Review Reports - Identification and Quantification of Lipopeptide Homologues Induced and Produced by Bacillus amyloliquefaciens

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study identified and quantified homologues of LPs biosynthesized by Bacillus amyloliquefaciens by inducing the synthesis of these secondary metabolites using different inducers. The manuscript organized well and the work is meaningful in this field. However, revisions needed to be performed before accepted for publication.

Main Concerns:
(1) How about the bio-activity of obtained secondary metabolites?

(2) In the discussion part, authors should analyze why this inducers work and

(3) Which inducers might be the most promising applied in the industry?

(4) Line 519, 53 should be removed.

Author Response

"Please see the attachment"

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In there manuscript Ley-Lopez et al. describe the identification of different lipopeptide homologoues in B. amyloliquefaciens. The results seem to be a start point for the investigation of lipopeptide production in this organism. Hence, I think it is not necessary to evaluate the bioactivity of the compounds, a concentration dependency of the inducers or a deeper investigation on the trigger within the inactivated cells that leads to induction of lipopeptide production in this study. Nevertheless, I have some comments that should be addressed to improve the paper.

Major comments:

Fig. 1: What is the difference between the 4 spectra? Have these single peaks already been isolated from the crude extract? How does the spectra from the real crude extract look like? (A spectra could be added to the supplementaries) Where does the 2^nd peak in the 3^rd spectrum (6.33 minutes) comes from? Please explain.

Fig. 2: Please add information in the figure and/or the figure legend on which of the isolated peaks resulted in which of these spectra. Moreover, an identification of all peaks (e.g. in a supplementary table) should be provided to verify the presence of the different substances. The same is true for the compounds in Fig. 4 and 6.

Fig. 1-6: What is the set point for 100 % of the Y-axis in these diagrams?

Tables 3-5: The letters behind the values are confusing. At least the letters should be written in superscript Nevertheless, a colored bar graph would be more descriptive. There is also no explanation about the letters given in the figure legend.

Line 266ff: Is there any explanation why most of the inducers, except inactivated cells, suppress production of bioactive compounds? Please explain.

The authors should try to avoid pagination for tables and figure legends.

Line 309ff: This part of the discussion should be placed more in context of the obtained results.

Line 361: Neither cellulose nor chitin or iron resulted in significant induction of production of any of the antimicrobial compounds. And glutamic acid induces only production of a single surfactin (even though this is highlighted in the table as insignificant). The only real inducer are the heat inactivated fungi.

Minor comments

General: Instead of using µg *mg^-1 I would suggest to use mg^-1 as this is a more common unit for the specific yield.

Line 15 : Instead of using “because” two sentences should be used.

Line 65: “These are some of the structural and biological characteristics of” can be removed.

Line 115: The table caption is in Spanish. Please change.

Line 211: There is a “-“ missing between Tyr and Ile in the fengycin A sequence.

Tables 3-5: The values should be rounded meaningfully. The determination of the masses is not nearly accurate enough to justify three decimal places.

Line 281-286: The font seems to be smaller than in the rest of the paper.

Author Response

"Please see the attachment"

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors responded adequately to most of my comments, which improved the comprehensibility and quality of the work. However, some of my comments have not been answered satisfactorily so far, so I still have some points to make.

1. In the first revision round I ask for identification of the labelled peaks in the spectra presented in Fig. 2, 4 and 6 as part of a supplementary figure or table. In deed some of the peaks are listed as "fragments" in table 2, however, listing fragments in a table is not identification of a fragment. Identifcation would mean to state which mass correspondes to which ion that could be generated from the proposed substance. It also means that All relevant peaks in a spectrum must be identified and assigned to the suspected substance. Moreover, not all peaks from the spectra can be found in table 2 and on the other hand some of the masses designated as fragments in table 2 are no actual fragments but isotope peaks (e.g. 1436.7892 is an isotope peak of the intact molecule with an exact mass of 1435.78). These isotope peaks should be removed from table 2.

Tables 3-5: As I have said before, I believe that the determination of the amounts of synthesised LP (as opposed to the determination of the atomic mass of a substance) is far from accurate enough to justify three decimal numbers and should therefore be meaningfully rounded (e.g. to one decimal number). I am well aware that in the case of fengycine, relatively small quantities were produced. Here, one could use the unit ng/mg instead of µg/mg. The use of rounded values would also have the advantage that Table 4 would fit on the page without reducing the font size. In addition, I still think that a column chart as an illustration would help to clarify the data.

Instead of using µg*mg-1 I would suggest to use mg*g-1. Espacially for larger scale production using mg*g is a more common unit for the specific yield.

Line 347: Please find a synonym for "feel".

Now Line 393 (prev. Line 361): This sentence has not been corrected. In this study homologues of different LPs have been identified and quantified. This is correct. However, these LPs are not produced upon the mentioned stresses as neither cellulose nor chitin or iron resulted in production of any of the compounds. Hence in this study different inducers for LP production have been tested and apart from one single surfactin that was induced by glutamic acid only inactivated fungi were found to be an inducer of LP production. This should be stated clearly.

