Twin Small RNAs and Divergent Fates: The Expansive Regulatory Networks of OmrA and OmrB

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Julia Konarska et al  present a review of the literature around regulatory networks of OmrA and OmrB, two homologous sRNAs that modulate outer membrane protein synthesis, motility, biofilm formation, iron uptake, and virulence. 

The main question of this review was to summarize the literature on two closely related sRNA, OmrA/B, and their role on bacterial adaptation and pathogenicity. This review focused not only on the similarities but also the differences on the molecular mechanisms of regulation and the final output of the balance of these mechanisms.
Given the complexity of the regulation of these two small RNAs, this review shows how OmrA/B are able to integrate not only the environmental information, but also to adapt to stress and fit all that information into protein expression and virulence. In summary, the topic of the manuscript is of interest, the content is well structured and clearly presents the information in a meaningful way to the reader. 

The review compares the regulatory mechanisms of these two sRNAs in different bacterial species and how this affects their adaptation to environmental conditions and pathogenicity.
In addition, it puts together a huge amount of information, and summarizes it for the reader, resulting in a well written, updated and valuable review for the journal. 

Conclusions summarize the main topics presented in the review, and also introduce which would be the focus of future research.

The literature reviewed is varied and up to date

Figures are very clear and give a summary of key concepts.
In addition, I recommend including a figure or scheme to resume all OmrA/B targets

Author Response

We would like to sincerely thank the Reviewer for the very positive and constructive feedback on our manuscript. We highly appreciate the encouraging comments regarding the clarity, structure, and relevance of our review. Below, we address the Reviewer’s suggestion in detail.

Comment:
“I recommend including a figure or scheme to resume all OmrA/B targets.”

Response:
We appreciate this excellent suggestion. In the revised version of the manuscript, we have added a new summary table (now Table 1), placed at the end of the manuscript and a figure (now Figure 1), which is placed after section “3. Target Specificity of OmrA/B” that provide a comprehensive schematic overview of all known OmrA and OmrB targets described in the literature. These new additions integrate information on regulatory interactions, cellular pathways affected, and phenotypic outcomes, facilitating a visual understanding of how OmrA/B coordinate bacterial adaptation and pathogenicity. We believe this addition significantly enhances the clarity and educational value of the review.

Reviewer 2 Report

Comments and Suggestions for Authors

In the manuscript entitled “Twin Small RNAs, Divergent Fates: The Expansive Regulatory Networks of OmrA and OmrB”, the authors comprehensively review different aspects of two sRNAs, OmrA and OmrB. This article is interesting, and it has a good structure and English style as well. I believe this article can be accepted after addressing some concerns, which are listed below;  

-The “Introduction” section is too short. Provide more data about different groups of small RNAs in bacteria (also their functions) before going through OmrA and OmrB.  

-It is highly recommended to add a table corresponding to the functions of the sRNAs in bacteria.  

-In line 11, Escherichia coli must be written italics.  

-In line 49, Escherichia coli must be written as E. coli since you already wrote its full name.

-The “Conclusions” section should be summarized, and a few sentences about the future perspective of this field of study.  

-Some references are too old. Replace them with the more novel ones.

Author Response

Comment 1:
The “Introduction” section is too short. Provide more data about different groups of small RNAs in bacteria (also their functions) before going through OmrA and OmrB.

Response:
We appreciate this valuable comment. In the revised version, we have expanded the Introduction section by including a broader overview of bacterial small RNAs. We now describe the general knowledge and functionality of sRNAs and their role in post-transcriptional regulation, stress response, and virulence. This additional background provides a stronger conceptual framework for introducing OmrA and OmrB.

Comment 2:

It is highly recommended to add a table corresponding to the functions of the sRNAs in bacteria.

Response:
We fully agree with this recommendation. A new Table 1 has been added, summarizing the that provides a comprehensive schematic overview of all experimentally confirmed OmrA and OmrB targets described in the literature. It is placed under the “13. Conclusions” section. This addition enhances the reader’s understanding of the information on regulatory interactions, cellular pathways affected, and phenotypic outcomes, facilitating a visual understanding of how OmrA/B coordinate bacterial adaptation and pathogenicity.

Comment 3:
In line 11, Escherichia coli must be written in italics.

Response:
We thank the Reviewer for noticing this formatting issue. “Escherichia coli” has now been italicized, as suggested.

Comment 4:
In line 49, Escherichia coli must be written as E. coli since you already wrote its full name.

Response:
This has been corrected accordingly. The second occurrence now appears as “E. coli” in the revised version.

