Selenocysteine Formation by Enterococcus faecium ABMC-05 Follows a Mechanism That Is Not Dependent on Genes selA and selD but on Gene cysK

Round 1

Reviewer 1 Report

In this experiment, the authors reported the presence of cysK gene but not SelA and SelD genes in Enterococcus faecium ABMC-05 isolated from a traditional Mexican fermented beverage. The work throws some lights on the mechanism underlying the Sec production

independent of the SelA and SelD pathways. However, there are some issues required to be addressed, and some of them are as follows:

1.     The sentence of the article is smooth, but there are still some problems with unclear descriptions, such as "absorption degree and biological conversion rate of selenocysteine" and "polymerase chain reaction (PCR) amplification determination by plasmid and chromosome DNA" in the abstract section.

2.     The diagram in the manuscript is not clear enough, so it is suggested to re-draw the diagram and adjust the clarity to facilitate reading, such as the ICONS in Figure 1, Figure 3 and Figure 5.

3.     There are many sentence errors in the article, such as CMI error in line 333, which is changed to MIC. Please check the sentence carefully to ensure the accuracy of abbreviations.

4.     Since there are less data in the chart of the article, supplementary data should be considered for support, such as Part 3.2.

5.     The writing logic of the article is not clear and coherent enough, and the language needs polishing. For example, "This critical point was 102 mg/L using the same graphical methodology" at line 342 is not clearly explained.

6.     In part 3.3, it is necessary to confirm that the strain used in this experiment has the activity of enzymes that reduce selenite to hydrogen selenide, so as to explain the tolerance to sodium selenite.

7.     Whether PCR amplification can completely show that the strain does not contain SelA and SelD genes, and whether other methods are needed to assist verification of the explanation remains to be considered. How about the possible isozymes for SelA and SelD with low homology?

Some sentences require to be polished.

Author Response

In this experiment, the authors reported the presence of cysK gene but not SelA and SelD genes in Enterococcus faecium ABMC-05 isolated from a traditional Mexican fermented beverage. The work throws some lights on the mechanism underlying the Sec production independent of the SelA and SelD pathways. However, there are some issues required to be addressed, and some of them are as follows:

The sentence of the article is smooth, but there are still some problems with unclear descriptions, such as "absorption degree and biological conversion rate of selenocysteine" and "polymerase chain reaction (PCR) amplification determination by plasmid and chromosome DNA" in the abstract section.

The phrase “absorption degree” was changed to “The concentration of selenium absorbed”. Line 25
The phrase “biological conversion rate of selenocysteine” was not found anywhere in the paper.
The sentence “polymerase chain reaction (PCR) amplification determination by plasmid and chromosome DNA” was rewritten to give clarity to the information. Lines 28-29

The diagram in the manuscript is not clear enough, so it is suggested to re-draw the diagram and adjust the clarity to facilitate reading, such as the ICONS in Figure 1, Figure 3 and Figure 5.

The phylogenetic tree was changed and Figures 3 and 5 were improved, according to reviewer suggestion

There are many sentence errors in the article, such as CMI error in line 333, which is changed to MIC. Please check the sentence carefully to ensure the accuracy of abbreviations.

The article was revised and the necessary changes were made in the abbreviations and acronyms. CMI was changed to MIC. Line 353

Since there are less data in the chart of the article, supplementary data should be considered for support, such as Part 3.2.

Data considered for supplementary material has been uploaded. However, the file of the supplementary table is disposable for the reviewer.

The writing logic of the article is not clear and coherent enough, and the language needs polishing. For example, "This critical point was 102 mg/L using the same graphical methodology" at line 342 is not clearly explained.

The sentence was rewritten to clarify the information and justify the study with the three different concentrations of sodium selenite. Lines 361-362

In part 3.3, it is necessary to confirm that the strain used in this experiment has the activity of enzymes that reduce selenite to hydrogen selenide, so as to explain the tolerance to sodium selenite.

As explained in the last paragraph of section 3.3, no studies were performed to determine the presence of this type of enzyme. The objective of the study was to identify an alternate route of selenocysteine ​​production not dependent on SelA and SelD, which was demonstrated with all the results obtained and with the identification of the cysK gene. It is important to determine the presence of glutathione (GSH) not only for the reduction to hydrogen selenide but also for the reduction of selenate to selenite since this enzyme participates in both processes. Thus, a perspective of the study in this sense has been placed in the conclusion. Lines 512-515

Whether PCR amplification can completely show that the strain does not contain SelA and SelD genes, and whether other methods are needed to assist verification of the explanation remains to be considered. How about the possible isozymes for SelA and SelD with low homology?

The reviewer's suggestion is correct and should be taken into account for future studies. However, there are no reports that give a precise explanation of the presence of isoenzymes with low homology related to SelD and SelA. That is why two sentences were added at the end of the results and discussions section that account for this fact. Lines 512-517

Reviewer 2 Report

As known, inorganic Se like selenate and selenite is often toxic. Inorganic selenium can be transformed to organic selenium by microorganism, such as LAB. The author introduced an Enterococcus faecium ABMC-05, which can accumulate Selenium in cells. As previous reports, selA and selD are main genes for selenocysteine in LAB. However, there is no selA and selD genes in ABMC-05. The study reported a new pathway (CysK) in ABMC-05 to synthesize selenocysteine. It has important research significance on the biotransformation of inorganic selenium into organic selenium, which can decrease toxicity. However, there are some doubts still need to answer and improve in this study.

1. The title should be improved. As description in the paper, cysK gene is the main pathway for selenocysteine synthesis. It should be expressed. And the introduction about cysK gene should be added in the part of “1. Introduction” and the explanation of differences about selA, selD and cysK. The highlight of this paper is not only independence of selA and selD, it should include the dependence of cysK.

