The CBS/H2S signalling pathway regulated by the carbon repressor CreA promotes cellulose utilization in Ganoderma lucidum

Cellulose is an important abundant renewable resource on Earth, and the microbial cellulose utilization mechanism has attracted extensive attention. Recently, some signalling molecules have been found to regulate cellulose utilization and the discovery of underlying signals has recently attracted extensive attention. In this paper, we found that the hydrogen sulfide (H2S) concentration under cellulose culture condition increased to approximately 2.3-fold compared with that under glucose culture condition in Ganoderma lucidum. Further evidence shown that cellulase activities of G. lucidum were improved by 18.2-27.6% through increasing H2S concentration. Then, we observed that the carbon repressor CreA inhibited H2S biosynthesis in G. lucidum by binding to the promoter of cbs, a key gene for H2S biosynthesis, at “CTGGGG”. In our study, we reported for the first time that H2S increased the cellulose utilization in G. lucidum, and analyzed the mechanism of H2S biosynthesis induced by cellulose. This study not only enriches the understanding of the microbial cellulose utilization mechanism but also provides a reference for the analysis of the physiological function of H2S signals.

The authors of this manuscript show that cellulose culture condition induces hydrogen sulfide (H2S) synthesis in Ganoderma lucidum, and carbon catabolite repressor CreA inhibits this H2S synthesis through repression of cystathionine β-synthase (CBS) gene expression.The authors also show that cellulase activity is increased by H2S.These are interesting phenomena.However, the mechanisms of how cbs gene expression is induced by cellulose and how H2S affects cellulase activity are not clear.These points are required for the readers of Communications Biology.In addition, there are various issues as indicated below.The authors describe that H2S concentration under cellulose culture condition increased by approximately1.25-foldcompared with that under glucose culture condition (lines 29-32 and 106-108).This description does not appear to be consistent with the results in Fig. 1b.

Fig. 1b
How many mycelia were examined?Fig. 1d and 1e The expression level of cbs gene should be examined.

Fig. 2a and 2b
The concentration of H2S in the presence of NaHS or HS should be examined.

Fig. 3a
Not enough information is provided about Y1H screening.If the cDNA library was used for screening, information about the cDNA library and the cDNA fragments that were introduced into the positive clones should be provided.Or if the cDNAs of the transcription factors are cloned for screening, information about the transcription factor genes being cloned should be indicated.The manuscript should be written more carefully because there are many minor errors.
Reviewer #2 (Remarks to the Author): In this study, Jiaolei Shangguan and colleagues set out to investigate the intracellular signaling mechanisms involved in the utilization of cellulose by the fungus Ganoderma lucidum.First of all, cellulose is a highly abundant carbohydrate and understanding the mechanisms that take place during its degradation or conversion have direct importance to understand ecological aspects as well as a direct link to the biotechnological utilization of cellulose for biobased materials such as biofuels.On the other hand, Ganoderma lucidum is a mushroom-forming fungus with an important role in research and folk medicine.Altogether, the topics under study in this manuscript are very relevant.
More specifically, the authors have demonstrated a link between cellulose utilization and hydrogen sulfide (H2S) production by fungi.This link involved the direct binding of the transcription factor CreA, known to be important for carbon catabolite repression in fungi, to the promoter of the cbs gene, repressing its expression.The binding site was determined too.
The production of H2S was increased on cellulose vs. glucose, but this increase was quite modest (1.25 folds), albeit significant.Exogenously added H2S did enhance cellulase activity, so the link seems to be solid.Furthermore, the authors conducted some validation experiments with chemicals that activate and inhibit H2S production as well as with silenced vs. overexpression cells.All data seem to fall into place and the rationale of the work is easy to understand.Nevertheless, some of the data is somewhat circumstantial and not necessarily indicative of a direct mechanism.
Fig. 1: It would be important to present more controls for the SF7-AM method to measure intracellular accumulation of H2S.Methods whose quantification is based on microscopy analysis strongly depend on appropriate controls so that autofluorescence is properly normalized.The same microscopy settings (e.g., exposure, gain, etc.) must be used for all samples to avoid situations of saturation, for instance.A non-staining control is missing, for instance.These controls could be a supplemental figure.Still related to this point, perhaps a more quantitative/robust method could have been tried, such as flow cytometry (in spores) or fluorimetry, using the SF7-AM probe.Fig. 2c: In the cbs-silenced lines, either the gene silencing efficiency is modest or how can the authors integrate their conclusion that cbs is so important for cellulose degradation but at the same time cbssilenced cells are able to increase CMCase activity when treated with NaHS?Perhaps justified because there are are other important pathways playing a role?I think this should be discussed.Fig. 2d: differences are quite modest, despite the statistical significance.Fig. 3b: the phylogenetic is not showing informative data.Many more protein accessions would have to be used to make a more detailed study of the relationship between these transcription factors.Furthermore, the description of this figure in the legend is "evolutionary tree", which is an incorrect oversimplification.Additionally, the values next to the tree branches are not defined in the legend.
Minor changes -Line 41: "maximum amount of renewable carbohydrates"; this phrase is unclear.What is meant by "maximum"?-Line 76: "H2S improved the tolerance of plants to Cu2+ and heat"; not clear or not specific enough.How did H2S improve tolerance?-Line 82: the word "well" seems unnecessary.
-Line 95: "a gene of H2S synthesis enzyme"; this phrase is not properly written.Perhaps "a gene encoding the H2S synthesis enzyme"?But could the authors be more specific about the role of this enzyme in the synthesis of H2S? -Line 97: "which promoting" is incorrect."which promotes"?-Fig.2E: the meaning of the red, horizontal line should be specified in the figure legend.
-Line 287-305 seems more appropriate for the Introduction rather than Discussion The manuscript is well written and easy to understand.However, an overall revision could be performed to fix minor incorrections.

