Genomic Analysis of a Newly Isolated Acidithiobacillus ferridurans JAGS Strain Reveals Its Adaptation to Acid Mine Drainage

Round 1

Reviewer 1 Report

The manuscript describes the genome, its comparison to other microorganisms and very few physiological properties of a strain of the acidophilic bacterium Acidithiobacillus ferridurans. Because of these reasons the manuscript is not recommended for publication:

1. Out of scope. This is a pure microbiological study without any minerals in the study. The only link to minerals is that the microorganism is used for bioleaching. The study does not fit in the journal “Minerals”
2. Lack of novelty. There are hundreds of strains of Acidithiobacillus species being isolated worldwide. This is just one more. Genomic features are interesting but do alone not justify any conclusions about adaptation to acid mine drainage. By the way all Acidithiobacillus species are adapted to acid mine drainage. The physiological growth data are weak and do no allow any conclusions about adaptation or comparison to other strains/species. Ferrous iron oxidation was just determined in shake flasks by measuring Fe(II) and Fe(III) concentrations, and ferrous iron concentration is just given as one percentage value in comparison to one other strain. This simple approach does not take into account changing physiologcial conditions such as pH and does not allow to determine Fe(II) oxidation rates as stated in the text.

Author Response

1. Out of scope. This is a pure microbiological study without any minerals in the study. The only link to minerals is that the microorganism is used for bioleaching. The study does not fit in the journal “Minerals”.

Response: This manuscript was submitted for the special issue “Novel and Emerging Strategies for Sustainable Mine Tailings and Acid Mine Drainage Management”. Bioleaching is a novel strategy to recover metals from minerals or mine tailings as well as AMD biotreatment. This study focused on the genomic analysis of an isolated strain A. ferridurans JAGS from AMD, which is a dominate strain (accounting for 92.6% of the enriched culture based on 16S rRNA gene sequence analysis) and has a great candidate for biomining. It provides information for researchers to better understand how it evolved and which genetic features might contribute to its growth advantage, and then offers insights for future studies, like genetic engineering strategies to improve Acidithiobacillus for bioleaching. Therefore, we believe this manuscript fits the scope of this journal, especially for the special issue.

2. Lack of novelty. There are hundreds of strains of Acidithiobacillus species being isolated worldwide. This is just one more. Genomic features are interesting but do alone not justify any conclusions about adaptation to acid mine drainage. By the way all Acidithiobacillus species are adapted to acid mine drainage. The physiological growth data are weak and do no allow any conclusions about adaptation or comparison to other strains/species. Ferrous iron oxidation was just determined in shake flasks by measuring Fe(II) and Fe(III) concentrations, and ferrous iron concentration is just given as one percentage value in comparison to one other strain. This simple approach does not take into account changing physiologcial conditions such as pH and does not allow to determine Fe(II) oxidation rates as stated in the text.

Response: This is the second genome information of species A. ferridurans in addition to A. ferridurans JCM18981. The A. ferridurans JAGS dominates the enriched consortium isolated from AMD and might be a great candidate for bioleaching. Through genomic analysis of A. ferridurans JAGS, we were able to better understand what were the differences between species A.ferridurans and A.ferrooxidans, how they evolved to adapt to AMD and which genetic features might contribute to its growth advantage, and then provided insights for future studies, like genetic engineering strategies to improve Acidithiobacillus strains for bioleaching. In a word, our work not only provides genomic information to better understand the new species A. ferridurans and its evolutionary relationship with species A. ferrooxidans, but also shares valuable genetic information with researchers in this field that might be useful to engineer bioleaching bacteria.  

Author Response File: Author Response.pdf

Reviewer 2 Report

The ms "Genomic and experimental analyses of a newly isolated Acidithiobacillus ferridurans JAGS strain reveals its adaptation to acid mine drainage" by Chen et al is an interesting microbiological work, although I do not see any close relation with minerals with the exception of the AMD from which was isolated. In any case authors will need to explain the selection of the genomes used for the analysis and perform an extensive editing of the microbial nomenclature before it could be accepted for publication.

