The Small RNA MicF Represses ObgE and SeqA in Escherichia coli
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe paper of Stibelman et al. is devoted to small noncoding RNA (sRNA). It is clear now that they play important role in gene expression especially in stress condition. As the result of the work the authors find out the novel gene targets of small MicF RNA - ObgE and SegA genes. The plan of the investigation is very logical. The obtained results are interesting and significant. All necessary control experiments were done.
However, I have some questions and suggestions to the authors to improve the manuscript.
1. Introduction. The main object of investigation is MicF. Addition information about this small RNA is necessary: the length of nucleotide sequence, secondary structure characterized one by chemical methods or with bioinformatics tools. If such information is absent in literature the authors could predict MicF secondary structure by themselves, e.g. with RNAfold. It is very important to understand secondary structure of sRNA to estimate their possible role in the cell.
2. Lines 148-149. The authors wrote "Repression of obgE::sfGFP was not observed unless both MicF and Hfq were present in the reaction (Figure 3)". Why they conclude later (line 151-152) that “Hfq is required for MicF’s inhibition of obgE … expression
3. The part concerning the application of synthetic hybrid RNAs looks very attractive. However, I was not found in Material and Methods the information where these RNA fragments were synthesized, what is their length, how the transfection was done. It should be special paragraph where all this information should be presented.
It is well known that exogenous RNA quickly degrade in cell. Therefore, the experiments with synthetic hybrid RNAs should be written in more details taking into account possible RNA degradation.
4. DNA sequence direction should be indicated in Table S2.
5. Discussion about possible role of MicF RNA in ObgE and SegA gene expression is not easy for understanding. Some hypothetic schemes could be useful in this case. E.g. see the figures in review Karpov et all (see attached file).
Comments for author File: 
Comments.pdf
Author Response
Comment 1: 1. Introduction. The main object of investigation is MicF. Addition information about this small RNA is necessary: the length of nucleotide sequence, secondary structure characterized one by chemical methods or with bioinformatics tools. If such information is absent in literature the authors could predict MicF secondary structure by themselves, e.g. with RNAfold. It is very important to understand secondary structure of sRNA to estimate their possible role in the cell.
Response 1: We agree with the reviewer's assessment. The secondary structure of MicF has been studied by previous researchers. We have added a sentence in line 39 to reference this work. "MicF is a 93-nucleotide sRNA with a well-studied secondary structure [9]."
Comment 2: 2. Lines 148-149. The authors wrote "Repression of obgE::sfGFP was not observed unless both MicF and Hfq were present in the reaction (Figure 3)". Why they conclude later (line 151-152) that “Hfq is required for MicF’s inhibition of obgE … expression
Response 2: We were not exactly sure what the reviewer was referring to. However, we do realize the text in those sentences could be written more clearly and have changed them as follows. Line 149-150 "Repression of obgE::sfGFP was only observed when both MicF and Hfq were present in the reaction (Figure 3)." Line 152-153 "Together these results suggest that like its other targets, MicF's inhibition of obgE and seqA expression is dependent on the presence of Hfq."
Comment 3: The part concerning the application of synthetic hybrid RNAs looks very attractive. However, I was not found in Material and Methods the information where these RNA fragments were synthesized, what is their length, how the transfection was done. It should be special paragraph where all this information should be presented.
It is well known that exogenous RNA quickly degrade in cell. Therefore, the experiments with synthetic hybrid RNAs should be written in more details taking into account possible RNA degradation.
Response 3: The synthetic sRNA constructs were cloned into plasmids and transformed into the cells. We did not synthesize the RNAs themselves. This method of analyzing sRNA binding was performed by Corcoran et al. (ref 11) and Holmqvist et al. (ref 18). Corcoran et al. confirmed that the synthetic sRNAs were equally expressed as the MicF sRNA. The sequences of all the synthetic or mutant MicF constructs can be found in Table S2. To help clarify, we added the words "plasmids expressing" to the sentence in line 190-192 "To test these constructs, we transformed E. coli BW25113 cells with plasmids expressing one of two MicF variants." We also added the p referring to plasmid in the text when referring to the different constructs to match what is shown in Figure 5C. Lines 211-213 "We built two new plasmid constructs where nucleotides 1-51 or 1-19 were fused to the 5' end of the SgrS scaffold, pMicF(1-51) SS and pMicF(1-19) SS, respectively."
Comment 4. DNA sequence direction should be indicated in Table S2.
Response 4: Thank you for this suggestion. We have changed the header in Table S2 to be "Sequence (5'-to-3')".
Comment 5: Discussion about possible role of MicF RNA in ObgE and SegA gene expression is not easy for understanding. Some hypothetic schemes could be useful in this case. E.g. see the figures in review Karpov et all (see attached file).
Response 5: We thank the reviewer for this suggestion. We have included Figure 8 to help summarize the discussion.
Reviewer 2 Report
Comments and Suggestions for AuthorsGood morning for all authors,
Analyzing with interest and attention the manuscript with ID: microorganisms-3316039-peer-review-v1, Type – Article, entitled " The small RNA MicF represses ObgE and SeqA in Escherichia coli", with authors: Aaron Y Stibelman, Amy Y Sariles, Melissa K Takahashi*, for a possible publication in the prestigious Journal – Microorganisms, Section – Molecular Microbiology and Immunology,
I consider that:
1. The article's authors proposed a much-discussed topic in today's medical scientific world: the involvement/role of sRNA and MicF in antibiotic resistance in Escherichia coli infections.
2. The article follows all the specific instructions of the journal presented in aims and scope, instructions for authors, and other information about the journal.
3. Chapter 1. Introduction: the authors present relevant information for the chosen subject.
4. Chapter 2. Results: the methods, statistical analysis, and results are well presented and easy to understand in the text of the manuscript.
- Figures: 1, 2(A-D), 3-3S, 4(A-B, S4), 5(A-C, S5), 6, 7(A-B) and Tables: 1, 2, S1-5 are suggestive of the study carried out and very well presented; thus, they can be easily analyzed and interpreted!
5. Chapter 3. Discussion: the authors exemplified and compared all the results obtained by them with the studies and assessments of other authors, according to the bibliography.
6. Chapter 4. Materials and Methods: the data presented in all nine subchapters are very well structured and coherent.
- The laboratory equipment/analysers and the kits with working reagents used are of high performance (using the following techniques: PCR and Sanger DNA sequencing, Western blot, cell and microbial cultures, specific staining).
- For the analysis of immunological, and biochemical markers, the authors used standard working protocols that respect all working times.
7. The bibliography chosen by the authors corresponds to the requirements and refers to the subject of this article.
8. All authors have made a fair contribution to the study.
9. The authors also received funding for this study (Award Number: TL4GM118977 to A.Y.S.-2) demonstrating professionalism and medical scientific relevance!
In conclusion:
I ACCEPT for publication in the prestigious Journal – Microorganisms, Section – Molecular Microbiology and Immunology, this article with ID: microorganisms-3316039-peer-review-v1, Type – Article, entitled " The small RNA MicF represses ObgE and SeqA in Escherichia coli", with authors: Aaron Y Stibelman, Amy Y Sariles, Melissa K Takahashi*!
Congratulations to all the authors of this article!
Comments for author File: Comments.pdf
Author Response
We thank the reviewer for their thorough analysis of our manuscript. We appreciate that they found it worthy of publication.
