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  • Gustavo Cuaxinque-Flores1,
  • Lorena Jacqueline Gómez-Godínez2 and
  • Alma Armenta-Medina2
  • et al.

Reviewer 1: Anonymous Reviewer 2: Srinivasan Sathiyaraj

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is my pleasure to review the manuscript of the research article on “Genome insights into Kocuria sp. KH4, a metallophilic bacterium harboring multiple biosynthetic gene clusters (BGCs)”.

This topic is interesting because Kocuria sp. KH4 combines two rare features—metallophilicity and the presence of multiple biosynthetic gene clusters (BGCs). Its genome reveals how the bacterium survives heavy-metal stress and simultaneously encodes diverse metabolic pathways that may produce novel antibiotics, pigments, antioxidants, or other bioactive compounds. This makes it a valuable organism for bioremediation, drug discovery, and understanding stress-adapted microbial evolution.

 

  1. In line 27, the term “biosynthetic clusters (BGCs)” is incomplete and should be corrected to “biosynthetic gene clusters (BGCs)” to follow proper genomic terminology
  2. In line 49, a proper citation is needed to support the claim that Kocuria strains tolerate high salt concentrations, variable pH, and limited nutrient availability.
  3. In line 61, the citation for “Kumar and Sujitha” is incomplete and not formatted properly
  4. In this line 109, the sentence “A representative colony exhibiting distinctive carotenoid pigmentation were subcultured…” shows a subject–verb agreement error. The noun “colony” is singular, so the verb should be corrected to “was subcultured.”
  5. In lines 143 and 144, the construction “core, soft-core, shell, and cloud genes was determined” contains a subject–verb agreement error. The subject is plural (“genes”) and therefore requires the plural verb form “were determined.” Please revise accordingly.
  6. In line 148, the phrase “Phylogenetic dendrogram were visualized” shows incorrect subject–verb agreement. The singular subject “dendrogram” should be paired with the singular verb “was visualized.” Please correct this for grammatical accuracy.
  7. In 159 and 160 lines, the clause “were considered valid to minimize false positives” is grammatically awkward. The infinitive “to minimize” creates ambiguity regarding which action is performing the minimization. To improve clarity, the sentence should be rephrased, for example: “…were considered valid, minimizing false positives.”
  8. In line 196, the term should be written as “16S rRNA gene.” Please correct the spacing and capitalization to follow standard scientific nomenclature.
  9. In line 235, the term “In-silico DNA–DNA hybridization” is incorrectly formatted. The correct expression is “in silico” (without a hyphen). Additionally, please remove italics, as in silico is not typically italicized in scientific writing.
  10. In line 257, contain an incorrect figure citation: the text refers to Figure 5b, but this panel is part of Figure 4 (core/pangenome curves). Please correct the in-text reference to Figure 4b
  11. In line 349, the phrase “Various strains of the genus Kocuria spp.” is redundant because “genus” and “spp.” convey the same taxonomic level. Please revise to either “genus Kocuria” or “Kocuria spp.” to maintain correct and concise scientific nomenclature.
  12. In line 361, the phrase “a set of features consistent with strong adaptation” is vague and lacks scientific precision. Please replace “strong adaptation” with a more specific expression, such as “consistent with adaptation to extreme conditions” or another clearly defined ecological context
  13. In line 398, the phrase “a mechanism also presents in the KH4 strain” contains a grammatical error. The correct construction is “a mechanism also present in strain KH4.” Please revise to ensure proper grammar and clarity.
  14. In line 413, the species designation “ rhizophila14ASP” is missing a required space between the species name and the strain identifier. Please correct the formatting to “K. rhizophila 14ASP.” to maintain proper taxonomic notation.
  15. In lines 311,337,416, the phrase “NRPS (non-ribosomal peptide synthases)” uses incorrect terminology. The correct form is “nonribosomal peptide synthetases,” not “synthases.” Please revise all occurrences accordingly and ensure consistent terminology throughout the manuscript.
  16. In line 572-575, Reference 38 is a duplicate of Reference 37 (Manaviparast et al., 2024). Duplicate citations should not appear in the reference list. Please remove the duplicate entry to maintain accuracy and consistency.
  17. In lines 542-543 and 550-551, these two references contain duplicate content, as both cite studies on the Kocuria flava NIO_001 genome mining published in 2025. This redundancy may confuse and is inconsistent with reference formatting standards. Please verify whether both entries are necessary or remove the duplicate to maintain clarity and accuracy in the reference list.

Author Response

We appreciate the review of our manuscript. We have attended to the recommendations you kindly shared with us. Thank you.

Reviewer 1:

This is my pleasure to review the manuscript of the research article on “Genome insights into Kocuria sp. KH4, a metallophilic bacterium harboring multiple biosynthetic gene clusters (BGCs)”.