Pedro Gonçalves
Reviewer #3 (Remarks to the Author): General Comments: The manuscript presents a well-executed study investigating the role of the hydrogen sulfide (H2S) signaling pathway in Ganoderma lucidum, particularly its impact on cellulase activity and cellulose utilization.The research is meticulously conducted, and the findings contribute valuable insights into the molecular mechanisms underlying microbial cellulose utilization.The presented data is wellsupported and convincingly demonstrates the involvement of the H2S signaling pathway, specifically regulated by the carbon repressor transcription factor CreA.

Specific Comments:
Clarity and Structure: The manuscript is well-structured, and the clarity of the presentation is commendable.The introduction effectively contextualizes the study, and the objectives are clearly defined.The results and discussion sections are logically organized, facilitating a smooth understanding of the experimental outcomes.

Experimental Design and Methods:
The experimental design is robust, and the methods are meticulously detailed, allowing for reproducibility.The use of both pharmacological and genetic experiments to probe the H2S signaling pathway, as well as the involvement of CreA, enhances the study's credibility.

Data Presentation:
The data presentation is clear and concise.Figures and tables are appropriately utilized to illustrate key findings, and the inclusion of statistical analyses adds rigor to the interpretation of results.The use of fluorescence probes and gene expression analysis provides a comprehensive view of H2S concentration and its regulatory mechanisms.

Discussion of Results:
The discussion of results is comprehensive, and the authors effectively relate their findings to existing literature.The identification of CreA as a negative regulator of H2S biosynthesis adds novelty to the study.Additionally, the discussion on the potential influence of CBS on cellulose utilization is insightful.

Language and Style:
The language used in the manuscript is generally clear and concise.However, attention to minor grammatical issues, as identified in the detailed suggestions, will enhance the overall polish of the manuscript.

