Decoding the Microcin J25 Biosynthetic Cluster: Modulation of the mcjA Promoter by the Novel Overlapping Gene mcjX

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript titled "Decoding the microcin J25 biosynthetic cluster: modulation of the mcjA promoter by the novel overlapping gene mcjX" primarily investigates a newly identified overlapping gene, mcjX, within the microcin J25 biosynthesis gene cluster in Escherichia coli, and explores its role in regulating the microcin J25 expression. The experimental design could be improved.

1. In Section 2.2, lines 129–131, no negative control was included in the Western blot experiment. Moreover, why are the panels in Figure 2D presented as separate images rather than as a single complete figure?
2. In Section 2.3, lines 134–136, this sentence should appear in the Acknowledgments section, not in the Results section.
3. In Section 2.5, lines 225–230, the activity of PmcjA69-35null (the -35 element proposed was deleted) is "greatly reduced," but it is not tested whether the activity can be restored.
4. In Section 2.6, lines 272–273, no data or figure are provided. What is the basis for the statement that mcjX does not confer resistance to exogenous the microcin J25?
5. Statistical analysis is lacking; no p-values are presented throughout the manuscript. Multiple experiments show only representative results, which substantially undermines the credibility of the findings.
6. Relying solely on Western blot and predicted structure for McjX is clearly insufficient. Purification and characterization of the protein should be performed.

Author Response

The manuscript titled "Decoding the microcin J25 biosynthetic cluster: modulation of the mcjA promoter by the novel overlapping gene mcjX" primarily investigates a newly identified overlapping gene, mcjX, within the microcin J25 biosynthesis gene cluster in Escherichia coli and explores its role in regulating the microcin J25 expression. The experimental design could be improved.

We thank the reviewer’s accurate review of the work and we hope this revision satisfactorily addresses the reviewer’s concerns.

Comment 1: In Section 2.2, lines 129–131, no negative control was included in the Western blot experiment. Moreover, why are the panels in Figure 2D presented as separate images rather than as a single complete figure?

Response 1: Figure 2D is presented in two panels because the molecular weight marker stays in the PVDF membrane, whereas the immunoreactivity is detected in an X-ray film. We included the raw data in the Supplementary information (Fig. S2 and Fig. S3).

Regarding the negative controls, they are present in the dot-blot figure (2C): control sample without McjX-polyHis and no His6x antibody.

Commet 2: In Section 2.3, lines 134–136, this sentence should appear in the Acknowledgments section, not in the Results section.

Response 2: The reviewer is absolutely correct. We apologize for that. The lines were moved to the Acknowledgments section as suggested.

Comment 3: In Section 2.5, lines 225–230, the activity of PmcjA69-35null (the -35 element proposed was deleted) is "greatly reduced," but it is not tested whether the activity can be restored.

Response 3: We thank the reviewer for this valuable comment. PmcjA69-35null was designed as a derivative of PmcjA69 in which the putative -35 element was specifically deleted. Therefore, PmcjA69 represents the corresponding intact promoter containing both the proposed -35 and -10 elements and serves as the reference control for promoter activity.

Consequently, restoration of the deleted -35 sequence in PmcjA69-35null would be expected to regenerate the original PmcjA69 construct and, accordingly, recover the promoter activity observed for PmcjA69. We apologize for not making this rationale sufficiently clear in the original manuscript and have now clarified this point in the revised version (Page 7, line 229-231).

Comment 4: In Section 2.6, lines 272–273, no data or figure are provided. What is the basis for the statement that mcjX does not confer resistance to exogenous microcin J25?

Response 4: The reviewer’s observation is well taken. We included the antimicrobial activity plates where it is demonstrated that McjX does not confer resistance to exogenous MccJ25 in the Supplementary Information as Figure S4. As it can be seen, the activity of MccJ25 on Escherichia coli BL21 bearing the pACYCDuet-mcjX plasmid when plated onto LB 0.6% glucose, i.e. with no expression of McjX, was comparable to the activity observed when this strain was plated onto LB containing 50 μM IPTG (Page 9, line 279).