Comments and suggestions:

• Title: the presented work is mainly genomic, I suggest to remove the experimental analysis from the title.
• All long the text. Authors use JAGS as a familiar nomenclature for the A. ferridurans isolate all along the text. From a microbiological point of view this is incorrect. I think in short could be mentioned as "isolate JAGS", although once in a while it will be good to use the correct name "A. ferridurans JAGS". 
• Abstract, see the comment above. Line 19: analyzed A. ferroxidans species. Line 25, I do not think that the ms deals with AMD biotreatments thus I suggest to remove it from the abstract.
• Line 40, "members of the Acidithiobacillus genus" better than known as Acidithiobacillus spp.
• Line 50, remove "the" in front of 1940.
• Line 88, pure strain is not a microbiological concept.
• Line 91, why only ferrous iron and nickel were measure? In biomining resistance to other metal cations will be convenient.
• Line 115, is not clear the variability in the genomes used for comparison used along the text. For instance for comparative genomics  four genomes are used and for Pan-Genome analysis nine genomes. Please clarify.
• Line 136, Results and "dicussion"
• Line 138, Acidithiobacillus genus, remove spp.
• Line 138, use mainly
• Line 147, clarify why tailings of Sudbury are important for this ms.
•  Fig 1, ATCC is not a nomenclature for an isolate
• Line 167, indirect tracking is not a good way to measure microbial growth.
• Line 170, which A. ferroxidans isolate?
• Line 171, use "were" insted of "are"
• Line 172, if precipitates are produced during growth, OD600 is not a good system to evaluate cell growth. 
• Lines 189-192. The suggestion does not allow authors to change the published and accepted nomenclature for isolate IO-2C, this should be done using an appropiate microbial nomenclature change system. Thus authors should maintain the original species name for the isolate IO-2C along the text.
• Line 202, with the results of Table 1 I do not understand how authors can talk about rich evolutionary history of A. ferridurans species.
• Line 219, IO-2C canot be considered a A. ferridurans strain.
• Lines 232 and 233, I suggest to remove the hypothesis. Underlining the genomic differences between both species should be sufficient. 
• Line 273, IO-2C is a A. ferrooxidans strain.
• Fig. 4, remove strain IO-2C from the A. ferridurans strains list.
• Line 320, isolate JAGS has been compared only with one A. ferridurans strain and eight A. ferrooxidans strains. Knowing that there are ten recognized species in this genus, authors can not say that the isolate has been compared to other Acidithiobacillus strains but to selected A. ferroxidans strains. See also the concern of the reviewer about the selection of only one species (A. ferrooxidans) for genomic comparison.
• Line 324, clarify what authors mean by "intermediate species".
• Line 359 for "intermediate strain" 

Author Response

Reviewer2

The ms "Genomic and experimental analyses of a newly isolated Acidithiobacillus ferridurans JAGS strain reveals its adaptation to acid mine drainage" by Chen et al is an interesting microbiological work, although I do not see any close relation with minerals with the exception of the AMD from which was isolated. In any case authors will need to explain the selection of the genomes used for the analysis and perform an extensive editing of the microbial nomenclature before it could be accepted for publication.

Response: This manuscript was submitted for the special issue “Novel and Emerging Strategies for Sustainable Mine Tailings and Acid Mine Drainage Management”. Bioleaching is a novel strategy to recover minerals from tailings and acid mine drainage as well as AMD treatment. This study focused on the genomic analysis of an isolated strain A. ferridurans JAGS from AMD, which is a dominate strain (accounting for 92.6% of the enriched culture based on 16S rRNA gene sequence analysis) and has a great candidate for bioleaching. It provides information for researchers to better understand how it evolved and which genetic features might contribute to its growth advantage, and then provides insights for future studies, like genetic engineering strategies to improve bioleaching bacteria. We described this in the introduction part (Line29-33, Line58-68) and also provide more details about A. ferridurans JAGS in the method part (Line86-87 ). We improved the microbial nomenclature in the text according to your great suggestions. We have thoroughly considered all the comments and tried our best to address all the concerns raised. We hope with your suggestions and our efforts, the revised manuscript is good for publication.