Comment 5:
The “Conclusions” section should be summarized, and a few sentences about the future perspective of this field of study.

Response:
We have revised the Conclusions section to make it more concise and focused. Additionally, we included a short paragraph outlining potential directions for future research, particularly regarding the design of programmable antisense oligonucleotides (ASOs) for species-specific RNA antibiotics.

Comment 6:
Some references are too old. Replace them with the more novel ones.

Response:
We have carefully reviewed and updated the reference list. Several older references are now accompanied by newer articles to ensure the review reflects the current state of knowledge.

Once again, we thank the Reviewer for their valuable comments, which have substantially improved the scientific quality, clarity, and completeness of our manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

In  their manuscript entitled “Twin Small RNAs, Divergent Fates: The Expansive Regulatory Networks of OmrA and OmrB “,  Konarska and colleagues review the molecular mechanisms involved in the regulation by the small non-coding RNAs OmrA and OmrB on a diversity of bacterial functions, including membrane transport and homeostasis, iron acquisition, motility and biofilm formation, pathogenicity, and other cellular functions. The authors also review the roles played by the chaperone Hfq on the stability and activity of the two sRNAs. An additional mechanism played by the sRNAs OmrA and OmrB and named as “sponge mechanism”, is also reviewed by the authors. The manuscript is well organized and written, with the expected details for such a review, which makes the review easy to read without losing rigorousness. A nice piece of work. The only issues raised is that throughout the text, names of genes and bacterial species are not always italicized. The text should be reviewed to solve this minor issue.

Author Response

We would like to sincerely thank the Reviewer for the positive and encouraging evaluation of our manuscript. We truly appreciate the kind remarks regarding the clarity, structure, and scientific rigor of our review.

Below, we address the Reviewer’s comment in detail.

Comment:
Throughout the text, names of genes and bacterial species are not always italicized. The text should be reviewed to solve this minor issue.

Response:
We thank the Reviewer for carefully pointing out this important formatting detail. The entire manuscript has been thoroughly revised to ensure consistent italicization of all bacterial species names (e.g., Escherichia coli, Salmonella enterica, Yersinia enterocolitica) and gene names where appropriate. We have carefully checked the text, figure legends, and tables to maintain proper formatting throughout.

Reviewer 4 Report

Comments and Suggestions for Authors

This review provides a detailed examination of the small RNAs OmrA and OmrB, their evolutionary context, regulatory mechanisms, and diverse roles in bacterial physiology, particularly in members of the Enterobacterales family, such as Escherichia coli and related pathogens. It covers the functional divergence of OmrA and OmrB, their target specificity, regulation by environmental signals, interactions with the RNA chaperone Hfq, and their impact on processes such as outer membrane protein synthesis, iron homeostasis, motility, biofilm formation, and virulence. It contains a wide range of literature.

The review is well written and requires only minor revision.

1. I propose shortening the description of the role of RelA in stringent response (lines 645-657) as excessively detailed descriptions of well-known mechanisms
2. Check and change the names of microorganisms, genes and mRNA to italics. (lines 412-443)
3. Figure 2 is not fully integrated into the text. Only panel 2A is described. Please improve the link between Figure 2 and section 10.
4. The section on synthetic small RNAs (Section 12) requires more detailed explanation. The description of OmrB as a synthetic mRNA scaffold is promising, but brief. Expanding this section with a description of its design principles and a comparison with other scaffolds would make it more informative. Perhaps a corresponding figure would make the presentation clearer.

Author Response

We would like to express our sincere gratitude to the Reviewer for the thoughtful and constructive feedback, as well as for the positive evaluation of our manuscript. We highly appreciate the acknowledgment of our work’s clarity, scope, and scientific relevance. Below, we address each of the Reviewer’s comments in detail.

Comment 1:
I propose shortening the description of the role of RelA in stringent response (lines 645–657) as excessively detailed descriptions of well-known mechanisms.

Response:
We appreciate this valuable suggestion. The section describing the role of RelA in the stringent response has been shortened to focus on the aspects directly relevant to OmrA/B regulation. We decided to combine this section with section now 5 (5. Regulation of Motility and Biofilm Formation). The revised text provides a concise summary of the mechanism while preserving the essential context needed for readers to understand its connection to OmrA/B activity.

Comment 2:
Check and change the names of microorganisms, genes and mRNA to italics. (lines 412–443)

Response:
We thank the Reviewer for drawing our attention to this formatting issue. The names of all microorganisms, genes, and mRNAs throughout the manuscript (including lines 412–443) have been carefully reviewed and consistently italicized according to journal guidelines.

