Identification of secondary metabolites containing a diketopiperazine core in extracts from myxobacterial strains with growth inhibition activity against a range of prey species
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Myxobacteria produce a variety of bioactive secondary metabolites, and with a wealth of under-researched species they hold vast potential for undiscovered compounds. With the ever-increasing need for new antibiotics, the development of novel therapeutics is vitally important. Therefore, this study aimed to extract and elucidate antimicrobial metabolites from the following myxobacteria: Myxococcus xanthus CA010 and AB022; Corallococcus exiguus DSM 14696 T ; Myxococcus stipitatus DSM 14675 T ; and Corallococcus aberystwythensis AB050A T . Metabolite mixtures were extracted in acetone from XAD-16 resin incubated in liquid cultures and analysed using GC-MS. Bioactivity was identified using a growth inhibition assay against a panel of clinically relevant prey species including Gram-positive and Gram-negative bacteria and a fungus. Growth of Klebsiella pneumoniae and Enterococcus faecalis was most affected by the metabolite mixtures and the mixtures from AB022 and AB050A T were effective against the most prey. GC-MS analysis revealed metabolites with roles in the synthesis and degradation of amino acids and fatty acids, but also identified compounds A and B with a diketopiperazine (DKP) core. With previously confirmed bioactivity of compound A, it is suggested that these DKP compounds are contributing to the antimicrobial activity observed. Furthermore, many compounds could not be identified and so these unknowns present further potential for novel bioactive compounds.
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The work presented is clear and the arguments well formed.
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Thank you for amending your manuscript as per the reviewer comments. I agree with the issue posed by a reviewer that the inclusion of an 18S sequence in a 16S tree is not necessary in this instance, and request this be removed. I am happy that no further changes would be required beyond this.
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Comments to Author
I don't think 16S and 18S should be combined in this way on a tree.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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The reviewers and I agree that this study is of interest and fits the scope of Access Microbiology. The reviewers raise some issues that I would like you to address in a revision. Please pay particular attention to comments from Reviewer 1 regarding the phylogenetic tree and the data presented in Figure 2.
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Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data Well written and reason for choice of prey and predators clearly explained, although re-arrangement of table 1 to have prey species organised to have Gram Stain results grouped together, would be logical. 2. Presentation of results BGC prediction section there is the use of % to describe the presence of BGCs, it may be easier for comparisons across other papers just to state the actual number of BGCs rather than as a percentage. Running BiGSCAPE with 'mixed' setting on the strains would have enabled a better prediction of the unknown novel metabolites - this would have allowed some level of prediction using similarities across other known species within the MiBIG database. Figure 2 explains the above text well, maybe using …
Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data Well written and reason for choice of prey and predators clearly explained, although re-arrangement of table 1 to have prey species organised to have Gram Stain results grouped together, would be logical. 2. Presentation of results BGC prediction section there is the use of % to describe the presence of BGCs, it may be easier for comparisons across other papers just to state the actual number of BGCs rather than as a percentage. Running BiGSCAPE with 'mixed' setting on the strains would have enabled a better prediction of the unknown novel metabolites - this would have allowed some level of prediction using similarities across other known species within the MiBIG database. Figure 2 explains the above text well, maybe using colours for colour blind people would also be more disability conscious. 3. How the style and organization of the paper communicates and represents key findings Organised in a logical manner showing the level of activity first followed by compound identification. 4. Literature analysis or discussion First paragraph of the discussion states that the method used was as efficient as others previously stated, however it is towards the lower end, only in the range by 0.002g/L, so a bold statement to say 'as good' There is also sign of no lytic activity, so growth inhibition was carried out - Maybe this could suggest bacteriostatic activity? Ustilago maydis and Candida albicans are not very closely related fungal species and so although DSM14675 has activity towards one fungal species, it should not be assumed to be active towards another. Nice hypothesis as to why S. saphyrophyticys and S. aureus growth may have been increased in the presence of predation metabolite. Agree on the idea of future work using LC-MS as you could then put the data into SIRIUS5 and allow for chemical structure predictions, using the CANOPUS software.This would then enable the prediction of the unknown compounds and enable further exploration into whether their function is likely to be antimicrobial. 5. Any other relevant comments Overall nicely written and in a logical manner - with evidence of further investigation/papers to come.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
