Aldehyde Dehydrogenase Diversity in Azospirillum Genomes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript: diversity-2701892-peer-review-v1.pdf

In the manuscript titled „Aldehyde dehydrogenase diversity in Azospirillum genomes” the Authors have investigated the phenomenon of cytoxicity of aldehydes, an ubiquitous organic compounds found in nature. In the manuscript it was stated that aldehyde levels can endanger bacterial viability (lines 14-15). Aldehyde dehydrogenases (ALDHs) are essential enzymes that mitigate aldehyde toxicity by converting them into less reactive compounds  (line 15). Therefore, the diversity of aldehyde dehydrogenases among different strains of Azospirillum can affect their adaptation and survival under various environmental conditions (lines 110-112).

Azospirillum bacteria can tolerate and adapt to a variety of stressful conditions, such as soils contaminated with oil, bacterial fuel cells, geysers, and sulfurous waters (lines 96-97). However, exposure to environmental stressors can increase aldehyde levels, which can endanger bacterial viability (lines 13-14). Other types of environmental stress that can affect the viability of these bacteria include changes in temperature, pH, salinity, and nutrient availability (lines 112-114). The diversity of aldehyde dehydrogenases in Azospirillum bacteria can play a role in their adaptation and survival under these stressful conditions (lines 110-112).

The findings of this study can potentially be used to improve agricultural production by enhancing the growth and productivity of crops. Azospirillum bacteria form a symbiotic relationship with plants and can promote plant growth by producing phytohormones such as indole-3-acetic acid (IAA) (lines 87-88). The diversity of aldehyde dehydrogenases in Azospirillum bacteria can affect their ability to produce IAA and other beneficial compounds that promote plant growth (lines 110-112). Therefore, understanding the genetic diversity of aldehyde dehydrogenases in Azospirillum can help identify strains that are more effective in promoting plant growth and can be used as biofertilizers to enhance agricultural productivity.

The outcome of this study is the identification and classification of 17 families and 31 subfamilies of aldehyde dehydrogenases (ALDHs) in fully assembled Azospirillum genomes (lines 20-22). This classification system provides a more comprehensive understanding of the diversity and redundancy of ALDHs across bacterial genomes, which can help elucidate the distinct characteristics and functions of each family (lines 17-19). The study also proposes the adoption of the ALDH19 family as a powerful phylogenetic marker due to its robust conservation and non-redundancy across various Azospirillum species. The findings of this study can potentially be used to improve agricultural production by identifying strains of Azospirillum that are more effective in promoting plant growth and can be used as biofertilizers to enhance agricultural productivity (lines 87-88).

The methods used are suitable for the task. I have not seen any scientific shortcomings in the reviewed manuscript. I am not a native, so I cannot judge the level of English used, but for me the clarity of reading is sufficient. The results are presented correctly and discussed using suitable scientific references. Therefore, I recommend the paper for publication. 

Comments on the Quality of English Language

I am not a native, so I cannot judge the level of English used, but for me the clarity of reading is sufficient.

Author Response

This is the new version of the manuscript. Thanks for your review of our work.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This paper brings insights by analyzing ALDH coding sequences in fully assembled Azospirillum genomes. The author used bioinformatics tools for phylogenetic and pangenomic analyses, thereby classifying the aldehyde dehydrogenase. To improve the quality of paper, the authors have to: 1) Organize the order of the references. For instance, in line 51, the reference 9 appears before 6-8. 2) In figure 1, it is necessary to change the representation color of A. thermophilum CFH70021, because the representation color of A. brasilense Sp7 is very similar and these points are far apart. 3) Improve the quality of the figures 4 and 5, there is hard to see and identify some items.

Author Response

Request 1: 1) Organize the order of the references. For instance, in line 51, the reference 9 appears before 6-8.

Response 1. The order of references was corrected.

Request 2: 2) In figure 1, it is necessary to change the representation color of A. thermophilum CFH70021, because the representation color of A. brasilense Sp7 is very similar and these points are far apart.

Response 2. The color of Figure 1 was modified as requested by reviewer 2.

Request 3: 3) Improve the quality of the figures 4 and 5, there is hard to see and identify some items.

Response 3. The quality of Figures 4 and 5 was improved to see and identify better the items non-visible.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Title :- Aldehyde dehydrogenase diversity in Azospirillum genomes

General comments:-

Written well, but presented bad. Results are good, M and M are standard, Some figures are good some are redundant with bad quality.

Specific Comments:-

Abstract :- Is it an essay ? off course not…out of 15 lines of abstract, more than half is the background the results are only given in 2 lines…This shows that authors don’t have the confidence on their own results and are not highlighting it. The abstract should focus on ALDH results.

Keywords:- hats off, 10 keywords are given. 5-6 are accepted by journals only but may be the authors are not aware of this being childish

Introduction:- presented bad

Line 33-39, support with more citations

Same for line 40-46, more citations needed

So many 2 line paragraphs, 3 line paragraphs in introduction without context. Authors even don’t care about basic writing methods, so careless

The whole introduction is not properly supported by citations. So many statements without support, why it is so?

