Transcriptional Regulation of the Genes Encoding Branched-Chain Aminotransferases in Kluyveromyces lactis and Lachancea kluyveri Is Independent of Chromatin Remodeling

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Reviewer’s Comments and Suggestions for Authors

 Journal: Microbiology Research, MDPI

Manuscript ID: microbiolres-3110927

Type: Article

Title: Transcriptional regulation of the genes encoding branched chain aminotransferases in Kluyveromyces lactis and Lachancea kluyveri is independent of chromatin remodeling

Authors: James Gonzalez, Hector Quezada, Jose Carlos Campero-Basaldua, Edgar Ramirez-Gonzalez, Lina Riego-Ruiz and Alicia Gonzalez*

The authors of the manuscript Manuscript ID: microbiolres-3110927 deciphered whether chromatin remodeling determines the transcriptional regulation of orthologous KlBAT1 and LkBAT1 genes in Kluyveromyces lactis and Lachancea kluyveri when cells are cultured under conditions in which the branched chain amino acids are synthesized or degraded. The authors reported that in K. lactis, the KlBAT1 expression is reduced under catabolic conditions, while in L. kluyveri, the LkBAT1 displays a constitutive expression profile. However, chromatin organization of KlBAT1 and LkBAT1 promoters did not change under the experimental conditions tested. The main determinant of transcriptional activation of the KlBAT1 and LkBAT1 genes is the availability of the α-IPM co-activator.

The manuscript was written clearly. However, essential amending should be performed as listed below. 

Major revisions 

1. In the Abstract section, the authors should describe the significance of the results obtained in this study. Also, the abstract is redundancy, please shorten it.

2. In the Materials and Methods section, certain essential information was missing, for example:

(1) In the “2.1 Growth conditions”, please provide the species information of the “strains”, and “cells” used in this study.   

(2) Lines 111-114: regarding the single mutants tested in this study, the authors should provide the detailed information of the deleted genes in previous reports.  

(3) Line 130: in the “deoxyoligonucleotides xx to xx (Table S1)”, what does the “xx” mean?

(4) In the “2.3 Phylogeny of yeast proteins”, no source information is available regarding the strains and proteins used for the phylogenetic tree construction. Please clarify.

(5) No information of the S. cerevisiae strain tested in this study was provided. Please clarify.

3. In the Results section, for example:

(6) Table 1: please add the information of species in the “Strain” column.

(7) Figure 4: the resolution of the c and d was low. please provide the high-resolution figures.

4. In the Discussion section, the authors should describe the limitations of this study.

Minor revisions

(8) Abbreviations and acronyms are typically defined the first time the term is used within the abstract and again in the main text and then used throughout the remainder of the manuscript. Please consider adhering to this convention, and check throughout the manuscript. For example, the “Nucleosome Free Region (NFR)”.

(9) All species’ names should be written in italics throughout the paper.

(10) Lines 63-64: please rephrase the sentence.

(11) Line 76: please change to the “when the culture medium is supplemented with an excess of VIL”.

(12) Line 209: please change to the “yeast cultures (OD600 = 0.5)...”.

(13) Line 381: please change to the “As expected, the presented results in this study show that...”.

(14) Please format the References section, and carefully check one by one.

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Reviewer 1

The authors of the manuscript Manuscript ID: microbiolres-3110927 deciphered whether chromatin remodeling determines the transcriptional regulation of orthologous KlBAT1 and LkBAT1 genes in Kluyveromyces lactis and Lachancea kluyveri when cells are cultured under conditions in which the branched chain amino acids are synthesized or degraded. The authors reported that in K. lactis, the KlBAT1 expression is reduced under catabolic conditions, while in L. kluyveri, the LkBAT1 displays a constitutive expression profile. However, chromatin organization of KlBAT1 and LkBAT1 promoters did not change under the experimental conditions tested. The main determinant of transcriptional activation of the KlBAT1 and LkBAT1 genes is the availability of the α-IPM co-activator.

The manuscript was written clearly. However, essential amending should be performed as listed below.

Major revisions 

1. In the Abstract section, the authors should describe the significance of the results obtained in this study. Also, the abstract is redundancy, please shorten it.

Many thanks for your review. The abstract has been modified. The significance of the results is expressed in a succinct form in the sentence:

“Comparison of the α-IPMS sensitivities to feedback inhibition, suggested that the main determinant of transcriptional activation of the KlBAT1 and LkBAT1 genes might be the availability of the α-IPM co-activator, as reported previously for the BAT1 gene of S. cerevisiae.” We want to point the possibility that the availability of the α-IPM co-activator may be a conserved key factor determining the transcriptional activation of the genes encoding branched-chain aminotransferases in K. lactis, L. kluyveri and S. cerevisiae, in contrast with the marginal roles of chromatin remodeling and binding of the Leu3 activator to the promoter regions.