Comment 5: Statistical analysis is lacking; no p-values are presented throughout the manuscript. Multiple experiments show only representative results, which substantially undermines the credibility of the findings.

Response 5: We thank the reviewer for this important observation and agree that the statistical analysis required further clarification and expansion. In response, we have incorporated a new subsection in the Materials and Methods section (Section 4.11, page 14, lines 527-531) describing in detail the statistical analyses performed, including the number of biological replicates, the statistical tests employed, and the criteria used to assess significance.

In addition, Figures 6 and 7 have been revised to include the corresponding statistical analyses and significance values. We also corrected an oversight in Figure 2B, which in the original version displayed data from a single representative experiment for clarity purposes and therefore lacked error bars. The revised figure has now been reconstructed using the complete dataset from independent experiments and appropriately includes error bars.

Furthermore, Table 1 has been fully updated following additional activity measurements performed to strengthen the robustness of the results. The revised table now includes mean values and standard deviations derived from independent experiments.

Comment 6: Relying solely on Western blot and predicted structure for McjX is clearly insufficient. Purification and characterization of the protein should be performed.

Response 6: We thank the reviewer for this valuable comment and agree that purification and biochemical characterization of McjX constitute important next steps toward understanding its biological function. The current study was primarily focused on identifying and providing initial functional evidence for this previously unrecognized open reading frame associated with the MccJ25 system. In this context, Western blot analysis and structural prediction were included as preliminary approaches to support the existence and expression of McjX.

We agree that a more comprehensive biochemical characterization would further strengthen the study. However, these experiments are beyond the scope of the present work and are currently part of ongoing research in our laboratory. In particular, we are planning to obtain McjX by solid-phase peptide synthesis in collaboration with Dr. Eduardo Cilli (UNESP, Brazil), an expert in peptide chemistry and a long-standing collaborator of our group. These future studies will allow us to investigate in greater detail the structural and functional properties of McjX and its potential role in the regulation of lasso peptide biosynthesis.

Reviewer 2 Report

Comments and Suggestions for Authors

Overall Assessment

This manuscript describes the identification of a previously unannotated overlapping ORF, mcjX, in the microcin J25 biosynthetic cluster and proposes a regulatory role in modulating mcjA expression. The topic is potentially interesting and relevant to the field of bacterial gene regulation.

However, the current data do not sufficiently support the main mechanistic conclusion that McjX acts as a transcriptional repressor of PmcjA. In particular, the study does not distinguish whether the observed effects are mediated by the peptide itself or by the overlapping RNA sequence. Several key controls and mechanistic experiments are also missing, and some conclusions appear overstated relative to the presented evidence.

Overall, the manuscript contains potentially valuable observations, but substantial revision and additional mechanistic support are required. I therefore recommend major revision before the manuscript can be considered for publication.

Major Comments

1. A major conceptual weakness is that the study does not distinguish whether the observed phenotype is mediated by the McjX peptide itself or by the overlapping RNA sequence. Since mcjX overlaps mcjA, the reported effects could arise from RNA-based mechanisms, including altered mRNA structure, translational coupling, ribosome interference, or RNA stability, rather than from the peptide product. Critical controls are missing, such as start codon mutations, Shine–Dalgarno disruption, synonymous substitutions, or premature stop codons. These experiments are necessary to determine whether the regulatory effect depends on translation of McjX or merely on the presence of the overlapping RNA sequence.
2. The central mechanistic claim that McjX functions as a transcriptional repressor of PmcjA is not sufficiently supported. The current data only demonstrate reduced GFP fluorescence and decreased antimicrobial activity, but do not directly show transcriptional repression. No evidence is provided for DNA binding, interaction with RNA polymerase, altered transcription initiation, or changes in mcjA transcript levels. Therefore, the observed effects could also result from indirect physiological or post-transcriptional mechanisms. Additional experiments such as EMSA, ChIP analysis, or in vitro transcription assays are needed to support the proposed mechanism. Without such evidence, the conclusions should be substantially moderated.