Conclusion:
The study significantly contributes to our understanding of the molecular mechanisms governing cellulose utilization in G. lucidum.The data is robust, the methodology is sound, and the results are well-discussed.With minor revisions for language and clarity, the manuscript is well-suited for publication in a peer-reviewed journal.
Here are some minor issues that needs to be addressed.Some references regarding CreA homologs in other organisms should be included for example PMID: 21980519, PMID: 21619626, PMID: 31040248.
Here are some minor points with language use.In line 104, "Ganoderma lucidum" could be italicized or written in a different format for emphasis on the species name.
In line 108, it should be "This result shows that" instead of "This result shown that."In line 111, "genes transcription levels" should be "gene transcription levels."In line 116, "cystathionine β-synthase (CBS)" could be introduced with "the" for better clarity, like "the cystathionine β-synthase (CBS)."In line 118, "cbs-silenced and cbs-overexpressed strains" could be rewritten as "cbs-silenced, cbsoverexpressed, and control strains" for better clarity.In line 123, "H2S biosynthesis in G. lucidum and CBS might be one" might read more smoothly as "H2S biosynthesis in G. lucidum, and CBS might be one."In line 126, "To explore" could be followed by a phrase indicating the action, such as "To explore the effect of H2S signal on cellulose utilization."In line 136, "These results of pharmacological experiments suggested that" could be more concise, like "Pharmacological experiments suggested that."In line 158, there's a space before the colon in "regulators of the cbs gene identifies diverse transcription."It should be "regulators of the cbs gene identifies diverse transcription:" In line 218, "which indicated that cellulose culture condition" could be revised for clarity, like "indicating that cellulose culture conditions."In line 234, "thereby improving cbs transcription and H2S biosynthesis under cellulose culture condition" could be simplified, such as "thereby enhancing cbs transcription and H2S biosynthesis under cellulose culture conditions."In line 270, "Microbial cellulose utilization contributes to the carbon cycle in the biosphere and is widely used in both agriculture and industry" could be split into two sentences for clarity.

Dear Reviewers:
Thank you for your comments concerning our manuscript entitled "CBS/H2S signalling pathway regulated by the carbon repressor CreA promotes cellulose utilization in Ganoderma lucidum" (COMMSBIO-23-3923).Those comments are valuable and very helpful for revising and improving our paper and providing important guidance for our research.We have read through the comments carefully and revised our manuscript according to your and three reviewers' suggestions.We hope that the revised manuscript will be appropriate for publication.
The revised sections are highlighted in red color in the revised manuscript.
Our point-by-point replies to the reviewers' comments are presented below.
Thank you very much for your attention and consideration.
Best regards.

< COMMENTS FOR THE AUTHOR >
Reviewer #1: The authors of this manuscript show that cellulose culture condition induces hydrogen sulfide (H2S) synthesis in Ganoderma lucidum, and carbon catabolite repressor CreA inhibits this H2S synthesis through repression of cystathionine β-synthase (CBS) gene expression.The authors also show that cellulase activity is increased by H2S.These are interesting phenomena.However, the mechanisms of how cbs gene expression is induced by cellulose and how H2S affects cellulase activity are not clear.These points are required for the readers of Communications Biology.In addition, there are various issues as indicated below.
Response: Thank you for your constructive comments during the review of the manuscript.Those comments are valuable and very helpful for our paper.
In this paper, we observed that the transcription of cbs genes is repressed by the transcription factor CreA. CreA was a recognized carbon catabolite repressor in filamentous fungi, such as Trichoderma reesei, Aspergillus nidulans, Ganoderma lucidum and Neurospora crassa 1-5 .In the case of repressing carbon sources, such as cellulose culture condition, CreA mRNA levels were decreased 3,6 .Therefore, we concluded that cellulose activities CBS/H2S signalling pathway by reducing the binding of the repressor CreA to the cbs promoter under cellulose culture condition.These data and conclusions explain the mechanism by which cellulose induces the transcription of cbs genes.We also have added the related points in the Discussion (Page 18, Line 369-374).
As to how H2S affects cellulase activity, we have only found this phenomenon.We have made some speculations in the Discussion (Page 15-16, Line 304-320).We also consider it is very important and necessary to assay the relevant mechanism.We will focus on the point in the future work.2958.1999.01341.x(1999).The authors describe that H2S concentration under cellulose culture condition increased by approximately1.25-foldcompared with that under glucose culture condition (lines 29-32 and 106-108).This description does not appear to be consistent with the results in Fig. 1b.
Response: Sorry for any confusion caused by our unclear description.We have revised our statement in Page 2, Line 28-29 and Page 6, Line 114.

Fig. 1d and 1e
The expression level of cbs gene should be examined.