Author Response

Reviewer 2

Comments and Suggestions for Authors

In the first revision round I ask for identification of the labelled peaks in the spectra presented in Fig. 2, 4 and 6 as part of a supplementary figure or table. In deed some of the peaks are listed as "fragments" in table 2, however, listing fragments in a table is not identification of a fragment. Identifcation would mean to state which mass correspondes to which ion that could be generated from the proposed substance. It also means that All relevant peaks in a spectrum must be identified and assigned to the suspected substance. Moreover, not all peaks from the spectra can be found in table 2 and on the other hand some of the masses designated as fragments in table 2 are no actual fragments but isotope peaks (e.g. 1436.7892 is an isotope peak of the intact molecule with an exact mass of 1435.78). These isotope peaks should be removed from table 2.

We are grateful for the reviewer's valuable suggestions and believe that we have improved the quality of the manuscript.

Response.

Three tables with information requested by the reviewer were added in supplementary materials, each table indicating the characteristics of the detected lipopeptide homologues of each family.

Instead of using µg*mg-1 I would suggest to use mg*g-1. Espacially for larger scale production using mg*g is a more common unit for the specific yield.

Response.

In accordance with the reviewer's suggestions, we decided to round to one decimal place the amounts obtained for the homologues of bacillomycin (table 3) and surfactin (table 5). In the case of table 4, the reviewer's proposal was considered and the unit was changed from "µg*mg^-1" to "ng*mg^-1".

Line 347: Please find a synonym for "feel".

Response.

the word "feel" was changed to "are".

Response.

Line 366 (formerly 393) was corrected, in line with the reviewer's suggestions.

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

I appreciate the authors efforts to improve the manuscript. The manuscript has improved and I have only a few minor comments.

1. Line 368: I would start this sentence with "In contrast, the antagonistic bacterium..."

Table 2: In the table some numbers are in bold (most likely the parent ions). this should be mentioned in the caption of the table.

Tables 3-5: I appreciate rounding of the numbers in these tables as this improved readability of the values. However, not only the values but also the deviations can be rounded. Moreover, in Table 4 the values have been switched to ng/mg but the deviations have not been switched to ng/mg but are still in µg/mg. This should be corrected and also rounded.

Supplementary tables: How did you identify these ions? The annotations in these tables seem to be incorrect.

In Table S3 the 227.1 Peak is present independent from the length of the fatty acid. Hence I would expect that this is a dipeptide rather than a part from the fatty acid. In contrast the peaks 754.4, 768.4, 782.4, 796.4 and 978.49, 992.5, 1007.5, 1020.5 roughly increase with 14 Da which seems to be the result of an increase in lenght of the fatty acid rather than the mentioned peptides. Especially the 978.5-1020.5 row is hardly the result of a Ser-Thr Dipeptide that has a molecular mass of about 206 Da. It is also very unlikely that the same ion results in two peaks (see table S1 & S2). The authors should try to explain these peaks or mention which loss (H2O, NH3...) resulted in these peaks.

Author Response

Reviewer 3

Comments and Suggestions for Authors

I appreciate the authors efforts to improve the manuscript. The manuscript has improved and I have only a few minor comments.

We appreciate the reviewer's valuable comments and criticisms, which have allowed us to improve this manuscript.

Line 368: I would start this sentence with "In contrast, the antagonistic bacterium..."

Response.

We have changed the sentence and started with the reviewer's recommended sentence.

Table 2: In the table some numbers are in bold (most likely the parent ions). this should be mentioned in the caption of the table.

Response.

Parent ions were incorporated into the title of table 2.

Response.

The reviewer's observation was considered and the standard deviation values in tables 3 and 5 were rounded to two decimal places. Also, the standard deviation values in table 5 were rounded and corrected from µg/mg to ng/mg.

Supplementary tables: How did you identify these ions? The annotations in these tables seem to be incorrect.

Response.

With regard to the comments made, we would like to express our appreciation for the reviewer's observation regarding the 227 ion, which could correspond to a dipeptide. After a thorough analysis, we confirmed that the reviewer was right, and the 227 ion is related to [Pro-Glu] dipeptides. We have made the necessary corrections and incorporated these possible compounds into our study.

In this context, we have clarified the relationship between the 754.43 ion and the 997.50 ion in the same spectrum, attributing it to the loss of the [Pro-Glu] dipeptide and a water molecule (H₂O). The calculation is based on the actual difference between the two values, i.e. 997.50 - 226 ([Pro-Glu]) - 18 (H₂O) = 753 + H.

It is important to note that we have included explanations for losses of water (H₂O) or methyl groups (CH₃) in the manuscript, as described in the supplementary material. We believe that these clarifications and corrections significantly strengthen the content of the manuscript, fulfilling the necessary requirements for the achievement of the objectives set out in this work. Furthermore, we believe that the detailed characterisation of the structure of each homologue will be addressed in future manuscripts, for further research purposes.

Thank you very much for your time and consideration.