Minor Comments

1. Figure quality needs improvement

Several figures are low resolution and difficult to interpret, especially Figure 2D, Figures 3 and 4. Higher-quality images and quantitative representations are needed.

2. The conclusion regarding “absence of toxicity” is not sufficiently supported

The manuscript states that deletion of mcjX does not induce toxicity. However: no viability assays or growth kinetics were provided.

Author Response

This manuscript describes the identification of a previously unannotated overlapping ORF, mcjX, in the microcin J25 biosynthetic cluster and proposes a regulatory role in modulating mcjA expression. The topic is potentially interesting and relevant to the field of bacterial gene regulation.

However, the current data do not sufficiently support the main mechanistic conclusion that McjX acts as a transcriptional repressor of PmcjA. In particular, the study does not distinguish whether the observed effects are mediated by the peptide itself or by the overlapping RNA sequence. Several key controls and mechanistic experiments are also missing, and some conclusions appear overstated relative to the presented evidence.

Overall, the manuscript contains potentially valuable observations, but substantial revision and additional mechanistic support are required. I therefore recommend major revision before the manuscript can be considered for publication.

We greatly appreciate the reviewer's detailed assessment and thank them for these valuable observations.

Major Comments

Comment 1: A major conceptual weakness is that the study does not distinguish whether the observed phenotype is mediated by the McjX peptide itself or by the overlapping RNA sequence. Since mcjX overlaps mcjA, the reported effects could arise from RNA-based mechanisms, including altered mRNA structure, translational coupling, ribosome interference, or RNA stability, rather than from the peptide product. Critical controls are missing, such as start codon mutations, Shine–Dalgarno disruption, synonymous substitutions, or premature stop codons. These experiments are necessary to determine whether the regulatory effect depends on translation of McjX or merely on the presence of the overlapping RNA sequence.

Response 1: We completely agree that, at this stage, we cannot definitively conclude that the McjX peptide is responsible for PmcjA regulation, even after confirming that mcjX is translated. It remains entirely plausible that the RNA sequence itself mediates this activity. Consequently, we have revised our conclusions throughout the manuscript, including the abstract, to avoid any overstatement.

Regarding the control experiments, we describe a setup in the manuscript where the entire microcin system was cloned, but with a deletion of the last nine codons of mcjX (Section 4.6). This modification generates a truncated sequence that lacks a stop codon for mcjX. Under these conditions, we observed a higher production of MccJ25 in the absence of the intact mcjX sequence (Table 1). While we agree this result is not entirely definitive, it strongly suggests that the McjX peptide (rather than the transcript alone) is responsible for the observed regulatory activity, given that the RNA sequence remains present. Introducing a mutation at the start codon or disrupting the ribosome binding site would indeed be elegant approaches to confirm this mechanism. However, we feel these detailed mechanistic assays are beyond the scope of the present study. We will certainly incorporate these excellent suggestions into the next phase of our project, where we plan to investigate the molecular mechanism of McjX in detail.

Comment 2: The central mechanistic claim that McjX functions as a transcriptional repressor of PmcjA is not sufficiently supported. The current data only demonstrate reduced GFP fluorescence and decreased antimicrobial activity, but do not directly show transcriptional repression. No evidence is provided for DNA binding, interaction with RNA polymerase, altered transcription initiation, or changes in mcjA transcript levels. Therefore, the observed effects could also result from indirect physiological or post-transcriptional mechanisms. Additional experiments such as EMSA, ChIP analysis, or in vitro transcription assays are needed to support the proposed mechanism. Without such evidence, the conclusions should be substantially moderated.