Fig. 2a and 2b
The concentration of H2S in the presence of NaHS or HS should be examined.
Response: Thank you for your attention and advice.We assume you're advising us to add the H2S concentration in the presence of NaHS or HT.
Therefore, we had added this data in the new Supplementary fig. 2  Response: Thank you for your suggestion.After careful thought, we also considered that it did not make sense to combine different transcription factors into one phylogenetic tree and have removed this result.
Line 351 "positive" Is this right?
Response: Sorry for any confusion caused by our description.We have revised it to a more detailed description in Page 18, Line 369-374.
The manuscript should be written more carefully because there are many minor errors.
Response: Thank you for your comments and helpful suggestions.We have revised the details of the article according to your advice.
Reviewer #2: In this study, Jiaolei Shangguan and colleagues set out to investigate the intracellular signaling mechanisms involved in the utilization of cellulose by the fungus Ganoderma lucidum.First of all, cellulose is a highly abundant carbohydrate and understanding the mechanisms that take place during its degradation or conversion have direct importance to understand ecological aspects as well as a direct link to the biotechnological utilization of cellulose for biobased materials such as biofuels.On the other hand, Ganoderma lucidum is a mushroom-forming fungus with an important role in research and folk medicine.Altogether, the topics under study in this manuscript are very relevant.
More specifically, the authors have demonstrated a link between cellulose utilization and hydrogen sulfide (H2S) production by fungi.This link involved the direct binding of the transcription factor CreA, known to be important for carbon catabolite repression in fungi, to the promoter of the cbs gene, repressing its expression.The binding site was determined too.
The production of H2S was increased on cellulose vs. glucose, but this increase was quite modest (1.25 folds), albeit significant.Exogenously added H2S did enhance cellulase activity, so the link seems to be solid.Furthermore, the authors conducted some validation experiments with chemicals that activate and inhibit H2S production as well as with silenced vs. overexpression cells.All data seem to fall into place and the rationale of the work is easy to understand.
Nevertheless, some of the data is somewhat circumstantial and not necessarily indicative of a direct mechanism.

Response:
We are appreciated with your suggestions.Sorry for our inappropriate descriptions about partial conclusions of results.We had revised the details of the article according to your and other reviewers' advice.We hope our revised manuscript provides appropriate descriptions.
Fig. 1: It would be important to present more controls for the SF7-AM method to measure intracellular accumulation of H2S.Methods whose quantification is based on microscopy analysis strongly depend on appropriate controls so that autofluorescence is properly normalized.The same microscopy settings (e.g., exposure, gain, etc.) must be used for all samples to avoid situations of saturation, for instance.A non-staining control is missing, for instance.These controls could be a supplemental figure.Still related to this point, perhaps a more quantitative/robust method could have been tried, such as flow cytometry (in spores) or fluorimetry, using the SF7-AM probe.