Indeed, the ideas in the abstract seem redundant because of the various BAT genes. However, as they belong to different species, we think that, for the sake of clarity, we should keep using the 200 words available to include the background information, our findings, and their significance.

2. In the Materials and Methods section, certain essential information was missing, for example:

(1) In the “2.1 Growth conditions”, please provide the species information of the “strains”, and “cells” used in this study.

Thanks for this pertinent correction. We have indicated that the growth conditions described in section 2.1 were used for the K. lactis, L. kluyveri and S. cerevisiae strains (Lines 103-111 in the version without tracked changes).

(2) Lines 111-114: regarding the single mutants tested in this study, the authors should provide the detailed information of the deleted genes in previous reports.

Some details about construction of single mutants have been added in the section “2.2 Strains” (Lines 115-132 in the version without tracked changes).

(3) Line 130: in the “deoxyoligonucleotides xx to xx (Table S1)”, what does the “xx” mean?

We apologize for this oversight. It has been corrected stating the names of the deoxyoligonucleotides used (Table S1; Lines 152-153 in the version without tracked changes).

(4) In the “2.3 Phylogeny of yeast proteins”, no source information is available regarding the strains and proteins used for the phylogenetic tree construction. Please clarify.

Thanks for your suggestion. The following text has been added to the “2.3 Phylogeny of yeast proteins” section: …PhylomeDB is a public database for complete catalogs of gene phylogenies (phylomes). It allows users to interactively explore the evolutionary history of genes through the visualization of phylogenetic trees and multiple sequence alignments. The proteomes used for the reconstruction of Saccharomycotina phylomes can be consulted at: http://phylomedb.org/phylome_206 (Lines 136-140 in the version without tracked changes).

(5) No information of the S. cerevisiae strain tested in this study was provided. Please clarify.

Indeed. The used S. cerevisiae strain was CLA11-700. This is indicated in the revised version in Table 1.

3. In the Results section, for example:

(6) Table 1: please add the information of species in the “Strain” column.

The species is now indicated for each strain in the first column of Table 1 and has been removed from the second column.

(7) Figure 4: the resolution of the c and d was low. please provide the high-resolution figures.

Thanks, panels C and D in Figure 4 have been replaced with high-resolution figures.

4. In the Discussion section, the authors should describe the limitations of this study.

Thank you for pointing this out. A paragraph addressing further functional studies that could be interesting to follow has been added in lines 442-445 in the version without tracked changes.

Minor revisions

(8) Abbreviations and acronyms are typically defined the first time the term is used within the abstract and again in the main text and then used throughout the remainder of the manuscript. Please consider adhering to this convention, and check throughout the manuscript. For example, the “Nucleosome Free Region (NFR)”.

Thanks for your correction. The text has been modified following the mentioned convention. Abbreviations are now defined the first time they are used and not again in the rest of the manuscript. Most of the corrections were for NFR, VIL and IC50. Some abbreviations are still defined in the Figure legends to make figures self-explanatory.

(9) All species’ names should be written in italics throughout the paper.

Thanks for your correction. The text has been modified accordingly.

(10) Lines 63-64: please rephrase the sentence.

The sentence “Conversely, BAT2 paralogue expression is not dependent on α-IPM availability; instead, transition from induced to repressed expression requires chromatin reconfiguration [8].” has been replaced by “Conversely, ScBAT2 paralogue expression is not dependent on α-IPM availability; instead, its transcriptional regulation requires chromatin remodeling.” (Lines 62-64 in the version without tracked changes).

(11) Line 76: please change to the “when the culture medium is supplemented with an excess of VIL”.

The sentence has been changed to:

Contrastingly, when the culture media is supplemented with high concentrations of BCAAs, transcription of the KlBAT1 gene is repressed [12]. (Lines 75-76 in the version without tracked changes).

A similar sentence has also been changed in the last paragraph of the introduction (Lines 88-89 in the version without tracked changes).

(12) Line 209: please change to the “yeast cultures (OD600 = 0.5)...”.

Thanks for this correction. The sign = (equal) has been added (Line 231 in the version without tracked changes).

(13) Line 381: please change to the “As expected, the presented results in this study show that...”.

Thanks. In the corrected version, the sentence is: “As expected, the presented results in this study show that the presence of high concentrations of BCAAs in the culture media reduced the rate of valine, isoleucine and leucine synthesis triggering repression of the KlBAT1 gene in K. lactis, confirming previous observations [12]” (Lines 414-417 in the version without tracked changes).

(14) Please format the References section, and carefully check one by one.

References have been checked and are now in the same format as a paper recently published in Microbiology Research.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors studied transcriptional regulation of the genes encoding branched chain aminotransferases in Kluyveromyces lactis and Lachancea kluyveri and relevant chromatin remodeling.