Comments and suggestions:

• Title: the presented work is mainly genomic, I suggest to remove the experimental analysis from the title.
• Response: Thanks for your suggestion. The title has been revisedaccordingly.
• All long the text. Authors use JAGS as a familiar nomenclature for the A. ferridurans isolate all along the text. From a microbiological point of view this is incorrect. I think in short could be mentioned as "isolate JAGS", although once in a while it will be good to use the correct name "A. ferridurans JAGS". 
• Response: Thanks for your professional advice. We improved our manuscript according your suggestion.
• Abstract, see the comment above. Line 19: analyzed A. ferroxidans species. Line 25, I do not think that the ms deals with AMD biotreatments thus I suggest to remove it from the abstract.
• Response: We revised the abstract according to your suggestions. We kept “AMD biotreatment”because dealing with the AMD is one of the most important goals of our research. From genome sequencing, genomic analysis, and then genome editing, our final goals are to stabilize mine wastes to prevent acid mine drainage and make the recovery process economic by recovering valuable metals through engineered bioleaching bacteria. More information can be found in our project website http://www.biomining.ca/.     
• Line 40, "members of the Acidithiobacillus genus" better than known as Acidithiobacillus spp.
• Response: Revised.
• Line 50, remove "the" in front of 1940.
• Response: Removed.
• Line 88, pure strain is not a microbiological concept.
• Response: Revised.
• Line 91, why only ferrous iron and nickel were measure? In biomining resistance to other metal cations will be convenient.
• Response: The minerals we are working on is the pyrrhotite tailings from Sudbury, Canana, which contains iron and nickel. We explained the reason in Line 148-151.  
• Line 115, is not clear the variability in the genomes used for comparison used along the text. For instance for comparative genomics  four genomes are used and for Pan-Genome analysis nine genomes. Please clarify.
• Response: Among the nine genomes, only 4 genome sequences are complete (see Table 1). The other five genome sequences have many contigs that not suitable for genome alignment.  
• Line 136, Results and "dicussion"
• Response: Revised.
• Line 138, Acidithiobacillus genus, remove spp.
• Response: Revised.
• Line 138, use mainly
• Response: Revised.
• Line 147, clarify why tailings of Sudbury are important for this ms.
• Response: The Acidithiobacillus ferriduransJAGS was isolated from AMD of Sudbury and will be used for the AMD treatment and bioleaching of the pyrrhotite tailings from Sudbury for now. It’s the background of our research.
• Fig 1, ATCC is not a nomenclature for an isolate
• Response: Revised.
• Line 167, indirect tracking is not a good way to measure microbial growth.
• Response: Yes, we agree. The pH or Fe²⁺changes may be affected by other factors, so we only used these as auxiliary references.
• Line 170, which A. ferroxidans isolate?
• Response: Revised.
• Line 171, use "were" insted of "are"
• Response: Revised.
• Line 172, if precipitates are produced during growth, OD600 is not a good system to evaluate cell growth. 
• Response: The absorbance method (OD) was reported by other papers (Barron and Lueking, 1990; Li, et al. 2014). The cells were collected and washed to get rid of precipitatesbefore the OD test (see Line 104-106). Although the OD₆₀₀ method is not quite accurate to evaluate cell growth, but it is good for cell storage with known numbers.   
• Lines 189-192. The suggestion does not allow authors to change the published and accepted nomenclature for isolate IO-2C, this should be done using an appropiate microbial nomenclature change system. Thus authors should maintain the original species name for the isolate IO-2C along the text.
• Response: Many strains are misclassified due to some reasonable and unreasonable reasons. For example, ferriduransJCM18981 was named as A. ferrooxidans ATCC33020 in the past (Miyauchi et al. 2018). Actually, until now the name A. ferrooxidans ATCC33020 is still being misused which may mislead new researchers in this field. If we can use the proper name in our paper, it will help more researchers realize this mistake. In addition, another paper (Li et al. 2019) also points out this misclassified strain and used A. ferridurans IO-2C as its name.
• Line 202, with the results of Table 1 I do not understand how authors can talk about rich evolutionary history of A. ferridurans species.
• Response: This part was talking about Figure 2 not Table 1.
• Line 219, IO-2C canot be considered a A. ferridurans strain.
• Response: Our results support this rename and it was also supported by another research (Li et al. 2019, Comparative Genomic Analysis Reveals the Distribution, Organization, and Evolution of Metal Resistance Genes in the Genus Acidithiobacillus).
• Lines 232 and 233, I suggest to remove the hypothesis. Underlining the genomic differences between both species should be sufficient. 
• Response: Revised.
• Line 273, IO-2C is a A. ferrooxidans strain.
• 4, remove strain IO-2C from the A. ferridurans strains list.
• Response: We explained these above.
• Line 320, isolate JAGS has been compared only with one A. ferridurans strain and eight A. ferrooxidans strains. Knowing that there are ten recognized species in this genus, authors can not say that the isolate has been compared to other Acidithiobacillus strains but to selected A. ferroxidans strains. See also the concern of the reviewer about the selection of only one species (A. ferrooxidans) for genomic comparison.
• Response: The studied genomes were not manually selected but chosen by Similar Genome Finder service (Line 112-113).
• Line 324, clarify what authors mean by "intermediate species".
• Line 359 for "intermediate strain" 
• Response: ferriduransJAGS showed a closer evolutionary relationship with other A. ferridurans species, but a higher similarity of genomic structure with the A. ferrooxidans strains. What’s more, as shown in Figure 4, part of gene clusters in A. ferridurans JAGS is like other A. ferridurans strains and part is like A. ferrooxidans strains. During the bacterial evolution, the species A. ferrooxidans gained or lose genes and evolved into A. ferridurans JAGS, then gained, lose or rearranged its genome and formed a different A. ferridurans strain, so we guessed maybe A. ferridurans JAGS is a "intermediate species" between A. ferrooxidans and A. ferridurans.