Response:
We are very appreciated with these important comments and suggestions.We have added the unstained control according your suggestions in the new Supplementary fig. 2.
In addition, we used the same fluorescence microscope and microscopy settings for all samples.Relative description has been added in the revised manuscript (Page 19, Line 405).
Moreover, Ganoderma lucidum is a multicellular filamentous fungus.
There is currently very difficult to quantify it using flow cytometry methods.The current method is according to the methods used in animal cells and our previous study 1,2 .We are also working on establishing even better methods to measure H2S. Reference: 1 Tian, J.  Response: Sorry for our inappropriate descriptions about the effect of H2S on CMCase activity.We had revised the descriptions the revised manuscript (Page 18, Line 369-374).
In addition, H2S was biosynthesized by Cystathionine β-synthase (CBS) 1,3 .The silence of cbs gene resulted in a decrease in the intracellular H2S content (Fig. 1d, e, f).NaHS is a direct donor of H2S.The exogenous addition of NaHS can directly increase the content of H2S without relying on the involvement of CBS.NaHS addition increased the CMCase activity in cbssilencied strains.These combined results suggest that H2S improved the cellulase activity of G. lucidum under cellulose culture condition.
We also added to the discussion other underlying regulators participate in the improvement of CBS to cellulose utilization in G. lucidum in Page 15-16, Line 304-320.We hope that this is conducive to understanding the effect of H2S in regulation of cellulose utilization. Reference: 1 Tian, J.  Response: We appreciate with your suggestions.We had revised the unclear descriptions in the revised manuscript (Page 9, Line 173).We hope our revised manuscript provides appropriate descriptions about the effect of H2S in regulation of cellulose utilization.
Fig. 3b: the phylogenetic is not showing informative data.Many more protein accessions would have to be used to make a more detailed study of the relationship between these transcription factors.Furthermore, the description of this figure in the legend is "evolutionary tree", which is an incorrect oversimplification.Additionally, the values next to the tree branches are not defined in the legend.
Response: Thank you very much for your advice.According your suggestion, we have deleted the phylogenetic tree.
Minor changes -Line 41: "maximum amount of renewable carbohydrates"; this phrase is unclear.What is meant by "maximum"?
Response: Thank you for your questions about our inaccurate description.We have revised according your suggestions (Page 3, Line 39-40).
-Line 76: "H2S improved the tolerance of plants to Cu 2+ and heat"; not clear or not specific enough.How did H2S improve tolerance?
Response: According to the suggestions, we have added a specific description of mechanism of H2S improve the tolerance of plants to Cu 2+ and heat in Page 5, Line 84-85.

Response:
We have removed "well" according your advice in Page 5, Line 91.
-Line 95: "a gene of H2S synthesis enzyme"; this phrase is not properly written.
Perhaps "a gene encoding the H2S synthesis enzyme"?But could the authors be more specific about the role of this enzyme in the synthesis of H2S?
Response: According your advice, we have revised the description in Page 5, Line101-102 and added the role of CBS in H2S synthesis in Page 4, Line 76-77.

Response:
We have revised accordingly in Page 6, Line 104.
-Fig.2E: the meaning of the red, horizontal line should be specified in the figure legend.

Response:
We have illustrated the meaning of the red horizontal line in the The manuscript is well written and easy to understand.However, an overall revision could be performed to fix minor incorrections.
Response: Thank you very much for your suggestions.Those comments are valuable and very helpful for our paper, and we have revised the details of the article according to your advice.

Pedro Gonçalves
Reviewer #3: General Comments: The manuscript presents a well-executed study investigating the role of the hydrogen sulfide (H2S) signaling pathway in Ganoderma lucidum, particularly its impact on cellulase activity and cellulose utilization.The research is meticulously conducted, and the findings contribute valuable insights into the molecular mechanisms underlying microbial cellulose utilization.The presented data is well-supported and convincingly demonstrates the involvement of the H2S signaling pathway, specifically regulated by the carbon repressor transcription factor CreA.

Specific Comments:
Clarity and Structure: The manuscript is well-structured, and the clarity of the presentation is commendable.The introduction effectively contextualizes the study, and the objectives are clearly defined.The results and discussion sections are logically organized, facilitating a smooth understanding of the experimental outcomes.
Experimental Design and Methods: The experimental design is robust, and the methods are meticulously detailed, allowing for reproducibility.The use of both pharmacological and genetic experiments to probe the H2S signaling pathway, as well as the involvement of CreA, enhances the study's credibility.
Fig. 1b I don't understand the y-axis label; what does 100% indicate?
Fig. 3b I do not understand the significance of this phylogenetic tree.Is there any sense in combining different transcription factors into one phylogenetic tree?

:
Thank you for your suggestion.According to the suggestions, we have added the cbs gene expression levels of wild-type (wt), sicontrol, cbssilenced and cbs-overexpressed strains under cellulose or glucose culture conditions (the new Fig.1f) and the description have been added in the revised manuscript (Page 7, Line 128-139) and the Figure legends (Page 23, Line 484-488). Fig.3a

Fig. 2c :
Fig.2c: In the cbs-silenced lines, either the gene silencing efficiency is modest or how can the authors integrate their conclusion that cbs is so important for cellulose degradation but at the same time cbs-silenced cells are able to increase CMCase activity when treated with NaHS?Perhaps justified because there are other important pathways playing a role?I think this should be discussed.