviewer has touched on a profound aspect of genetic evolution. A chapter has been added to the ms addressing the evolutionarily conserved role of rNTP incorporation and its modulating effects on the DNA repair systems in bacteria (and across the Tree of Life). A detailed analysis has been omitted, however, as being out of scope.

 

 

From the Editorial point of view, the article is well written and readable with correctly cited references. The figures are correct in their context, but I recommend making them more reader-friendly. Also, the captions for figures and tables should be useful for their correct understanding.

 

Author: Admittedly the legends are detailed but I believe that benefits the reader and aids in elaborating and clarifying the text to which the legends and figures refer. I hope the Reviewer will agree.

 

 

I have som ecritical relmarcke:

The article is partially hypothetical, which requires its separation from experimental (previous data)  with references.

 

Author: I have gone over the references to ensure their accuracy and support of the hypothetical parts of the ms. No claims are being made beyond what has been previously published either as experimental observation or the experimentally supported hypotheses of other reseachers.

 

 

Please check the text in the linguistic and stylistic correction – the sentences are too long, which makes it difficult to follow.

 

Author: the text has been re-read and reworked, hopefully to make it more readable.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

Authors have done a great job in this deep review and mechanistic exploration of the connection between bacterial metabolism, cell size, and DNA replication. Here are a few suggestions that can help improve the manuscript.

1. Title of manuscript can be replaced with a description that presents a clearer description of review. Eg, DNA/Call mass homeostasis: Coordinating Bacterial metabolism and its replication. Or DNA/Call mass homeostasis: Coordinating Cell size and DNA replication during bacterial growth.
2. Table 1. Last line calculation is wrong. Kindly check all calculations and make sure there is no error.
3. Table 1. .caption says, A 3X increase in the C-period corresponds to an approximately 2X increase in nucleoide complexity. Line 174. This does not match to table 1 results but to table 2.
4. Table 2 . what is Zaritsky Fig. 1?
5. Formula in line 104, 105 should be written more clear to remove confusion of exponential and multiplication. Like 2ⁿ      

Not 2n.

 

6. Line 243 , the growth rate parameter τ is missing. Check the formula again.
7. Line 55. Check the time 40 min or 44min.
8. Line 310. These genes are not present in figure 2. Remake figure 2. Also few genes mentioned in figure 2 is not mentioned in text. There should be no discrepancies between figures and texts.
9. Authors have used many short terma for complete words like aa for amino acids. This types of errors should be thoroughly checked in the whole manuscript.
10. Figure 4 is very misleading and conceptually wrong. Terminology should be refined and there should be clearity in ON\OFF. Redraw the figure.
11. There are multiple errors found in refrences and bibliography. Ref 39 lack journal name, volume, pg numbr, ref52 has typo error. Ref 79, 80 are wrongly written. Some refrences are duplicate like 59, 82. Check year of refre nce 7.
12. There are multiple mismatch of refrecnes and citaiotns that do not match to the text written n the manuscript.
13. Table 2. Check all refrences . is the study provide same information? Si et al. study to show a correlation between mass and fork rate.
14. Although there is no clear evidence of AI writing but lack of technical and formatting artifacts suggest that there should be proofreading of each and every table, figure s, formulas. It should be applied to reduce errors of automated writing process.

 

Author Response

Reviewer 2: Authors have done a great job in this deep review and mechanistic exploration of the connection between bacterial metabolism, cell size, and DNA replication. Here are a few suggestions that can help improve the manuscript.

1. Title of manuscript can be replaced with a description that presents a clearer description of review. Eg, DNA/Call mass homeostasis: Coordinating Bacterial metabolism and its replication. Or DNA/Call mass homeostasis: Coordinating Cell size and DNA replication during bacterial growth.

 

Author: the author agrees. This has been done.

 

 

2. Table 1. Last line calculation is wrong. Kindly check all calculations and make sure there is no error.

 

Author: the author would like to thank the reviewer for the reviewer’s careful handling of the manuscript. The corrections have been made.

 

3. Table 1. .caption says, A 3X increase in the C-period corresponds to an approximately 2X increase in nucleoide complexity. Line 174. This does not match to table 1 results but to table

 

 

Author: this has been clarified in the legend.

 

 

4. Table 2 . what is Zaritsky Fig. 1?

 

 

Author: the Zaritsky 2015 has been added to the references. This was an oversight.

 

 

5. Formula in line 104, 105 should be written more clear to remove confusion of exponential and multiplication. Like 2ⁿ      

Not 2n.

 

 

Author: this has been clarified.

 

 

6. Line 243 , the growth rate parameter τ is missing. Check the formula again.

 

 

Author: this has been corrected.

 

 

7. Line 55. Check the time 40 min or 44min.

 

 

Author: this has been specified.

 

 

8. Line 310. These genes are not present in figure 2. Remake figure 2. Also few genes mentioned in figure 2 is not mentioned in text. There should be no discrepancies between figures and texts.

 

 

Author: the figure has been corrected and the text clarified in the figure legend.

 

 

9. Authors have used many short terma for complete words like aa for amino acids. This types of errors should be thoroughly checked in the whole manuscript.

 

 

Author: this has been noted and corrected.