Author Response File: Author Response.pdf

Reviewer 3 Report

Line 90 – “…or with 2:2 solid medium in an incubator at 30 °C…”

It is not clear what the 2:2 ratio refers to. Please clarify.

 

Lines 95-96 – “…and calculated by using consumed Fe2+ divided its initial concentration as in a previous study…”

 

What was calculated? Is it the Fe³⁺ formation?

 

 

Lines 101-102 “Three cell counting methods were tested, including direct cell counting, optical density (OD600) measurements, and plate counting.”

Should be:

“Three cell counting methods were tested: direct cell counting, optical density (OD600) measurements, and plate counting.”

 

 

Lines 124-125 - “were mapped into various COG categories and KEGG pathways”

What bioinformatic tools were used for this?

 

Line 147 - “there is 0.5–1% of Ni existing in the pyrrhotite tailings of Sudbury”.

Why the pyrrhotite tailings of Sudbury are mentioned?

 

Lines 247-248 - “In response to acidic heavy-metal stress, acidophiles have developed different genetic mechanisms to survive and is described by Mirete et al [36], which can be very complex [2,37].”

Shouldn’t it be like this?

“In response to acidic heavy-metal stress, acidophiles have developed different genetic mechanisms to survive, which can be very complex [2,36,37].”

 

Line 256 – “The mechanisms responsible for acid stress resistance are complex.”

This sentence should be deleted; this idea is already stated in lines 247-248.

 

Lines 256-257 – “We tried to find relevant genes in A. ferridurans JAGS and listed them in Table S2 (Supplementary Material).”

Shouldn’t it be?

“We tried to find relevant genes responsible for acid stress resistance in A. ferridurans JAGS and listed them in Table S2 (Supplementary Material).”

Moreover, it is not clear what was the strategy or method used to search for the so-called relevant genes. This information must be added to the manuscript.

 

Lines 280-281 – "As shown in Figure 4 and Table S2, genes involved in heavy metal resistance pathways in A. ferridurans JAGS were analyzed.”

Shouldn’t it be?

“We also tried to find genes involved in heavy metal resistance pathways in A. ferridurans JAGS (Figure 4 and Table S2).”

Again, it is not clear what was the strategy or method used to search for these genes,

 

 

 

Author Response

Reviewer3

 

1. Line 90– “…or with 2:2 solid medium in an incubator at 30 °C…” It is not clear what the 2:2 ratio refers to. Please clarify.

Response: The name “2:2 solid medium” was created by former researchers (Peng et al, 1994). I guess 2:2 may be the concentrations of FeSO₄ * 7H₂0 (2g/L) and Na₂S₂0₃* 5H₂0 (2 g/L). 

