Systematic Analysis of Two Tandem GGDEF/EAL Domain Genes Regulating Antifungal Activities in Pseudomonas glycinae MS82
Round 1
Reviewer 1 Report
The manuscript lays out some findings related to gene deletions in Pseudomonas glycinae in the cyclic diguanylate signaling pathway. This pathway is best known for regulating interconversion of bacteria between planktonic and biofilm lifestyles, but this manuscript emphasizes the regulation of production of antifungal metabolites. The cited motivation is that Pseudomonas glycinae is presented as a natural protective agent against fungal pathogens in the cultivation of mushrooms.
The manuscript is difficult to read.
The introduction is inadequate and confusing.
Line 57: "In the study of antagonistic bacteria, c-di-GMP in Pseudomonas fluorescens could improve the effect of biological control through promoting the formation of biofilm". The term "antagonistic" does not appear in the reference cited. The cited study shows that high c-di-GMP corresponds to biofilm formation.
Line 59: "The antifungal substance HSAF
secreted by Lysobacter enzymogenes OH11 is closely related to the regulation of c-di-GMP". Presumably they mean that the secretion is regulated by c-di-GMP. The cited study actually states that high c-di-GMP inhibits secretion of HSAF, the opposite of the overall gist of the manuscript.
Line 61: "Furthermore, identical results occurred in P. gylcinae [sic] MS82. When the tandem GGDEF/EAL domain gene PafR was mutated, the antifungal activity of P. gylcinae [sic] MS82 against T. viride and M. perniciosa was lost [10]" Ref 10 is their prior isolation of a mutation in PafR. It's hard to say that's an identical result, since they don't know if PafR is primarily a synthetase or a phosphodiesterase. In this paper they show that the PafR deletion inhibits biofilm formation, suggesting it is primarily a synthetase, and apparently the deletion decreases antifungal activity, which is the opposite of the result said to be identical in the previous sentence.
I'm not suggesting that a major review be written as an introduction; the paper is an appropriately short report. But there are only about three sentences in the introduction that a reader can use to anticipate what the paper is going to be about, and they disagree with each other.
Starting with that basis of confusion, we go to the results. There both PafR, and PafQ (another gene with both synthetase and phosphodiesterase domains) were individually deleted. Both mutations reduced overall antifungal activity. Reduction was more prominent for PafR, which makes sense since that's how it was selected in the first place. That seems clear enough.
Then there is qPCR of nine transcripts said to be involved in synthesizing antifungal substances. These are said to be identified by id numbers in GenBank entry CP028826. There are no such id numbers in CP028826, and CP028826 is said to be P. fluorescens in GenBank, not P. glycinae. There are also no PafQ, PafR, or hcnA or others of the identifiers listed in the manuscript found in that GenBank entry. Their former manuscript about the isolation of PafR also called MS82 P. fluorescens, not P. glycinae. If I use the PCR primers listed in a blastN search of CP028826, I do find what are plausibly the indicated genes, so I guess they have decided to reclassify the bacteria, as opposed to giving the wrong accession number in the manuscript. But if they want to change the name of the bacteria, we either need a citation or an explanation, because as it stands I'm not 100% sure I can find any of the genes discussed in the manuscript. And they need to use identifiers that actually exist in the cited GenBank file.
There is a table in the methods that gives some names for these genes. The first one named is cysC. That's usually thought to be a gene of cysteine synthesis. What's the supposed antifungal pathway involving cysC? For each of these nine genes we need to see either a citation or an explanation of why they might be considered antifungal.
There are some results on biofilm formation and motility measurements that seem reasonable.
The discussion does make an attempt to sort out some of the complexities of the interpretation by comparison to homologous genes in other bacteria, so that was helpful.
Author Response
We appreciate positive comments, suggestions and constructive criticisms made by the experts and editor on to the manuscript. We have revised the manuscript according to these excellent reviews. The following are the details of our responses.
The manuscript lays out some findings related to gene deletions in Pseudomonas glycinae in the cyclic diguanylate signaling pathway. This pathway is best known for regulating interconversion of bacteria between planktonic and biofilm lifestyles, but this manuscript emphasizes the regulation of production of antifungal metabolites. The cited motivation is that Pseudomonas glycinae is presented as a natural protective agent against fungal pathogens in the cultivation of mushrooms.
The manuscript is difficult to read.
The introduction is inadequate and confusing.
Response: Thanks for your comments. We have extensively revised this manuscript. Please see the following responses to specific comments and questions.
（1）Line 57: "In the study of antagonistic bacteria, c-di-GMP in Pseudomonas fluorescens could improve the effect of biological control through promoting the formation of biofilm". The term "antagonistic" does not appear in the reference cited. The cited study shows that high c-di-GMP corresponds to biofilm formation.
