PONE-D-25-07489Analysis and comparison of the bacterial σ54 regulon: evidence of phylogenetic trends in gene regulationPLOS ONE

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors present a study titled “Analysis and comparison of the bacterial σ54 regulon: evidence of phylogenetic trends in gene regulation.” This work provides a comprehensive analysis of the presence of Sigma54 binding sites across a diverse range of bacterial species. The results are original, as the authors effectively demonstrate the distribution of these regulons across phylogenetic classes using their own regulon identification method. This approach offers valuable insights into evolutionary trends in gene regulation.

1. Comprehensive and review style introduction to Sigma54.

2. l.200 The R script used to calculate COG overrepresentation is not provided. Please include it to ensure reproducibility.

3. l.207 Identification of EBPs is based on the presence of the PF00158 domain. However, it is unclear how specific this domain is. Could there be potential false positives? Provide an error analysis supporting the specificity of this domain

4. l.233 Please add the webpage link here: https://biocomputo.ibt.unam.mx/arpondb/. The web server is up and running, and the data is easily accessible.

5. l.240 Phylogenetic distribution of σ54: The phylogenetic group Actinomycetota appears to have only four organisms with a Sigma54. Could these 4 be false positives? How do these four organisms differ? Additionally, how confident are you that the AID, CBD, and DBD domains are not misidentified (are false positives)?

6. l.286 Identifying protein function based on a single domain is problematic. While the approach is generally valid, it is likely to result in a significant number of false positives and false negatives. How specific is the Pfam Sigma54_activat motif (PF00158)? The results indicate up to 100 EBPs per genome—how realistic is this estimate? See also point 3

7. l.321 The Matthew correlation coefficient (MCC) of 0.778 appears to be based on E. coli. Given the reported precision and specificity of 100%, this suggests an overfitted model for E. coli. How can such a model be reliably applied to genomes that are not closely related to E. coli? Error analysis is not provided

8. l.332 The values of the four parameters for the hypergeometric distribution depend heavily on the number of false positives and false negatives detected. If genomes from different taxonomic groups are included, the DNA and protein motifs will likely introduce more errors, which will impact the hypergeometric results. How confident are you that Sigma54 is being analyzed specifically, rather than general sequence diversity? Would a similar analysis with Sigma70 yield comparable trends? Was this tested?

9. l.1.4 – l.1.8 The list of pathways is extensive. While some have literature references to support their regulation by Sigma54, in many cases, it is unclear whether these pathways are directly regulated. Providing additional context or evidence would improve clarity.

10. l.477 The same issue applies to Cellular processes and signaling.

11. l.683 Similar concerns apply to Information storage and processing.

12. Several web servers and computational tools are already available for mining Sigma54 regulons. However, the authors do not provide a comparison to existing tools or demonstrate whether their method offers any improvements. A comparative analysis would strengthen the study.

13. A clear overview is missing that directly compares known regulon-associated genes/proteins with those identified in this study. Additionally, there is no clear indication of genes previously unknown to be influenced by Sigma54. Such a summary would enhance the impact of the findings.

14. l.834 "Through rigorous statistical analysis": The cross-organism application of PSSMs for motif identification in DNA is highly unreliable. The manuscript does not explain how errors were quantified or how confident the authors are in their results. Please provide details on this analysis.

15. l.840 "Our study revealed unreported cellular processes": Could these findings be due to false positives? Additionally, expected genes are not explicitly discussed—were they identified as expected, or were some missed? These results should be included to clarify the reliability of the method.

Reviewer #2: This is a very interesting survey of the sigma54 regulon across a very large set of genome sequences. It makes an important contribution to the field. The accompanying website is also a useful contribution.

However, I have some suggestions and comments that the authors may wish to consider.

The website at https://biocomputo.ibt.unam.mx/arpondb/ is a good contribution. However, previous experience suggests that websites often "disappear" after publication as the authors move on to new projects, funding for webhosting dries up, etc. I strongly urge the authors to deposit a snapshot of the webpage in Zotero or as supplementary data attached to the manuscript.

I am surprised that the authors do not cite this paper:

Barrios, H., Valderrama, B., & Morett, E. (1999). Compilation and analysis of sigma(54)-dependent promoter sequences. Nucleic acids research, 27(22), 4305–4313. https://doi.org/10.1093/nar/27.22.4305

This is a seminal paper that represents the first major attempt to systematically catalogue sigma54 promoter sequences and underpins much of the subsequent computer-based predictive studies of the sigma54 regulon.

The authors should also discuss this recent work and how it is similar/dissimilar the theirs:

Achterberg, T., & de Jong, A. (2025). ProPr54 web server: predicting σ54 promoters and regulon with a hybrid convolutional and recurrent deep neural network. NAR genomics and bioinformatics, 7(1), lqae188. https://doi.org/10.1093/nargab/lqae188

Lines 117-125. When selecting genome sequences for their analyses, did they consider the quality/completeness/reliability of the genome sequences? If yes, then what quality criteria did they apply. If not, then why not?