This topic is interesting because Kocuria sp. KH4 combines two rare features—metallophilicity and the presence of multiple biosynthetic gene clusters (BGCs). Its genome reveals how the bacterium survives heavy-metal stress and simultaneously encodes diverse metabolic pathways that may produce novel antibiotics, pigments, antioxidants, or other bioactive compounds. This makes it a valuable organism for bioremediation, drug discovery, and understanding stress-adapted microbial evolution

  1. In line 27, the term “biosynthetic clusters (BGCs)” is incomplete and should be corrected to “biosynthetic gene clusters (BGCs)” to follow proper genomic terminology

R: Thank you for your comment. We have corrected the error.

 

  1. In line 49, a proper citation is needed to support the claim that Kocuria strains tolerate high salt concentrations, variable pH, and limited nutrient availability.

R: We have incorporated a proper citation.

 

  1. In line 61, the citation for “Kumar and Sujitha” is incomplete and not formatted properly

R: We have incorporated the corresponding reference.

 

  1. In this line 109, the sentence “A representative colony exhibiting distinctive carotenoid pigmentation were subcultured…” shows a subject–verb agreement error. The noun “colony” is singular, so the verb should be corrected to “was subcultured.”

R: Thanks for the observation, we have corrected the sentence.

 

  1. In lines 143 and 144, the construction “core, soft-core, shell, and cloud genes was determined” contains a subject–verb agreement error. The subject is plural (“genes”) and therefore requires the plural verb form “were determined.” Please revise accordingly.

R: We have corrected the lines.

 

  1. In line 148, the phrase “Phylogenetic dendrogram were visualized” shows incorrect subject–verb agreement. The singular subject “dendrogram” should be paired with the singular verb “was visualized.” Please correct this for grammatical accuracy.

R: Thank you, the error has been corrected.

 

  1. In 159 and 160 lines, the clause “were considered valid to minimize false positives” is grammatically awkward. The infinitive “to minimize” creates ambiguity regarding which action is performing the minimization. To improve clarity, the sentence should be rephrased, for example: “…were considered valid, minimizing false positives.”

R: Excellent suggestion, the change has been made.

 

  1. In line 196, the term should be written as “16S rRNA gene.” Please correct the spacing and capitalization to follow standard scientific nomenclature.

R: The change was made.

 

  1. In line 235, the term “In-silico DNA–DNA hybridization” is incorrectly formatted. The correct expression is “in silico” (without a hyphen). Additionally, please remove italics, as in silico is not typically italicized in scientific writing.

R: Thank you for the suggestion, it has been implemented.

 

 

  1. In line 257, contain an incorrect figure citation: the text refers to Figure 5b, but this panel is part of Figure 4 (core/pangenome curves). Please correct the in-text reference to Figure 4b

R: Excellent observation, the text has been corrected.

 

  1. In line 349, the phrase “Various strains of the genus Kocuria spp.” is redundant because “genus” and “spp.” convey the same taxonomic level. Please revise to either “genus Kocuria” or “Kocuria spp.” to maintain correct and concise scientific nomenclature.

R: We have corrected the line.

  1. In line 361, the phrase “a set of features consistent with strong adaptation” is vague and lacks scientific precision. Please replace “strong adaptation” with a more specific expression, such as “consistent with adaptation to extreme conditions” or another clearly defined ecological context

R: Good observation. The changes have been made.

 

  1. In line 398, the phrase “a mechanism also presents in the KH4 strain” contains a grammatical error. The correct construction is “a mechanism also present in strain KH4.” Please revise to ensure proper grammar and clarity.

R: Thank you, the comment has been resolved.

 

  1. In line 413, the species designation “ rhizophila14ASP” is missing a required space between the species name and the strain identifier. Please correct the formatting to “K. rhizophila 14ASP.” to maintain proper taxonomic notation.

R: Thank you, the comment has been resolved.

 

  1. In lines 311,337,416, the phrase “NRPS (non-ribosomal peptide synthases)” uses incorrect terminology. The correct form is “nonribosomal peptide synthetases,” not “synthases.” Please revise all occurrences accordingly and ensure consistent terminology throughout the manuscript.

R: Excellent observation, we have modified the term in all cases.

 

  1. In line 572-575, Reference 38 is a duplicate of Reference 37 (Manaviparast et al., 2024). Duplicate citations should not appear in the reference list. Please remove the duplicate entry to maintain accuracy and consistency.

R: Thanks, we have modified the references following the journal's author guidelines.

 

  1. In lines 542-543 and 550-551, these two references contain duplicate content, as both cite studies on the Kocuria flava NIO_001 genome mining published in 2025. This redundancy may confuse and is inconsistent with reference formatting standards. Please verify whether both entries are necessary or remove the duplicate to maintain clarity and accuracy in the reference list.

R: Thank you, we have removed one of the references and organized everything according to the authors' guidelines.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is scientifically sound, methodologically up to date, and offers valuable genomic insights into a metallophilic Kocuria strain, including comparative pangenome and BGC analyses. I do not identify any major flaws that undermine the core results. My comments relate to tempering some claims, improving clarity, and tightening the link between the data and the broader conclusions.