Comment 3:
Figure 2 is not fully integrated into the text. Only panel 2A is described. Please improve the link between Figure 2 and section 10.

Response:
We thank the Reviewer for pointing out that Figure 2 was not fully integrated into the text and that only panel 2A was discussed. In response to this valuable suggestion, we have decided to remove the entire Section 10 and incorporate its relevant content into the preceding paragraphs to improve coherence and narrative flow. Additionally, we have removed the original Figure 2. Instead, we now provide a new Figure 1, which presents the predicted secondary structure of OmrA along with its functional subzones. Figure 1 is placed under section “3. Target Specificity of OmrA/B”. To further enhance clarity and accessibility of the information previously contained in Section 10 and Figure 2, we have also included a summary table at the end of the manuscript that compiles all experimentally validated targets of OomrA and OomrB, which is placed at the end of the manuscript.

We believe these modifications significantly improve the integration of visual elements with the text and strengthen the overall organization of the manuscript. Thank you again for your constructive feedback.

Comment 4:
The section on synthetic small RNAs (Section 12) requires more detailed explanation. The description of OmrB as a synthetic mRNA scaffold is promising, but brief. Expanding this section with a description of its design principles and a comparison with other scaffolds would make it more informative. Perhaps a corresponding figure would make the presentation clearer.

Response:
We fully agree with this excellent suggestion. Section 12 has been expanded to provide a more comprehensive overview of synthetic small RNAs, including design strategies, modular organization, and comparison with other synthetic RNA scaffolds described in recent literature. We believe these additions significantly enhance the depth and clarity of this section.

Reviewer 5 Report

Comments and Suggestions for Authors

The manuscript entitled “Twin Small RNAs, Divergent Fates: The Expansive Regulatory Networks of OmrA and OmrB”, the authors Konarska et al., provides a thorough review of expression dynamics and different modes of function and functional consequences of the small RNAs OmRA and OmrB. OmrA/B regulate the outer membrane protein synthesis, motility, biofilm formation, iron uptake and virulence. While this is a very interesting review, multiple sections get into a level of detail that distract from the main points of the authors. The key points are buried in extensive background information, which, while they’re still very informative and well written, it is making it difficult for the reader to follow the main points and the scope of the manuscript. While this is seen throughout the manuscript, I will highlight a few sections below.

Comments:

• The current organization of sections is affecting readability, as some sections are overlapping and the structure is to some extend unclear. For instance, while the authors go into lengthy detail about “Iron Homeostasis and Regulation of Siderophore Uptake” in section 5 and explain how OmrA/B controls expression of OMPs, the author later add in section 10 “The Role of Hfq in OmrA/B Function”, how Hfq likely stabilizes OmrA to promote its base-pairing with target transcripts to regulate iron homeostasis. The fragmented structure makes it challenging for the reader to grasp the full regulatory picture. It would be helpful to restructure.
• Similar to my comment above, section 6 is very information dense and lengthy, and the structure of this section appears to me unclear and led to confusion. The authors describe the role of OmrA/B in regulating motility and biofilm formation, while simultaneously describing different regulatory mechanisms of OmrA/B. As a result, the key regulatory insights are buried, and the conceptual flow of this section is unclear. The authors could shorten and condense this section, and structure it more clearly, which would help the reader navigate the complexity and retain the main messages.
• The issue regarding structure is also present in following sections, where dense descriptions and layered mechanisms are presented without a structured framework. I’d suggest revisiting the overall structure to help the reader stay focused and to understand the main concepts more effectively without getting lost in the details.
• Section 2, line 61 – 79: This is a well-written section. To enhance the clarity and support the readers understanding, I suggest visualizing transcript regions of OmrA/B, indicating structural elements and corresponding target sequences by these regions in a figure. This could also be combined with a figure for section 3, where the authors could visualize expression regulation by the two different systems, EnvZ/OmpR and RpoS.
• Figure 2: While most panels of Figure 2 are discussed in the text, only Fig. 2A is specifically referenced, even though, for instance, Fig. 2C could be referenced in line 577. Please make sure that all figure panels are discussed and are referenced in respective sections.

Author Response

We would like to sincerely thank the Reviewer for their careful and insightful evaluation of our manuscript and for the constructive feedback provided. We highly appreciate the recognition of the review’s scientific relevance and the detailed suggestions that helped us improve the clarity, structure, and readability of our work. Below, we respond to each of the Reviewer’s comments in detail.