See line 88-98 and so many, you can go on and on and on

What is the hypothesis of the study..not presented, objectives are very flimsy, not presented well

M and M. if the data was downloaded, why not submitted to a repository? And why not provided as supplementary files? Data statement says not applicable, while authors says they used NCBI data

Results:- Too long, give significant results only

Figure 8 and 9 are not readable with bad color choices

Discussion:- This is the worst part of the article, no proper reasons and probabilities provided for several results, it is written as it is a prospective review,

Line 493 – 522 presents so many important results but non of them discussed, all of these copy pasted again from results but not discussed in line with previous findings and probabilities of importance of these results not given at all.

Line 536-547 and line 607-621, line 631-640 are other similar examples with no probabilities discussed.

Conclusion:- repetitive with no importance, delete it

Comments on the Quality of English Language

ok

Author Response

Request 1 Abstract :- Is it an essay ? off course not…out of 15 lines of abstract, more than half is the background the results are only given in 2 lines…This shows that authors don’t have the confidence on their own results and are not highlighting it. The abstract should focus on ALDH results.

Response 1.  We changed the Abstract in agreement with Reviewer 3

Lines 13-31 Aldehyde dehydrogenases (ALDHs) are indispensable enzymes that play a pivotal role in mitigating aldehyde toxicity by converting them into less reactive compounds. Despite the availability of fully sequenced Azospirillum genomes in public databases, a comprehensive analysis of the ALDH superfamily within these genomes has yet to be undertaken. This study presents the identification and classification of 17 families and 31 subfamilies of ALDHs in fully assembled Azospirillum genomes. This classification system framework provides a more comprehensive understanding of the diversity and redundancy of ALDHs across bacterial genomes, which can aid in elucidating the distinct characteristics and functions of each family. The study also proposes the adoption of the ALDH19 family as a powerful phylogenetic marker due to its remarkable conservation and non-redundancy across various Azospirillum species. The diversity of ALDHs among different strains of Azospirillum can influence their adaptation and survival under various environmental conditions. The findings of this study could potentially be used to improve agricultural production by enhancing the growth and productivity of crops. Azospirillum bacteria establish a mutualistic relationship with plants and can promote plant growth by producing phytohormones such as indole-3-acetic acid (IAA). The diversity of ALDHs in Azospirillum can affect their ability to produce IAA and other beneficial compounds that promote plant growth and can be used as biofertilizers to enhance agricultural productivity.

Request 2 Keywords:- hats off, 10 keywords are given. 5-6 are accepted by journals only but may be the authors are not aware of this being childish

Response 2 We changed the Keywords  Line 33 Azospirillum; aldehyde dehydrogenases; phylogeny; pangenomics; abiotic stress

Request 3 Introduction:- presented bad

Response 3   Introduction was revised and add more references, in lines  36-42, and lines 43-52. References included are: 1) Deza-Ponzio, R.; Herrera, M.L.; Bellini, M.J.; Virgolini, M.B.; Hereñú, C.B. Aldehyde Dehydrogenase 2 in the Spotlight: The Link between Mitochondria and Neurodegeneration. Neurotoxicology 2018, 68, 19–24, doi:10.1016/j.neuro.2018.06.005.

2) Muzio, G.; Maggiora, M.; Paiuzzi, E.; Oraldi, M.; Canuto, R.A. Aldehyde Dehydrogenases and Cell Proliferation. Free Radical Biology and Medicine 2012, 52, 735–746, doi:10.1016/j.freeradbiomed.2011.11.033.

3) Shortall, K.; Djeghader, A.; Magner, E.; Soulimane, T. Insights into Aldehyde Dehydrogenase Enzymes: A Structural Perspective. Front. Mol. Biosci. 2021, 0, doi:10.3389/fmolb.2021.659550.

Request 4. Line 33-39, support with more citations

Response 4

in lines  36-42, and lines 43-52. References included are: 1) Deza-Ponzio, R.; Herrera, M.L.; Bellini, M.J.; Virgolini, M.B.; Hereñú, C.B. Aldehyde Dehydrogenase 2 in the Spotlight: The Link between Mitochondria and Neurodegeneration. Neurotoxicology 2018, 68, 19–24, doi:10.1016/j.neuro.2018.06.005.

2) Muzio, G.; Maggiora, M.; Paiuzzi, E.; Oraldi, M.; Canuto, R.A. Aldehyde Dehydrogenases and Cell Proliferation. Free Radical Biology and Medicine 2012, 52, 735–746, doi:10.1016/j.freeradbiomed.2011.11.033.

3) Shortall, K.; Djeghader, A.; Magner, E.; Soulimane, T. Insights into Aldehyde Dehydrogenase Enzymes: A Structural Perspective. Front. Mol. Biosci. 2021, 0, doi:10.3389/fmolb.2021.659550.