 

1.      The objectives of the study are not described clearly, please rephrase.

2.      The authors do not make clear what is the novelty of their work and its advantage over similar, previously published studies. Please add.

3.      Methodology. Description of controls is missing. Please add details about control chemicals and control strains. The control strains are the most important point to address in correcting this manuscript.

4.      Transcript levels were determined for BAT1, BAT2, KlBAT1 and LkBAT1 using deoxyoligonucleotides xx to xx (Table S1). Sorry, but this raises suspicions about the integrity of the study.

5.      Visualization. Please colourize the graphs to improve the artistic aspect of the manuscript. No figures in Discussion please.

6.      Tables. Please summarize results by adding tables in the manuscript and reducing length of text.

7.      Conclusions. These are not in line with the findings of the study and must be corrected.

 

Overall. The manuscript has significant flaws. I do not support publication, but I believe that the authors should be given a chance to correct them and improve the manuscript. If this is not done correctly, then the recommendation will be to reject the revised manuscript.

Author Response

Reviewer 2

The authors studied transcriptional regulation of the genes encoding branched chain aminotransferases in Kluyveromyces lactis and Lachancea kluyveri and relevant chromatin remodeling.

1. The objectives of the study are not described clearly, please rephrase.

Thanks for your careful revision. The last paragraph in the Introduction has been changed to describe the objectives clearly (Lines 81-85 in the version without tracked changes).

2. The authors do not make clear what is the novelty of their work and its advantage over similar, previously published studies. Please add.

Thanks for this pertinent suggestion. New lines have been added to the first paragraph of the Discussion section to make clear what the novelty of this work is (Lines 385-393 in the version without tracked changes).

3. Description of controls is missing. Please add details about control chemicals and control strains. The control strains are the most important point to address in correcting this manuscript.

We appreciate this suggestion. In the experiment shown in Figure 2, the transcriptional regulation of the BAT1 and BAT2 genes from S. cerevisiae, were used as controls. It has been reported that transcript levels of the BAT1 gene are lower on VIL than on glutamine, and that those of the BAT2 gene show the opposite expression pattern (reference 8). This is now indicated in the Results section 3.2 KlBAT1 and LkBAT1 expression profiles are not dependent on chromatin remodeling (Lines 233-237 in the version without tracked changes).

In the nucleosome scanning experiments, the control of nucleosome localization is implicit in the methodology. In Figure S1, it is shown that nucleosome positioning within the KlVCX1 and LkVCX1 promoters is equivalent in Gln or VIL as nitrogen sources. The VCX1 genes encode for vacuolar calcium exchangers and have been used as controls of nucleosome positioning in S. cerevisiae, K. lactis and L. kluyveri (https://doi.org/10.1098/rsos.231209, https://doi.org/10.1534/genetics.117.300290, https://doi.org/10.3389/fmicb.2017.01150, respectively). With this controls, the corresponding ratios were calculated to analyze nucleosome positioning in the promoter regions of the KlBAT1 and LKBAT1 genes. This is indicated in lines 171-173 in the version without tracked changes.

4. Transcript levels were determined for BAT1, BAT2, KlBAT1 and LkBAT1 using deoxyoligonucleotides xx to xx (Table S1). Sorry, but this raises suspicions about the integrity of the study.

We apologize for this oversight. It has been corrected stating the names of the deoxyoligonucleotides used (Table S1; Lines 152-153 in the version without tracked changes).

5. Please colourize the graphs to improve the artistic aspect of the manuscript. No figures in Discussion please.

Thanks for this suggestion. In the new version, Figure 2 and panels a and b of Figure 3 are shown in colors (green for K. lactis, blue for L. kluyveri, and dark red for S. cerevisiae).

Figure 6 (which was in the Discussion section) has been removed.

6. Please summarize results by adding tables in the manuscript and reducing length of text.

In the manuscript, results are mainly non-numeric (i.e. repressed Vs non repressed, chromatin remodeled Vs non-remodeled, highly sensitive to inhibition Vs slightly sensitive). We think that a table would help if data were mainly numerical. Results are shown in five figures, we believe that they present our main findings in an accessible way for readers. Thus, we decided to forego the use of tables in the Results section.

7. These are not in line with the findings of the study and must be corrected.

Thanks for this pertinent comment. Conclusions section has been modified to limit the conclusions to the obtained results and cautiously present the idea that the availability of the α-IPM co-activator could modulate the transcription levels of the KlBAT1 and LkBAT1 genes.

 

Overall. The manuscript has significant flaws. I do not support publication, but I believe that the authors should be given a chance to correct them and improve the manuscript. If this is not done correctly, then the recommendation will be to reject the revised manuscript.

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed the previous comments and have improved the manuscript.