Lin 159. "propensity of some sigma factors for self-transcription". Is there any published evidence that sigma54 initiates transcription of itself? Given the uniqueness of this transcription factor, it is dangerous to extrapolate what we know about other sigma factors to sigma54. It would make more sense to start the seed matrix using a set of confirmed promoter sequences or a datasets such as that of Barrios et al. (1999).

Line 165. "cutoff value of 0.001". Value of what? Is this an E-value cut-off?

Line 247. Please be consistent in taxonomic names. Cereibacter sphaeroides is a synonym of Rhodobacter sphaeroides. So, use one name or the other. Don't use one synonym for one strain and another synonym for the other. Both names refer to precisely the same entity, so it makes no sense to use both. See https://lpsn.dsmz.de/species/rhodobacter-sphaeroides for full taxonomic information about this species.

Line 260. "Pfam motifs AID, DBD, and CBD". Are you sure that these are "motifs" in Pfam? Pfam has families, domains and clans. I don't think it has motifs. Also, please provide the Pfam accession numbers for these entities.

Line 273. "We hypothesize that this overlap in high GC content ...". To what extent has this been previously explored in the literature and to what extent does previous work support the hypothesis? For example, see:

Calistri, E., Livi, R., & Buiatti, M. (2011). Evolutionary trends of GC/AT distribution patterns in promoters. Molecular phylogenetics and evolution, 60(2), 228–235. https://doi.org/10.1016/j.ympev.2011.04.015

Line 277. "Tree of Life [with capital initial letters]". What is this? Can the authors provide a reference or URL? Do they mean https://www.evogeneao.com/en/explore/tree-of-life-explorer? Or https://itol.embl.de/?

Line 353. "Nitrogen metabolism has long been recognized as one of the key processes that is regulated by σ54, initially leading to the association of this sigma factor with the name σN. However, our study's findings challenge the notion that the regulation of nitrogen metabolism, which is indicated by the red circles in Fig 5, is a universal trend across bacteria.". Here, the authors are setting up a "straw man". I am not aware of any recent reviews that posit that sigma54's role in nitrogen regulation is universal. Can the authors cite any specific papers with which they disagree on this point?

Line 403. "Interestingly, as previously documented, RNAP-σ54 complex also regulates the transcription of PTS component genes in β-Proteobacteria. Also gamma-proteobacteria.

See:

Studholme D. J. (2002). Enhancer-dependent transcription in Salmonella enterica Typhimurium: new members of the sigmaN regulon inferred from protein sequence homology and predicted promoter sites. Journal of molecular microbiology and biotechnology, 4(4), 367–374.

Line 507. When considering the sigma54 regulon in planctomycetes, the authors may wish to read this paper:

Studholme, D. J., & Dixon, R. (2004). In silico analysis of the sigma54-dependent enhancer-binding proteins in Pirellula species strain 1. FEMS microbiology letters, 230(2), 215–225. https://doi.org/10.1016/S0378-1097(03)00897-8.

I note that the authors' predictions at https://biocomputo.ibt.unam.mx/arpondb/tables/rba_Rhodopirellula_baltica.html are not entirely consistent with the results in that paper. The authors may or may not wish to consider that. Furthermore, I was unable to locate in the authors' web database their predictions for "Aquifex aeolicus" to compare those against the published work the sigma54 regulon in that organism.

Throughout the manuscript, please use a superscript for 54 when writing sigma54. And do the same for other sigma factors; e.g. sigma70 should have superscripted 70.

Line 23 and elsewhere. Rather than saying "bacterial organisms", it is more straightforward to just say "bacteria".

Line 25. Instead of "corresponds to" simply write "is".

Line 33. Delete "lacking sequence homology". First, it is unclear how sequence homology is different from simply "homology". Second, the lack of homology is discussed in subsequent sentences, so does not need to be stated here.

Line 38. The word "instances" does not make sense here. Please consider using a different word.

Line 44. "ubiquitously present". No. As the authors show later in this manuscript, this sigma factor is NOT ubiquitous. There are several bacterial taxa where this sigma factor is absent.

Line 57. "class Clostridiales". This is an order, not a class.

Line 79. "matrices.". These are not matrices. They are profile-HMMs.

Line 78. Do not use italics for Pfam.

Line 112. "1,249 organisms". Here, and many other times in the manuscript the authors refer to numbers of organisms. What does this mean? Does it mean numbers of species? Strains? How do the authors count organisms? Are two strains counted as two different organisms?

Line 289. The use of italics has gone wrong on this line.

Line 481. Use lowercase 'c' for cellular.

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Reviewer #1: No

Reviewer #2: Yes:  David Studholme

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Analysis and comparison of the bacterial σ54 regulon: evidence of phylogenetic trends in gene regulation

PONE-D-25-07489R1

Dear Dr. Merino,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have significantly improved the quality of the manuscript. In particular, the refinement of the search strategy and the inclusion of new supplementary data on phylogenetic distribution have strengthened the study and made the manuscript suitable for publication.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: Yes:  David Studholme

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