Comment 1:

The current organization of sections is affecting readability, as some sections are overlapping and the structure is to some extent unclear. For instance, while the authors go into lengthy detail about “Iron Homeostasis and Regulation of Siderophore Uptake” in section 5 and explain how OmrA/B controls expression of OMPs, the authors later add in section 10 “The Role of Hfq in OmrA/B Function”, how Hfq likely stabilizes OmrA to promote its base-pairing with target transcripts to regulate iron homeostasis. The fragmented structure makes it challenging for the reader to grasp the full regulatory picture. It would be helpful to restructure.

Response:
We thank the Reviewer for this insightful comment regarding the manuscript’s organization. We agree that the separation of mechanistic details into Section 10 contributed to fragmentation and impaired readability. In response, we have removed Section 10 entirely and integrated all relevant information on Hfq-mediated regulation into the preceding sections, particularly those addressing iron homeostasis and OmrA/B control of outer membrane proteins. This restructuring provides a more coherent and continuous explanation of the regulatory mechanisms. Additionally, we have removed Figure 2, which was previously linked to the deleted section.

Comment 2:

Similar to my comment above, section 6 is very information dense and lengthy, and the structure of this section appears unclear and led to confusion. The authors describe the role of OmrA/B in regulating motility and biofilm formation, while simultaneously describing different regulatory mechanisms of OmrA/B. As a result, the key regulatory insights are buried, and the conceptual flow of this section is unclear. The authors could shorten and condense this section, and structure it more clearly, which would help the reader navigate the complexity and retain the main messages.

Response:
We appreciate this thoughtful suggestion. Section 6 has been substantially revised for clarity and conciseness. This section is now “5. Regulation of Motility and Biofilm Formation”. We shortened redundant descriptions, and emphasized the key regulatory interactions of OmrA/B. This restructuring helps the reader follow the sequence of regulatory mechanisms more intuitively and retain the main conclusions.

Comment 3:
The issue regarding structure is also present in following sections, where dense descriptions and layered mechanisms are presented without a structured framework. I’d suggest revisiting the overall structure to help the reader stay focused and to understand the main concepts more effectively without getting lost in the details.

Response:
We thank the Reviewer for pointing this out. In response, we reviewed the structure of the entire manuscript to enhance coherence and reader guidance. Each major section now begins with a short overview paragraph summarizing its focus and ends with a concise summary, beside improving sections mentioned in previous comments we also introduced changes to sections describing the btuB gene regulation, OmrA/B as potential synthetic RNAs and expanded conclusion section. We also adjusted subheadings to better reflect the logical flow of the text. These revisions collectively improve navigation and help readers follow the core narrative more easily.

Comment 4:
Section 2, lines 61–79: This is a well-written section. To enhance the clarity and support the reader’s understanding, I suggest visualizing transcript regions of OmrA/B, indicating structural elements and corresponding target sequences by these regions in a figure. This could also be combined with a figure for section 3, where the authors could visualize expression regulation by the two different systems, EnvZ/OmpR and RpoS.

Response:
We sincerely thank the Reviewer for this excellent suggestion. In the revised version, we have added a new Figure 1, which illustrates the transcript architecture of OmrA and OmrB, highlighting conserved structural elements and known target interaction regions. Figure 1 is placed under section “3. Comparative Genomics and Target Specificity of OmrA/B”. We believe this new figure significantly enhances clarity and visual understanding.

Comment 5:
Figure 2: While most panels of Figure 2 are discussed in the text, only Fig. 2A is specifically referenced, even though, for instance, Fig. 2C could be referenced in line 577. Please make sure that all figure panels are discussed and are referenced in respective sections.

Response:
We thank the Reviewer for noticing this inconsistency. As previously mentioned, we decided to remove section describing the role of Hfq and integrate all relevant information on Hfq-mediated regulation into the preceding sections. We have also removed Figure 2, which was previously linked to the deleted section and instead we added new Figure 1 and Table 1 that provides a comprehensive schematic overview of all known OmrA and OmrB targets described in the literature. Figure 1 is placed under section “3. Comparative Genomics and Target Specificity of OmrA/B” and Table 1 is placed at the end of the manuscript.

Once again, we sincerely thank the Reviewer for their insightful and constructive feedback. The suggested revisions have greatly improved the scientific quality, precision, and overall readability of our manuscript.

Round 2

Reviewer 5 Report

Comments and Suggestions for Authors

Thank you to the authors for addressing all of my comments. The revised version of the manuscript greatly enhanced the clarity and readability and I recommend the manuscript for publication.