Response: The more appropriate reference was revised in the manuscript.
（2）Line 59: "The antifungal substance HSAFsecreted by Lysobacter enzymogenes OH11 is closely related to the regulation of c-di-GMP". Presumably they mean that the secretion is regulated by c-di-GMP. The cited study actually states that high c-di-GMP inhibits secretion of HSAF, the opposite of the overall gist of the manuscript.
Response: The sentence “The antifungal substance HSAF secreted by Lysobacter enzymogenes OH11 is closely related to the regulation of c-di-GMP” was changed as “But the higher intracellular levels of c-di-GMP is the inhibitory signal for the antifungal substance HSAF secreted by Lysobacter enzymogenes OH11”.
（3）Line 61: "Furthermore, identical results occurred in P. gylcinae [sic] MS82. When the tandem GGDEF/EAL domain gene PafR was mutated, the antifungal activity of P. gylcinae [sic] MS82 against T. viride and M. perniciosa was lost [10]" Ref 10 is their prior isolation of a mutation in PafR. It's hard to say that's an identical result, since they don't know if PafR is primarily a synthetase or a phosphodiesterase. In this paper they show that the PafR deletion inhibits biofilm formation, suggesting it is primarily a synthetase, and apparently the deletion decreases antifungal activity, which is the opposite of the result said to be identical in the previous sentence.
Response: Thanks for the comment. We apologize for the confusion. These sentences have been revised and please see the details in the revised version.
（4）I'm not suggesting that a major review be written as an introduction; the paper is an appropriately short report. But there are only about three sentences in the introduction that a reader can use to anticipate what the paper is going to be about, and they disagree with each other.
Response: Thanks for the comment. The introduction to the research has been extensively revised as shown in the revised version.
（5）Starting with that basis of confusion, we go to the results. There both PafR, and PafQ (another gene with both synthetase and phosphodiesterase domains) were individually deleted. Both mutations reduced overall antifungal activity. Reduction was more prominent for PafR, which makes sense since that's how it was selected in the first place. That seems clear enough.
Response: Thanks for the positive comment.
（6）Then there is qPCR of nine transcripts said to be involved in synthesizing antifungal substances. These are said to be identified by id numbers in GenBank entry CP028826. There are no such id numbers in CP028826, and CP028826 is said to be P. fluorescens in GenBank, not P. glycinae. There are also no PafQ, PafR, or hcnA or others of the identifiers listed in the manuscript found in that GenBank entry. Their former manuscript about the isolation of PafR also called MS82 P. fluorescens, not P. glycinae. If I use the PCR primers listed in a blastN search of CP028826, I do find what are plausibly the indicated genes, so I guess they have decided to reclassify the bacteria, as opposed to giving the wrong accession number in the manuscript. But if they want to change the name of the bacteria, we either need a citation or an explanation, because as it stands I'm not 100% sure I can find any of the genes discussed in the manuscript. And they need to use identifiers that actually exist in the cited GenBank file.
Response: Based on the preliminary identification results, the strain MS82 was P. fluorescens, which was shown in our previous publications. However, after in-depth research on the classification of this strain, Jia et al. confirmed the strain is a member of the novel species P. glycinae, and this new species was validated and cited in the Approved Lists of Bacterial Names in the International Journal of Systematic and Evolutionary Microbiology (IJSEM). The Paf genes were named according to their functions, and these new names were not reflected in the whole genome information.
（7）There is a table in the methods that gives some names for these genes. The first one named is cysC. That's usually thought to be a gene of cysteine synthesis. What's the supposed antifungal pathway involving cysC? For each of these nine genes we need to see either a citation or an explanation of why they might be considered antifungal.
Response: When we performed the genome-wide analysis, seven databases were used for general function annotation: KEGG (Kyoto Encyclopedia of Genes and Genomes), COG (Clusters of Orthologous Groups), NR (Non-Redundant Protein database), Swiss-Prot, GO (Gene Ontology), TrEMBL and EggNOG. All of the genes showed in table 3 were selected based on the genomic annotation and function predictions. These genes were also showed in the references [11]. The gene cysC is homologous to a biosynthetic gene of phytotoxin phaseolotoxin.
There are some results on biofilm formation and motility measurements that seem reasonable. The discussion does make an attempt to sort out some of the complexities of the interpretation by comparison to homologous genes in other bacteria, so that was helpful.
Response: Thanks for the positive comments on the research.