2. Lines 95-96– “…and calculated by using consumed Fe2+ divided its initial concentration as in a previous study…” What was calculated? Is it the Fe3+ formation?

Response: Revised. The ferrous iron oxidation rate was calculated. We revised this sentence to make it clear (Line 97-98).

3. Lines 101-102“Three cell counting methods were tested, including direct cell counting, optical density (OD600) measurements, and plate counting.” Should be: “Three cell counting methods were tested: direct cell counting, optical density (OD600) measurements, and plate counting.”

 Response: Revised. 

4. Lines 124-125- “were mapped into various COG categories and KEGG pathways”, What bioinformatic tools were used for this?

 Response: Bacterial Pan-genome Analysis tool (BPGA ver. 1.2) (Line 122-124). 

5. Line 147- “there is 0.5–1% of Ni existing in the pyrrhotite tailings of Sudbury”. Why the pyrrhotite tailings of Sudbury are mentioned?

Response: The Acidithiobacillus ferridurans JAGS was isolated from AMD of Sudbury and will be used for the AMD treatment and bioleaching of the pyrrhotite tailings from Sudbury for now. It’s the background of our research.  

6. Lines 247-248- “In response to acidic heavy-metal stress, acidophiles have developed different genetic mechanisms to survive and is described by Mirete et al [36], which can be very complex [2,37].” Shouldn’t it be like this? “In response to acidic heavy-metal stress, acidophiles have developed different genetic mechanisms to survive, which can be very complex [2,36,37].”

Response: Thank you. Revised accordingly. 

7. Line 256– “The mechanisms responsible for acid stress resistance are complex.” This sentence should be deleted; this idea is already stated in lines 247-248. 

Response: We hope we can keep it here as this sentence states the point of this paragraph.  

8. Lines 256-257– “We tried to find relevant genes in A. ferridurans JAGS and listed them in Table S2 (Supplementary Material).”

Shouldn’t it be?  “We tried to find relevant genes responsible for acid stress resistance in A. ferridurans JAGS and listed them in Table S2 (Supplementary Material).” Moreover, it is not clear what was the strategy or method used to search for the so-called relevant genes. This information must be added to the manuscript.

Response: I kept the sentence “The mechanisms responsible for acid stress resistance are complex”, so adding “responsible for acid stress resistance” will make the sentence a little repetitive. I checked these genes manually by referring to relevant information in other reviews or research articles.

9. Lines 280-281– "As shown in Figure 4 and Table S2, genes involved in heavy metal resistance pathways in A. ferridurans JAGS were analyzed.” Shouldn’t it be? “We also tried to find genes involved in heavy metal resistance pathways in A. ferridurans JAGS (Figure 4 and Table S2).” Again, it is not clear what was the strategy or method used to search for these genes.

Response: Thank you, revised accordingly. 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

There are only a few minor revisons beining done. I still think that the manuscript lacks novelty related to biomining and is out of scope for the journal.

Author Response

Reviewer 1

The manuscript describes the genome, its comparison to other microorganisms and very few physiological properties of a strain of the acidophilic bacterium Acidithiobacillus ferridurans. Because of these reasons the manuscript is not recommended for publication:

1. Out of scope. This is a pure microbiological study without any minerals in the study. The only link to minerals is that the microorganism is used for bioleaching. The study does not fit in the journal “Minerals”.

Response: This manuscript was submitted for the special issue “Novel and Emerging Strategies for Sustainable Mine Tailings and Acid Mine Drainage Management”. With all reviewers’ suggestions, we revised the introduction part to make the connection of this work to this journal and the special issue more clear (P1L 28-P2L52).

 

2. Lack of novelty. There are hundreds of strains of Acidithiobacillus species being isolated worldwide. This is just one more. Genomic features are interesting but do alone not justify any conclusions about adaptation to acid mine drainage. By the way all Acidithiobacillus species are adapted to acid mine drainage. The physiological growth data are weak and do no allow any conclusions about adaptation or comparison to other strains/species. Ferrous iron oxidation was just determined in shake flasks by measuring Fe(II) and Fe(III) concentrations, and ferrous iron concentration is just given as one percentage value in comparison to one other strain. This simple approach does not take into account changing physiologcial conditions such as pH and does not allow to determine Fe(II) oxidation rates as stated in the text.