Response 2: We thank the reviewer for this important and insightful comment. We fully agree that the current data do not provide definitive mechanistic evidence demonstrating that McjX directly acts as a transcriptional repressor of PmcjA. At present, our results support an inhibitory effect of McjX on PmcjA activity; however, the precise molecular mechanism underlying this effect remains to be elucidated.

As correctly pointed out by the reviewer, additional experiments such as EMSA, ChIP analyses, in vitro transcription assays, and transcriptomic approaches will be necessary to determine whether McjX directly interacts with DNA, affects RNA polymerase recruitment, or modulates mcjA expression through indirect mechanisms. In this regard, ongoing work in our laboratory is focused on clarifying the mechanism of action of McjX. For example, RNA-seq and EMSA experiments are currently being planned. For the latter, we have established a collaboration with Dr. Eduardo Cilli (UNESP, Brazil), an expert in peptide solid-phase synthesis.

Since these experiments are still in progress and the corresponding results are not yet available, we have substantially moderated our conclusions throughout the revised manuscript in accordance with the reviewer’s suggestion (Page 10, lines 332-338).

Minor Comments

Comment 1: Figure quality needs improvement

Several figures are low resolution and difficult to interpret, especially Figure 2D, Figures 3 and 4. Higher-quality images and quantitative representations are needed.

Response 1: Figure 2D is presented as two panels to facilitate visualization: the right panel shows the molecular weight marker, whereas the left panel displays the corresponding Western blot. To ensure full transparency, the raw, uncropped images have now been included in the Supplementary Information. These figures were prepared at 300 dpi and comply with all journal requirements regarding image quality and resolution. These experiments were performed in glass Petri dishes, and the visible marks correspond to natural imperfections and scratches on the glass surface. While we agree with the reviewer that the images might appear more aesthetically pleasing in the absence of these irregularities, we believe that their presence reflects the authenticity of the original experimental plates and does not affect the interpretation of the results. Nonetheless, western-blot (Figure 2D) and Figures 3 and 4 were improved with the assistance of a graphic designer expert in image editing (she is mentioned in the Acknowledgments section).

Comment 2: The conclusion regarding “absence of toxicity” is not sufficiently supported

The manuscript states that deletion of mcjX does not induce toxicity. However: no viability assays or growth kinetics were provided.

Response 2: The reviewer raises a very important point. We agree that the statement regarding the absence of toxicity was insufficiently supported in the original version of the manuscript, as bacterial growth analyses were not included.

To address this issue, we have now incorporated growth kinetics experiments in the revised manuscript (Figure S5). Specifically, we compared the growth of E. coli carrying the pTUC346woX plasmid with that of E. coli transformed with the empty pUC18 vector, which does not support MccJ25 production. Under the conditions tested, no significant differences in bacterial growth were observed, supporting the conclusion that deletion of mcjX does not result in detectable toxicity.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript presents an interesting and potentially important discovery of the overlapping gene mcjX within the microcin J25 biosynthetic cluster and provides evidence for its role as a negative regulator of PmcjA. The work is original, well organized, and relevant to the fields of microbial genetics, peptide biosynthesis, and regulatory biology. 

The manuscript is well-written, and the experimental design is generally sound. However, I have several comments and recommendations that the authors should address to improve the mechanistic impact and clarity of the paper before publication.

• The authors demonstrate that mcjX negatively affects MccJ25 production and promoter activity, but the molecular mechanism remains unclear. The Discussion section would benefit from a more balanced interpretation of the data, emphasizing that the regulatory mechanism is still hypothetical.
• The promoter analysis section is informative but difficult to follow in some parts. A simplified schematic summarizing all promoter constructs, deletions, and corresponding GFP activities would improve readability.
• Statistical analysis should be described more clearly throughout the manuscript. The authors should indicate the number of biological replicates, statistical tests used, and significance values for quantitative experiments.
• The possibility that mutations or truncations in mcjX alter mRNA stability or secondary structure should be discussed, particularly because mcjX overlaps with mcjA.
• The authors mention that direct interaction between McjX and the PmcjA would be highly unlikely because of the low isoelectric point. To strengthen the mechanistic insights, it would be helpful to expand the discussion whether McjX works indirectly by interacting with host transcription factors that regulate PmcjA (such as ppGpp, IHF, or Lrp mentioned on lines 55-57)  
• Some sentences require grammatical revision for clarity. Examples include:

1. • “downstream PmcjA” should be changed to “downstream of PmcjA”.
   • “β-beta-galactosidase” should be corrected to “β-galactosidase”.

Author Response

The manuscript presents an interesting and potentially important discovery of the overlapping gene mcjX within the microcin J25 biosynthetic cluster and provides evidence for its role as a negative regulator of PmcjA. The work is original, well organized, and relevant to the fields of microbial genetics, peptide biosynthesis, and regulatory biology. 

The manuscript is well-written, and the experimental design is generally sound. However, I have several comments and recommendations that the authors should address to improve the mechanistic impact and clarity of the paper before publication.

We sincerely thank the reviewer’s insightful observation. All suggested recommendations were considered to improve the manuscript.

Comment 1: The authors demonstrate that mcjX negatively affects MccJ25 production and promoter activity, but the molecular mechanism remains unclear. The Discussion section would benefit from a more balanced interpretation of the data, emphasizing that the regulatory mechanism is still hypothetical.

Response 1: The reviewer raises a very important point. We added a line in the Discussion to clarify that the regulatory mechanism remains hypothetical based on our experimental data (Page 9, line 291 and Page 10, lines 332-338).

Comment 2: The promoter analysis section is informative but difficult to follow in some parts. A simplified schematic summarizing all promoter constructs, deletions, and corresponding GFP activities would improve readability.

Response 2: We acknowledge that the original promoter schematic may not have been sufficiently clear. To improve clarity and facilitate interpretation of the experimental design, we have now included the complete sequences of all promoter variants in the Supplementary Information (Page 15, lines 553-554). We hope that this additional information will provide greater transparency, improve the readability of the manuscript, and help readers follow the rationale underlying the promoter analyses.

Comment 3: Statistical analysis should be described more clearly throughout the manuscript. The authors should indicate the number of biological replicates, statistical tests used, and significance values for quantitative experiments.

Response 3: We fully agree with the reviewer’s comment. In the Materials and Methods section, a specific section 4.11 entitled Statistical analysis was added with all the required information (page 14, lines 527-531). In addition, statistical significance values and error bars have now been incorporated into the corresponding figures. 

Comment 4: The possibility that mutations or truncations in mcjX alter mRNA stability or secondary structure should be discussed, particularly because mcjX overlaps with mcjA.

Response 4: We agree with the reviewer's insightful comment regarding the genetic context of these genes. Because mcjX overlaps with mcjA, any mutations or truncations could potentially impact MccJ25 production by altering the overlapping region. To address this, we performed an experiment where the complete microcin J25 system was cloned, but with a deletion of the final 9 codons of mcjX. Consequently, the native McjX peptide is not expressed. Under these conditions, the absence of the intact McjX peptide was still associated with an increase in MccJ25 production (Table 1). This observation suggests that the peptide itself, rather than structural changes to the transcript alone, is primary to the observed regulatory effect. However, we fully acknowledge that this result does not constitute conclusive proof, which is why we have tempered our conclusions accordingly in the revised manuscript, including the abstract. We have now addressed this important possibility in the Discussion section of the revised manuscript (Page 10, lines 332-338). We believe this addition significantly strengthens the Discussion.