Reviewer 2 Report
The manuscript ”Systematic analysis of two tandem GGDEF/EAL domain genes regulating antifungal activities in Pseudomonas glycinae MS82” is written well. The theme of the manuscript is good but there are many duplications of the work with their previous works and other works. The authors published the same types of data several times with the same bacterial strain Pseudomonas glycinae MS82 such as reference numbers 10, 11, 23, 24. This study focused on the antifungal characteristics of PafQ or PafR gene of Pseudomonas glycinae MS82 that have antifungal properties. The authors have shared the same hypothesis, published earlier “The PafR gene is required for antifungal activity of strain MS82 306 against Mycogone perniciosa”.
Besides these I have several minor comments:
ABSTRACT: The abstract is adequate, but need to revise the keywords. All the keywords
are in the title. Keywords should be different from the title.
INTRODUCTION: The introduction is written well with the problem, hypothesis and aim.
The literature review in the introduction section must be improved with some new
references.
METHODS: This section includes all the needed information. Please describe how the
sampling was performed in detail, including the time duration of sampling though the
authors have mentioned their earlier references. How the bacterial strains were isolated?
RESULTS: What could be the rationale for getting different results at different time points?
I suggest performing the test with a lower concentration (0.1 or 0.2%) of crystal violet
DISCUSSION: Though an adequate number of references are added it could be improved
by using more updated articles on similar studies in the discussion section.
CONCLUSION: The conclusion part should be summarized with more results
Author Response
We appreciate positive comments, suggestions and constructive criticisms made by the experts and editor on to the manuscript. We have revised the manuscript according to these excellent reviews. The following are the details of our responses.
The manuscript ”Systematic analysis of two tandem GGDEF/EAL domain genes regulating antifungal activities in Pseudomonas glycinae MS82” is written well. The theme of the manuscript is good but there are many duplications of the work with their previous works and other works. The authors published the same types of data several times with the same bacterial strain Pseudomonas glycinae MS82 such as reference numbers 10, 11, 23, 24. This study focused on the antifungal characteristics of PafQ or PafR gene of Pseudomonas glycinae MS82 that have antifungal properties. The authors have shared the same hypothesis, published earlier “The PafR gene is required for antifungal activity of strain MS82 306 against Mycogone perniciosa”.
Besides these I have several minor comments:
（1）ABSTRACT: The abstract is adequate, but need to revise the keywords. All the keywords are in the title. Keywords should be different from the title.
Response: we have changed the keywords that are different from the title.
（2）INTRODUCTION: The introduction is written well with the problem, hypothesis and aim. The literature review in the introduction section must be improved with some new references.
Response: We have changed the new references.
（3）METHODS: This section includes all the needed information. Please describe how the sampling was performed in detail, including the time duration of sampling though the authors have mentioned their earlier references. How the bacterial strains were isolated?
Response: The isolated information of MS82 was described in the reference [10], which has been cited in the manuscript.
（4）RESULTS: What could be the rationale for getting different results at different time points?I suggest performing the test with a lower concentration (0.1 or 0.2%) of crystal violet.
Response: Yes, it may show different results at different time points. But we just conducted it as described in the peer-reviewed publication (Ref. 26) for the assay.
（5）DISCUSSION: Though an adequate number of references are added it could be improved by using more updated articles on similar studies in the discussion section.
Response: We have updated the references cited in the manuscript.
（6）CONCLUSION: The conclusion part should be summarized with more results.
Response: We have revised the conclusion section with addition of the key findings of this research.
Round 2
Reviewer 1 Report
In the resubmission:
The confusion between P. glycinae and P. florescens has now been clarified.
The confusion about the gene numbers has not. The legend to fig. 3 says the id numbers come from CP028826, and they do not. If the problem is that you've used these numbers in other papers and don't want to be inconsistent, then put another column in table 3 with the CP02886 locus_tags, and change the legend to fig. 3 to say the gene IDs are as defined in table 3. That way you can keep your numbering system and us readers can use the locus_tags to look up the genes. You should also state somewhere the locus_tags in CP028826 for PafR and PafQ.
You have replied that the information implicating the genes in Fig. 3 in anti-fungal activity is to be found in ref. 11. Then state in the legend that these genes were chosen based on ref. 11.
Author Response
Thank you very much for the detailed revision suggestions from the experts again. This recommendation mainly focused on the consistency of gene names and NCBI gene locus_tags. This time I understood the experts' concerns. In this revision, I modified the numbers of all relevant genes, using the numbers of corresponding genes in the whole genome data on NCBI. It is convenient for readers to find out more relevant information. Meanwhile, the gene names in Table 3 are removed to avoid misunderstandings.
Reviewer 2 Report
the manuscript has been edited as per comments. It can be accepted for publication.