Request 5. Same for line 40-46, more citations needed

Response 5

 lines 43-52. References included are: 1) Deza-Ponzio, R.; Herrera, M.L.; Bellini, M.J.; Virgolini, M.B.; Hereñú, C.B. Aldehyde Dehydrogenase 2 in the Spotlight: The Link between Mitochondria and Neurodegeneration. Neurotoxicology 2018, 68, 19–24, doi:10.1016/j.neuro.2018.06.005.

2) Muzio, G.; Maggiora, M.; Paiuzzi, E.; Oraldi, M.; Canuto, R.A. Aldehyde Dehydrogenases and Cell Proliferation. Free Radical Biology and Medicine 2012, 52, 735–746, doi:10.1016/j.freeradbiomed.2011.11.033.

3) Shortall, K.; Djeghader, A.; Magner, E.; Soulimane, T. Insights into Aldehyde Dehydrogenase Enzymes: A Structural Perspective. Front. Mol. Biosci. 2021, 0, doi:10.3389/fmolb.2021.659550.

Request 6. So many 2 line paragraphs, 3 line paragraphs in introduction without context. Authors even don’t care about basic writing methods, so careless

Response 6 We revised the introduction and deleted paragraphs without context

Request 7. The whole introduction is not properly supported by citations. So many statements without support, why it is so?

Response 7 we revised the whole introduction and added references

Request 8. See line 88-98 and so many, you can go on and on and on

Response 8 We revised the introduction and deleted this paragraph; “Various studies indicate that the Azospirillum genus possesses multiple metabolic pathways, such as metabolizing carbon sources such as glucose, sucrose and organic acids, fixing atmospheric nitrogen, and producing siderophores and hormones [20]. Thus, the Azospirillum genus strains have a wide range of metabolic pathways, which results in the production of a variety of intermediates, including aldehydes.”

Request 9. What is the hypothesis of the study..not presented, objectives are very flimsy, not presented well

Response 9. Aldehyde dehydrogenases (ALDHs) are indispensable enzymes that play a pivotal role in mitigating aldehyde toxicity by converting them into less reactive compounds. Despite the availability of fully sequenced Azospirillum genomes in public databases, a comprehensive analysis of the ALDH superfamily within these genomes has yet to be undertaken. This study presents the identification and classification of 17 families and 31 subfamilies of ALDHs in fully assembled Azospirillum genomes. This classification system framework provides a more comprehensive understanding of the diversity and redundancy of ALDHs across bacterial genomes, which can aid in elucidating the distinct characteristics and functions of each family.

Request 10. M and M. if the data was downloaded, why not submitted to a repository? And why not provided as supplementary files? Data statement says not applicable, while authors says they used NCBI data

Response 10. We added the accession numbers of each Azospirillum genome analyzed in Data Availability Statement section: Genomic data was obtained from the NCBI database. The strains and their corresponding RefSeq accession numbers are listed below:

• Azospirillum baldaniorum Sp245, GCF_003119195.2
• Azospirillum brasilense Sp 7, GCF_008274945.1
• Azospirillum brasilense MTCC4039, GCF_005222205.1
• Azospirillum brasilense MTCC4038, GCF_005222145.1
• Azospirillum brasilense Cd, GCF_008274965.1
• Azospirillum lipoferum 4B, GCF_000283655.1
• Azospirillum oryzae KACC 14407, GCF_013347285.1
• Azospirillum ramasamyi M2T2B2, GCF_003233655.1
• Azospirillum sp. B510, GCF_000010725.1
• Azospirillum sp. TSA2s, GCF_004923315.1
• Azospirillum sp. TSH100, GCF_004923295.1
• Azospirillum sp. TSH58, GCF_003119115.1
• Azospirillum thermophilum CFH 70021, GCF_003130795.1
• Azospirillum thiophilum BV-S, GCF_001305595.1

Request 11. Results:- Too long, give significant results only

Response 11. We intend to present the results in this format to capture the attention of students and researchers new to ALDH study. I hope the Reviewer 3 understand us.  

Request 12. Figure 8 and 9 are not readable with bad color choices

Response 12.  We changed the resolution of figures 8 and 9

Request 13. Discussion:- This is the worst part of the article, no proper reasons and probabilities provided for several results, it is written as it is a prospective review,

Response 13. We improve the discussion section. Lines 473- 480 and 480- 489

Request 14. Line 493 – 522 presents so many important results but non of them discussed, all of these copy pasted again from results but not discussed in line with previous findings and probabilities of importance of these results not given at all.

Response 14. We improved the discussion section. Lines 508- 514

Request 15. Line 536-547 and line 607-621, line 631-640 are other similar examples with no probabilities discussed.

Response 15. We improved the discussion section. Lines 537 -542 and 643-648

Request 16. Conclusion:- repetitive with no importance, delete it

Response 16. We deleted the conclusion section.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

MS is improved after revision.

Comments on the Quality of English Language

OK