Comment 5: The authors mention that direct interaction between McjX and the PmcjA would be highly unlikely because of the low isoelectric point. To strengthen the mechanistic insights, it would be helpful to expand the discussion whether McjX works indirectly by interacting with host transcription factors that regulate PmcjA (such as ppGpp, IHF, or Lrp mentioned on lines 55-57)  

Response 5: We thank the reviewer for this insightful suggestion. We agree that the possibility of McjX acting indirectly through interactions with host regulatory factors represents an interesting alternative mechanistic scenario and have now expanded the Discussion section accordingly (Page 10, lines 307-311).

As previously reported by Chiuchiolo et al., three potential IHF binding sites and one putative Lrp binding site were identified upstream of the mcjA promoter region. However, these regulatory elements are located outside the promoter fragments analyzed in the present study, and therefore the PmcjA variants characterized here do not contain the proposed IHF or Lrp binding sequences.

Regarding ppGpp, we initially considered a direct interaction with McjX to be unlikely because of the low isoelectric point of the peptide and the absence of abundant positively charged residues typically associated with ppGpp binding. Nevertheless, prompted by the reviewer’s comment, we performed preliminary docking analyses based on AlphaFold structural predictions. Interestingly, these analyses suggested a potential interaction between the N-terminal region of McjX and ppGpp. The best-ranked model displayed a predicted local distance difference test (pLDDT) score of 89.6, indicating high confidence in the structural prediction itself, although the interface predicted template modeling (ipTM) score was only 0.539, suggesting that the interaction remains tentative.

Although these observations are clearly preliminary, we believe they represent an interesting hypothesis that deserves further investigation. In this regard, future studies using synthetic McjX peptide will allow us to experimentally evaluate this potential interaction.

Comment 6: Some sentences require grammatical revision for clarity. Examples include:

• “downstream PmcjA” should be changed to “downstream of PmcjA”.
• “β-beta-galactosidase” should be corrected to “β-galactosidase”.

Response 6: The reviewer is absolutely correct, the corrections were made to the manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

The current manuscript titled “Decoding the microcin J25 biosynthetic cluster: modulation of 2 the mcjA promoter by the novel overlapping gene mcjX” by Masias et. al. provides evidence where 31 amino acid peptides translated by mcjX via +1 frameshift involved in transcriptional repressor of PmcjA. The study establishes a rare overlap of 53 nucleotides at 3’ end of mcjA in the Escherichia coli and demonstrates regulatory mechanism mediated by an overlapping gene within a lasso peptide operon. All the studies carried were very conclusive and satisfactory. The manuscript was well written and can be accepted to publish with a minor revision mentioned below. 

Comments/suggestions:

1. In Figure 2, I would suggest the authors to provide a single western blot/gel image showing both the cjX-polyHis with the ladder in one image. If possible, I would include all the raw data in the SI.  
2. In Figure 6, do the authors validate the promoter activities in triplicate? If yes, is it possible to provide its statistical significance?

Author Response

The current manuscript titled “Decoding the microcin J25 biosynthetic cluster: modulation of 2 the mcjA promoter by the novel overlapping gene mcjX” by Masias et. al. provides evidence where 31 amino acid peptides translated by mcjX via +1 frameshift involved in transcriptional repressor of PmcjA. The study establishes a rare overlap of 53 nucleotides at 3’ end of mcjA in the Escherichia coli and demonstrates regulatory mechanism mediated by an overlapping gene within a lasso peptide operon. All the studies carried were very conclusive and satisfactory. The manuscript was well written and can be accepted to publish with a minor revision mentioned below. 

We sincerely thank the reviewer for the careful evaluation of our work and for the insightful comments provided. 

Comment 1: In Figure 2, I would suggest the authors to provide a single western blot/gel image showing both the cjX-polyHis with the ladder in one image. If possible, I would include all the raw data in the SI.  

Response 1: We thank the reviewer for the suggestion. Figure 2D is presented in two images because the molecular weight marker stays in the PVDF membrane, whereas the immunoreactivity is detected in an X-ray film. The raw data were included in the Supplementary Information as required (Figures S2 and S3).

Comment 2: In Figure 6, do the authors validate the promoter activities in triplicate? If yes, is it possible to provide its statistical significance?