Response: We summarized our previous work and described the importance and novelty of this study at P2L81-89. A brand new strain was studied, we analyzed its genome information, which is the second genome information of species A. ferridurans in addition to A. ferridurans JCM18981. The A. ferridurans JAGS dominates the enriched consortium isolated from AMD and might be a great candidate for bioleaching. Through genomic analysis of A. ferridurans JAGS, we were able to better understand what were the differences between species A.ferridurans and A.ferrooxidans, how they evolved to adapt to AMD and which genetic features might contribute to its growth advantage, and then provided insights for future studies, like genetic engineering strategies to improve Acidithiobacillus strains for bioleaching. In a word, our work not only provides genomic information to better understand the new species A. ferridurans and its evolutionary relationship with species A. ferrooxidans, but also shares valuable genetic information with researchers in this field that might be useful to engineer bioleaching bacteria.  

Author Response File: Author Response.docx

Reviewer 2 Report

Ms has been improved. Actual version is publishable.

Author Response

Reviewer2

The ms "Genomic and experimental analyses of a newly isolated Acidithiobacillus ferridurans JAGS strain reveals its adaptation to acid mine drainage" by Chen et al is an interesting microbiological work, although I do not see any close relation with minerals with the exception of the AMD from which was isolated. In any case authors will need to explain the selection of the genomes used for the analysis and perform an extensive editing of the microbial nomenclature before it could be accepted for publication. 

Response: This manuscript was submitted for the special issue “Novel and Emerging Strategies for Sustainable Mine Tailings and Acid Mine Drainage Management”. With reviewers’ suggestions, we revised the introduction part to make the connection of this work to this journal and the special issue more clear (P1L29-P2L47). Bioleaching is a novel strategy to recover minerals from tailings and acid mine drainage as well as AMD treatment. This study focused on the genomic analysis of an isolated strain A. ferridurans JAGS from AMD, which is a dominate strain (accounting for 92.6% of the enriched culture based on 16S rRNA gene sequence analysis) and has a great candidate for bioleaching (P2L80-83). It provides information for researchers to better understand how it evolved and which genetic features might contribute to its growth advantage, and then provides insights for future studies, like genetic engineering strategies to improve bioleaching bacteria. We improved the microbial nomenclature in the text according to your great suggestions. We have thoroughly considered all the comments and tried our best to address all the concerns raised. We hope with your suggestions and our efforts, the revised manuscript is good for publication.

 

Comments and suggestions:

1. Title: the presented work is mainly genomic, I suggest to remove the experimental analysis from the title.

Response: Thanks for your suggestion. The title has been revised accordingly.

2. All long the text. Authors use JAGS as a familiar nomenclature for the A. ferridurans isolate all along the text. From a microbiological point of view this is incorrect. I think in short could be mentioned as "isolate JAGS", although once in a while it will be good to use the correct name "A. ferridurans JAGS". 

Response: Thanks for your professional advice. We improved our manuscript according your suggestion.

3. Abstract, see the comment above. Line 19: analyzed A. ferroxidans species. Line 25, I do not think that the ms deals with AMD biotreatments thus I suggest to remove it from the abstract.

Response: We revised the abstract according to your suggestions. We hope to keep “AMD biotreatment” because dealing with the AMD is one of the most important goals of our research. From genome sequencing, genomic analysis, and then genome editing, our final goals are to stabilize mine wastes to prevent acid mine drainage and make the recovery process economic by recovering valuable metals through engineered bioleaching bacteria. More information can be found in our project website http://www.biomining.ca/.     

4. Line 40, "members of the Acidithiobacillus genus" better than known as Acidithiobacillus spp.

Response: We revised the whole sentence as “Acidithiobacillus is a group of Gram-negative, chemoautotrophic, acidophilic aerobes, which dominates in all types of extremely acidic habitats, suggesting its tremendous potential in bioleaching process ”

5. Line 50, remove "the" in front of 1940.

Response: Removed.

6. Line 88, pure strain is not a microbiological concept.

Response: We removed “pure”.