  1. Please slightly temper the strength of the claim that KH4 is a “novel genomospecies” and keep this clearly at the genome-based level. The text already acknowledges that formal species description requires phenotypic characterization. I suggest consistently using “novel Kocuria genomospecies” or “putative new Kocuria species based on genome-level criteria”
  2. The ecological and biotechnological inferences based on metal-resistance genes and BGCs are interesting, but at some points the it reads as if physiological traits and metabolite production have been experimentally demonstrated. For example, the discussion strongly links gene repertoires to “ecological success” and heavy-metal bioremediation potential. Please state in the Results and Discussion that these are genome-based predictions and avoid over-stating functional capabilities that have not been tested experimentally.
  3. The assembly and annotation section is generally clear, but some key genome statistics that readers expect are not yet fully summarized in one place. Consider adding N50, number of contigs, maximum contig size, sequencing coverage, and GenBank accession(s) to either the text.
  4. It would help to add 2–3 sentences in the Results (or in the Discussion) that highlight what is special about KH4 relative to other Kocuria species: for example, whether KH4 is unusually rich in mobile elements, regulators, or certain functional categories that plausibly relate to adaptation to metal-rich tailing.
  5. I recommend adding (i) one sentence explicitly stating which BGC class(es) in KH4 are rare or unique among the analyzed strains, and (ii) brief mention of the closest known clusters (if available from KnownClusterBlast) and any predicted or putative metabolites, even if tentative. This will help support the claim that KH4 is a “promising source of bioactive compounds”.

Author Response

Thank you very much for your pertinent comments and suggestions regarding the content and analysis. We have taken your observations into account.

Reviewer 2:

The manuscript is scientifically sound, methodologically up to date, and offers valuable genomic insights into a metallophilic Kocuria strain, including comparative pangenome and BGC analyses. I do not identify any major flaws that undermine the core results. My comments relate to tempering some claims, improving clarity, and tightening the link between the data and the broader conclusions.

  1. Please slightly temper the strength of the claim that KH4 is a “novel genomospecies” and keep this clearly at the genome-based level. The text already acknowledges that formal species description requires phenotypic characterization. I suggest consistently using “novel Kocuria genomospecies” or “putative new Kocuria species based on genome-level criteria”

R: Thank you very much for your comment. You are absolutely right. Since there is no phenotypic evaluation, we have used the term “genomospecies,” which has been established based on genomic criteria and is widely used in taxonomy in the genomic era. We have included the definition of “genomospecies” for better understanding and to avoid confusion.

“A genomospecies is a bacterial taxonomic group defined exclusively on the basis of its genomic relationship, i.e., by the degree of overall similarity between its complete genomes. Unlike novel species traditionally defined by phenotypic, physiological, or ecological characteristics, a genomospecies does not require or consider phenotypic traits for its delimitation (Chun et al., 2018)”.

  1. The ecological and biotechnological inferences based on metal-resistance genes and BGCs are interesting, but at some points the it reads as if physiological traits and metabolite production have been experimentally demonstrated. For example, the discussion strongly links gene repertoires to “ecological success” and heavy-metal bioremediation potential. Please state in the Results and Discussion that these are genome-based predictions and avoid over-stating functional capabilities that have not been tested experimentally.

R: We agree with the comments and have clarified the statements, indicating that they are assumptions based on genomic information and the physicochemical characteristics of mining tailings. In other cases, we have removed the line so as not to fall into exaggeration.

  1. The assembly and annotation section is generally clear, but some key genome statistics that readers expect are not yet fully summarized in one place. Consider adding N50, number of contigs, maximum contig size, sequencing coverage, and GenBank accession(s) to either the text.

R: We have included this information in the results. With regard to GenBank, this information is in the “Data Availability Statement” section.

  1. It would help to add 2–3 sentences in the Results (or in the Discussion) that highlight what is special about KH4 relative to other Kocuria species: for example, whether KH4 is unusually rich in mobile elements, regulators, or certain functional categories that plausibly relate to adaptation to metal-rich tailing.

R: Excellent suggestion. We have now reported genomic islands (GIs) and mobile genetic elements (MGEs) using IslandViewer4 based on three search algorithms (IslandPath-DIMOB, SIGI-HMM, and IslandPick) (Bertelli et al., 2017). The data are described in a new section in the results, 3.5 Genomic islands (GIs) in Kocuria sp. KH4.

  1. I recommend adding (i) one sentence explicitly stating which BGC class(es) in KH4 are rare or unique among the analyzed strains, and (ii) brief mention of the closest known clusters (if available from KnownClusterBlast) and any predicted or putative metabolites, even if tentative. This will help support the claim that KH4 is a “promising source of bioactive compounds”.

R: Thank you for your comment. We have clarified this information in the results. In our strain, we did not identify exclusive BGCs, but we did identify others that are shared with other species. We consider it to be a promising source of bioactive compounds because it has different types of BGCs. It is possible that even though the type of BGCs is similar (genetic level), the metabolite produced may have different chemical substitutions or biological activity. This is a future prospect for our work and is mentioned in the conclusion.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Author implemented all the suggested corrections.

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript has addressed all previous concerns effectively; the manuscript is now suitable for acceptance.