Response 2: The reviewer is right to highlight this aspect. The results presented in this figure are the means and standard deviations of three independent experiments. This information is now present in the revised version of the manuscript in a new subsection of Materials and Methods (4.11). Figures 6 and 7 have been updated with the statistical analysis.

Reviewer 5 Report

Comments and Suggestions for Authors

Authors haver reported the identification of a cryptic overlapping gene, mcjX, within the MccJ25 biosynthetic locus. The study presents evidence that its product acts as a negative regulator of the mcjA promoter. Since overlapping genes with regulatory function are rare in E. coli, this would be the first such gene described in a lasso peptide operon. The authors use multiple complementary approaches (lacZ fusions, His-tag detection, Western blot, GFP reporter assays, antimicrobial bioassays) and the promoter deletion analysis is methodical.

Some comments:

1. Page 2, line 53: "β-beta-galactosidase" is redundant, instead should be “β-galactosidase"

2. Section 4.10 discloses that "the graphical abstract was generated using Gemini Pro," but no graphical abstract is present in the submission.

3. The authors identify two potential "TG" motifs that could contribute to extended -10 function, one at positions 14, 15 and another at 16, 17. Only the 14, 15 TG is mutagenized. The 16, 17 position is mentioned but not tested, despite the authors noting it "could also have an impact." Authors should address this briefly as to how it does not affect full characterization of PmcjA.

Author Response

Authors have reported the identification of a cryptic overlapping gene, mcjX, within the MccJ25 biosynthetic locus. The study presents evidence that its product acts as a negative regulator of the mcjA promoter. Since overlapping genes with regulatory function are rare in E. coli, this would be the first such gene described in a lasso peptide operon. The authors use multiple complementary approaches (lacZ fusions, His-tag detection, Western blot, GFP reporter assays, antimicrobial bioassays) and the promoter deletion analysis is methodical.

We sincerely thank the reviewer for the positive evaluation of our work and for recognizing the novelty and significance of our findings.

Some comments:

Comment 1: Page 2, line 53: "β-beta-galactosidase" is redundant, instead should be “β-galactosidase"

Response 1: The reviewer is absolutely correct, the correction was made to the manuscript.

Comment 2: Section 4.10 discloses that "the graphical abstract was generated using Gemini Pro," but no graphical abstract is present in the submission.

Response 2: We apologize for the confusion regarding this. The graphical abstract was uploaded through the journal submission system under the corresponding section; however, we were not aware that it would not be visible within the manuscript file provided for review. To facilitate evaluation, we have now attached the graphical abstract to the present response.

Comment 3: The authors identify two potential "TG" motifs that could contribute to extended -10 function, one at positions 14, 15 and another at 16, 17. Only the 14, 15 TG is mutagenized. The 16, 17 position is mentioned but not tested, despite the authors noting it "could also have an impact." Authors should address this briefly as to how it does not affect full characterization of PmcjA.

Response 3: We thank the reviewer for this thoughtful observation. As correctly pointed out, two potential TG motifs are present in the promoter region, located at positions −14/−15 and −16/−17 relative to the transcription start site. In the present study, we specifically focused on the TG motif at positions −14/−15 because previous work by Burr et al. (2000) demonstrated that this motif constitutes the canonical stimulatory extended −10 element and that promoter enhancement critically depends on these positions.

In contrast, the TG motif located at positions −16/−17 has not been shown to independently confer extended −10 promoter activity. Rather, available evidence suggests that this upstream TG sequence may only contribute synergistically when present together with the canonical −14/−15 TG motif. For this reason, we prioritized mutational analysis of the latter, which is considered the primary determinant of extended −10 function.

We agree, however, that the possible contribution of the −16/−17 motif represents an interesting aspect that deserves further investigation. To clarify this point for the reader, we have now expanded the Conclusions in the revised manuscript (Page 15, lines 541-546).