7. Line 91, why only ferrous iron and nickel were measure? In biomining resistance to other metal cations will be convenient.

Response: The minerals we are working on is the pyrrhotite tailings from Sudbury, Canana, which contains iron and nickel. We explained the reason in Line 148-151.  

8. Line 115, is not clear the variability in the genomes used for comparison used along the text. For instance for comparative genomics  four genomes are used and for Pan-Genome analysis nine genomes. Please clarify.

Response: Among the nine genomes, only 4 genome sequences are complete (see Table 1). The other five genome sequences have many contigs that not suitable for genome alignment.  

9. Line 136, Results and "dicussion"

Response: We added “discussion”.

10. Line 138, Acidithiobacillus genus, remove spp.

Response: We removed “spp.”.

11. Line 138, use mainly

Response: We added “mainly”.

Line 147, clarify why tailings of Sudbury are important for this ms.

Response: The Acidithiobacillus ferridurans JAGS was isolated from AMD of Sudbury and will be used for the AMD treatment and bioleaching of the pyrrhotite tailings from Sudbury for now. It’s the background of our research.

12. Fig 1, ATCC is not a nomenclature for an isolate

Response: We corrected JAGS and ATCC to “A. ferridurans JAGS” and “A. ferrooxidans ATCC23270”, respectively.

13. Line 167, indirect tracking is not a good way to measure microbial growth.

Response: Yes, we agree. The pH or Fe²⁺ changes may be affected by other factors, so we only used these as auxiliary references.

14. Line 170, which A. ferroxidans isolate?

Response: We added “ATCC23270”.

15. Line 171, use "were" insted of "are"

Response: We changed “are” to “were”.

16. Line 172, if precipitates are produced during growth, OD600 is not a good system to evaluate cell growth. 

Response: The absorbance method (OD) was reported by other papers (Barron and Lueking, 1990; Li, et al. 2014). The cells were collected and washed to get rid of precipitates before the OD test (P3L121-123). Although the OD₆₀₀ method is not quite accurate to evaluate cell growth, but it is good for cell storage with known numbers.   

17. Lines 189-192. The suggestion does not allow authors to change the published and accepted nomenclature for isolate IO-2C, this should be done using an appropiate microbial nomenclature change system. Thus authors should maintain the original species name for the isolate IO-2C along the text.

Response: Many strains are misclassified due to some reasonable and unreasonable reasons. For example, A. ferridurans JCM18981 was named as A. ferrooxidans ATCC33020 in the past (Miyauchi et al. 2018). Actually, until now the name A. ferrooxidans ATCC33020 is still being misused which may mislead new researchers in this field. If we can use the proper name in our paper, it will help more researchers realize this mistake. In addition, another paper (Li et al. 2019) also points out this misclassified strain and used A. ferridurans IO-2C as its name.

18. Line 202, with the results of Table 1 I do not understand how authors can talk about rich evolutionary history of A. ferridurans species.

Response: This part was talking about Figure 2 not Table 1.

19. Line 219, IO-2C canot be considered a A. ferridurans strain.

Response: Our results support this rename and it was also supported by another research (Li et al. 2019, Comparative Genomic Analysis Reveals the Distribution, Organization, and Evolution of Metal Resistance Genes in the Genus Acidithiobacillus).

20. Lines 232 and 233, I suggest to remove the hypothesis. Underlining the genomic differences between both species should be sufficient. 

Response: The hypothesis was removed.

21. Line 273, IO-2C is a A. ferrooxidans strain. 4, remove strain IO-2C from the A. ferridurans strains list.

Response: We explained these above.

22. Line 320, isolate JAGS has been compared only with one A. ferridurans strain and eight A. ferrooxidans strains. Knowing that there are ten recognized species in this genus, authors can not say that the isolate has been compared to other Acidithiobacillus strains but to selected A. ferroxidans strains. See also the concern of the reviewer about the selection of only one species (A. ferrooxidans) for genomic comparison.

Response: The studied genomes were not manually selected but chosen by Similar Genome Finder service (P3L129-131).