 

 

10. Figure 4 is very misleading and conceptually wrong. Terminology should be refined and there should be clearity in ON\OFF. Redraw the figure.

 

 

Author: the reviewer is correct. The figure has been remade to align more closely and exactly with the text, avoiding unnecessary speculations of the previous figure.

 

 

 

11. There are multiple errors found in refrences and bibliography. Ref 39 lack journal name, volume, pg numbr, ref52 has typo error. Ref 79, 80 are wrongly written. Some refrences are duplicate like 59, 82. Check year of refre nce 7
12. There are multiple mismatch of refrecnes and citaiotns that do not match to the text written n the manuscript.
13. Table 2. Check all refrences . is the study provide same information? Si et al. study to show a correlation between mass and fork rate.
14.  

 

Author: the references have been corrected and verified. The Si et al. study does not claim a mechanistic relationship between fork rate and initiation mass. It is a phenomenological observation that supports that mechanistic explanation. The author did not want to go into such detail as being out of scope (the Si et al. paper does not address specifically the issue of the relationship between nucleoid complexity and replication fork rate).

 

 

15. Although there is no clear evidence of AI writing but lack of technical and formatting artifacts suggest that there should be proofreading of each and every table, figure s, formulas. It should be applied to reduce errors of automated writing process.

 

Author: the paper has since been copy-edited. Any remaining errors/typos etc. are the author’s responsibility.

Round 2

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

The author made the effort to improve manuscript. Therefore, I can recommend it for publication.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

Author has fulfilled previous given comments.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript, titled “Nucleoid Complexity, the Nucleotypic Effect, and DNA/Cell Mass Homeostasis during Bacterial Growth,” addresses an interesting and yet unsolved problem in bacterial cell biology concerning the relationship between nucleoid complexity, the nucleotypic effect, DNA/cell mass homeostasis, nucleotide metabolism, replication initiation, and membrane biogenesis. The author proposes an integrative conceptual model combining classical concepts (Donachie, Cooper-Helmstetter, DnaA, RNR) with the more recent literature on bacterial cell metabolism and spatial organization. The concept is original. The work is enriched by useful diagrams (especially Figs. 2-5) that aid understanding of the text and its reasoning.

 

However, in its current version, the article has significant limitations:

Main remarks:

1. The author does not distinguish clearly enough between what is already known (proven) and what is proposed through a synthesis of the literature. In my opinion, each chapter should be restructured into three sections: Established evidence, Plausible interpretation, and Author's speculative model/hypothesis.
2. The main hypothesis/model is interesting, but currently too weakly "proven" based on the literature. The author presents the model more as an intuitive narrative than as a model grounded in a hierarchy of evidence. This picture requires much stronger anchoring in experimental data. It should be clearly indicated which elements of the model are supported by experimental results and which are only indirectly consistent with the literature. A table containing the data: Claim - Evidence - Strength of support - can help resolve this issue.

 

3. The author repeatedly formulates claims in an overly universalistic manner, as eukaryotes and bacteria are talking about very different replication control systems.
   
   
4. The author extends the classic, primarily eukaryotic, term "nucleotypic effect" to bacteria, arguing that cell size depends on the number of active forks/ori/"chromosomal equivalents." It should be explained why the term "nucleotypic effect" was used at all in bacteria, how its use differs from its classical meaning in eukaryotic biology, and what the limitations of this analogy are.
   
   
5. In the paragraph discussing DnaA and RNR, the author refers to an observation described as "unpublished." The review should be based on available, verifiable sources.
   
   
6. The conclusions go far beyond what is actually critically presented in the text.
   The manuscript is, in fact, primarily concerned with E. coli/bacteria, DNA replication, nucleotide metabolism, and cell cycle organization. The Conclusion should be narrowed to what the paper actually discusses.
   
   
7. Regarding the paper's structure, the content should be more consistent with the title. 8. Paragraphs 6-8 absolutely require logical ordering: Known evidence, Interpretation, Hypothesis, Outstanding questions.
   
   
8. A thorough editing review is necessary - typos and errors:

- Conculsion instead of Conclusion,

- Nuceloid instead of nucleoid,

- Phopholipid instead of phospholipid,

- Methonine / S-adenosylmethione instead of Methionine / S-adenosylmethionine,

- are tightly coupled,

- Ammino acid, etc.

 

10. Figure captions are too long and contain interpretations. Figure 1 and Figure 4 in particular have captions that are essentially mini-discussions/mini-hypotheses. Interpretations should be moved to the main text.

 

11. It is worth clarifying and maintaining consistent terminology:

- initiation mass (Mi),

- nucleoid complexity (NC),

- nucleotypic effect (NE),

- DNA/cell mass homeostasis,

- fork rate vs. replication rate,

- initiation frequency vs. origin firing.

Some terms are used quite loosely in the text.

Overall, the manuscript has conceptual promise, but it needs significant restructuring and editorial refinement.

 

 

Comments on the Quality of English Language

Needs careful language and editorial polishing.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses a relevant and conceptually interesting topic; however, in its current form it remains largely descriptive and does not provide sufficient critical synthesis or clearly defined novelty. The proposed framework, particularly regarding membrane-associated “hyperstructures,” is intriguing but appears largely speculative and would benefit from stronger support and clearer formulation. Substantial development would be required to meet the standards for publication.