2. There are some spelling mistakes, as line 141, it should be “Amplification of the 16S rDNA gene”. Another, capital/lower-case and italics should be also checked in the text. For example, Line 241, the “cysk” should be “cysK”.

3.The full name of “Enterococcus faecium” in the text only need appear in the first time, and the following ones should be abbreviation “E. faecium”.

4. Figure 1a and Figure 1b are not clear. The clearer pictures should be provided.

5. Table 2, there is no difference significance analysis of the content of “Selenium in cells”. Please give the explanation. Or add the detailed information.

 Minor editing of English language required

Author Response

As known, inorganic Se like selenate and selenite is often toxic. Inorganic selenium can be transformed to organic selenium by microorganism, such as LAB. The author introduced an Enterococcus faecium ABMC-05, which can accumulate Selenium in cells. As previous reports, selA and selD are main genes for selenocysteine in LAB. However, there is no selA and selD genes in ABMC-05. The study reported a new pathway (CysK) in ABMC-05 to synthesize selenocysteine. It has important research significance on the biotransformation of inorganic selenium into organic selenium, which can decrease toxicity. However, there are some doubts still need to answer and improve in this study.

The title should be improved. As description in the paper,cysK gene is the main pathway for selenocysteine synthesis. It should be expressed. And the introduction aboutcysK gene should be added in the part of “1. Introduction” and the explanation of differences about selA, selD and cysK. The highlight of this paper is not only independence of selA and selD, it should include the dependence of cysK.

The title was changed according to the reviewer's suggestion.
One more reference was added to explain the importance of the presence of CysK in the biotransformation of selenium to selenocysteine. In this way, further explanation is given to the differences between the traditional route and the route in which CysK is involved.

There are some spelling mistakes, as line 141, it should be “Amplification of the 16S rDNA gene”. Another, capital/lower-case and italics should be also checked in the text. For example, Line 241, the “cysk” should be “cysK”.

The article was revised, and all the errors of lack of italics and combination of upper and lower case letters were reviewed and corrected where appropriate.

The full name of “Enterococcus faecium” in the text only need appear in the first time, and the following ones should be abbreviation “E. faecium”.

The corrections requested by the reviewer were made according to suggestions, highlighting all changes.

Figure 1a and Figure 1b are not clear. The clearer pictures should be provided.

Other visually clearer ones replaced the figures.

Table 2, there is no difference significance analysis of the content of “Selenium in cells”. Please give the explanation. Or add the detailed information.

Results of statistical analysis were plotted to denote significant differences in selenium concentration in cells. Placed id on table

Reviewer 3 Report

I revised the manuscript in titled (Selenocysteine formation by Enterococcus faecium ABMC-05 2 follows a mechanism not dependent on genes selA and selD), it contains very interesting information and a I have some suggestions to improve it

- Line 62, Never start with abbreviation

- Lin 74-76, re-write the aim of work in clear sentence to describe the importance of the manuscript and what is the scientific addition

- Line 89, (Add manufacture, town, country) in all chemical, solvents, apparatus and machine

- Lines 118, 121, 127, At which centrifuge (Add manufacture, town, country)

- Lines 156, 157, 161, 166, 176, 186, 190, 195, 197, 226 (Add manufacture, town, country)

- Line 186, determined by what

- Line 226, Which standard used

- In discussion section add updated references at lat two years

- Line 327, How GSH protect the cell (add sentence and updated reference)

- In conclusion section, Add a sentence to clarify the future vision of this work and who will benefit from it

Minor editing of English language required

Author Response

I revised the manuscript in titled (Selenocysteine formation by Enterococcus faecium ABMC-05 2 follows a mechanism not dependent on genes selA and selD), it contains very interesting information and a I have some suggestions to improve it

Line 62, Never start with abbreviation

In line 62 the origin of the recommendation was not found. The entire article was reviewed and no sentences beginning with an abbreviation were found

Lin 74-76, re-write the aim of work in clear sentence to describe the importance of the manuscript and what is the scientific addition

A sentence was added that completes the information required to identify the importance of the study and its contribution to science. Lines 83-85

Line 89, (Add manufacture, town, country) in all chemical, solvents, apparatus and machine

All reagents and materials were reviewed, inconsistencies were completed or corrected

Lines 118, 121, 127, At which centrifuge (Add manufacture, town, country)

Lines 156, 157, 161, 166, 176, 186, 190, 195, 197, 226 (Add manufacture, town, country)

Missing information was placed in each of the comments indicated by the reviewer

Line 186, determined by what

A previous sentence specifies that the study is done on biomass. We decided to leave the sentences intact because it would seem like redundant wording. Lines 196-198

Line 226, Which standard used

The standard used is specified in the carboxymethylation section. The conditions for preparing both the standard and the sample are specified. The authors do not consider placing the information again in this section because it would be redundant.

In discussion section add updated references at lat two years

It was added 5 different and current references were according to the discussion

Line 327, How GSH protect the cell (add sentence and updated reference)

The authors consider that within the article, there is sufficient information with which it is understood that the detoxification process is how the cell protects itself from the damage caused by the presence of selenium and that GSH is involved as a factor. main in the process of reducing selenate to selenite and from there to hydrogen selenide and elemental selenium. In any case, this information has been added again in one sentence, and a reference in the introduction was used. Lines 332-336

In conclusion section, Add a sentence to clarify the future vision of this work and who will benefit from it

Two sentences were added specifying the job prospects, benefits and future uses of organic selenium. Lines 512-517

Round 2

Reviewer 3 Report

The authors made all suggestions and the manuscript is fit for publication