23. Line 324, clarify what authors mean by "intermediate species".Line 359 for "intermediate strain" 

Response: A. ferridurans JAGS showed a closer evolutionary relationship with other A. ferridurans species, but a higher similarity of genomic structure with the A. ferrooxidans strains. What’s more, as shown in Figure 4, part of gene clusters in A. ferridurans JAGS is like other A. ferridurans strains and part is like A. ferrooxidans strains. During the bacterial evolution, the species A. ferrooxidans gained or lose genes and evolved into A. ferridurans JAGS, then gained, lose or rearranged its genome and formed a different A. ferridurans strain, so we guessed maybe A. ferridurans JAGS is a "intermediate species" between A. ferrooxidans and A. ferridurans.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors have revised the manuscript according to my suggestions and in some few cases of sentences they have preferred to keep the original form, but they have given appropriate justifications. In addition, they have answered to all my questions and my doubts were clarified. Therefore, I think the paper can now be published and it will add information to the state of the art of this issue.

Author Response

Reviewer3

 

1. Line 90– “…or with 2:2 solid medium in an incubator at 30 °C…” It is not clear what the 2:2 ratio refers to. Please clarify.

Response: The name “2:2 solid medium” was created by former researchers (Peng et al, 1994). I guess 2:2 may be the concentrations of FeSO₄ * 7H₂0 (2g/L) and Na₂S₂0₃* 5H₂0 (2 g/L). 

2. Lines 95-96– “…and calculated by using consumed Fe2+ divided its initial concentration as in a previous study…” What was calculated? Is it the Fe3+ formation?

Response: Revised. The ferrous iron oxidation rate was calculated. We revised this sentence to make it clear (P2L111-112).

3. Lines 101-102“Three cell counting methods were tested, including direct cell counting, optical density (OD600) measurements, and plate counting.” Should be: “Three cell counting methods were tested: direct cell counting, optical density (OD600) measurements, and plate counting.”

 Response: We revised this sentence as you suggested. 

4. Lines 124-125- “were mapped into various COG categories and KEGG pathways”, What bioinformatic tools were used for this?

 Response: Bacterial Pan-genome Analysis tool (BPGA ver. 1.2) (P3L139). 

5. Line 147- “there is 0.5–1% of Ni existing in the pyrrhotite tailings of Sudbury”. Why the pyrrhotite tailings of Sudbury are mentioned?

Response: We revised the sentence on P3L100-102. The Acidithiobacillus ferridurans JAGS was isolated from AMD of Sudbury and will be used for the AMD treatment and bioleaching of the pyrrhotite tailings from Sudbury for now. It’s the background of our research.  

6. Lines 247-248- “In response to acidic heavy-metal stress, acidophiles have developed different genetic mechanisms to survive and is described by Mirete et al [36], which can be very complex [2,37].” Shouldn’t it be like this? “In response to acidic heavy-metal stress, acidophiles have developed different genetic mechanisms to survive, which can be very complex [2,36,37].”

Response: Thank you. We revised this sentence as you suggested.  

7. Line 256– “The mechanisms responsible for acid stress resistance are complex.” This sentence should be deleted; this idea is already stated in lines 247-248. 

Response: We hope to keep it here as this sentence states the point of this paragraph.  

8. Lines 256-257– “We tried to find relevant genes in A. ferridurans JAGS and listed them in Table S2 (Supplementary Material).”

Shouldn’t it be?  “We tried to find relevant genes responsible for acid stress resistance in A. ferridurans JAGS and listed them in Table S2 (Supplementary Material).” Moreover, it is not clear what was the strategy or method used to search for the so-called relevant genes. This information must be added to the manuscript.

Response: We hope to keep the sentence “The mechanisms responsible for acid stress resistance are complex”. If we add “responsible for acid stress resistance”, it will make the sentence a little repetitive. I checked these genes manually by referring to relevant information in other reviews or research articles.

9. Lines 280-281– "As shown in Figure 4 and Table S2, genes involved in heavy metal resistance pathways in A. ferridurans JAGS were analyzed.” Shouldn’t it be? “We also tried to find genes involved in heavy metal resistance pathways in A. ferridurans JAGS (Figure 4 and Table S2).” Again, it is not clear what was the strategy or method used to search for these genes.

Response: Thank you, we revised this sentence as you suggested.  

Author Response File: Author Response.docx