The authors present an interesting story of the isolation and characterisation of bioactive metabolites from Myxobacteria. Here, they demonstrate using bacterial growth assays, that crude extracts from DSM14696 reduce maximum growth rates in E. coli and E. faecalis. They then use GCMS alongside library comparison to elucidate chemical structures from bioactive extracts. Overall this is a nice paper which fits the themes of Access Microbiology. I have made some suggestions which will improve the overall flow and impact of this manuscript. 1. Methodological rigour, reproducibility and availability of underlying data In regards to Figure 2, I have concerns about the size of the error bar for S. aureus 'prey only'. I appreciate that the authors have presented their findings accurately but the varibilty …
Comments to Author
The authors present an interesting story of the isolation and characterisation of bioactive metabolites from Myxobacteria. Here, they demonstrate using bacterial growth assays, that crude extracts from DSM14696 reduce maximum growth rates in E. coli and E. faecalis. They then use GCMS alongside library comparison to elucidate chemical structures from bioactive extracts. Overall this is a nice paper which fits the themes of Access Microbiology. I have made some suggestions which will improve the overall flow and impact of this manuscript. 1. Methodological rigour, reproducibility and availability of underlying data In regards to Figure 2, I have concerns about the size of the error bar for S. aureus 'prey only'. I appreciate that the authors have presented their findings accurately but the varibilty here may be masking the significance of other results such as AB022 for Staph. Could you possibly repeat this? Perhaps you could run outlier analysis? Could you add the specific individual data points for each bar chart so that the reader can assess variability in context? Raw GCMS data should be made available to comply with FAIR principles. In addition, your antiSMASH parameters should be defined in the methods. 2. Presentation of results The results are generally presented well however in Figure 2 and Figure 4C, the bar charts are of low resolution and there should be solid axes on the graphs. If figure 4C represents a mean then it should also have an error bar. For the phylogenetic tree, there are better ways to represent species relatedness than to use an 18S sequence from Candida in a 16S tree. They are both sequences for components of different ribosomal subunits and therefore will differ greatly. This tree does not provide any information about the relation of Candida to these bacteria. In section 6.1, could you add a tree/table showing how the strains you have chose cluster based on their predation profile? In figure 4C I am unsure what the a, b, c, and d are representing. Are these the comparisons or significance values? "these significant differences are represented by different letters adjacent to the bars?" suggest the letters represent a P value but do they really represent which strain they show significance with? This should be clarified. 3. How the style and organization of the paper communicates and represents key findings Using 16S alone is not a robust enough method of phylogenetic analysis to conclude that metabolism/predatory activity and phylogeny are not linked. A conclusion that relatedness based on 16S-based phylogeny is not a good predictor of secondary metabolism or biosynthetic/predatory potential may be more suitable. Whole genome phylogeny or MLST may support your conclusion further. Perhaps AutoMLST (Nadine Ziemert's group) may be of use to the authors as it links phylogeny and biosynthetic potential. 4. Literature analysis or discussion In lines 340/341 it is stated that strains were chosen due to their 'good' predatory activity. The use of good here is objective and a definition of good should be given. Following this, it would aid the discussion to indicate whether these 'good' predators have more biosynthetic potential at a genomic level than other myxobacteria. I also feel that the discussion is very lengthy and the authors should be more selective in terms of which results to discuss in the context of the literature. It would also add to your discussion to speculate which on which gene clusters from your antismash results are most likely to encode your molecules of interest. 5. Any other relevant comments In your introduction you should introduce the idea of 'predator and prey' interactions rather than just stating that Myxobacteria is predatory (line 45). It would help the flow of the introduction for you to introduce the concept. In line 47 please change 'is not a good predictor' to 'does not accurately predict' Line 61-63 needs to be referenced. In line 84 define what you mean by 'in the low nanomolar concentrations'. Could you give numeric values here? In line 86 'undoubtedly yet to be revealed' should be changed to 'which may have untapped potential' or something less conclusive. In line 93 do you mean all metabolites? Secondary metabolites? All bacteria make metabolites. Perhaps something like 'tailored metabolite production' is more suitable. In Table 1, why is Candida Gram positive? I understand that Candida may appear purple in a Gram stain but this is confusing for readers. N/A in this column would be more suitable. Table one should have genome accession/contigs/referenced for prey or this table should be separated in to two tables for predator and prey. In line 196, what is a 'top' predator? Again, this adjective should be defined clearly. This initial paragraph of results is unclear. By this do you mean that high levels of predatory activity against one species are is associated with the ability of a strain to predate multiple species? Did you screen 112 isolates for their ability to predate ten species and chose the best predators? I feel this would be more suited to your methods section. In line 204, what is 'good predatory activity'? Can you define this by how many species it can predate or a predation rate? Section 6.4 would be more suited to the methods section. In line 235, are there clusters which are common to all species tested? This would be useful information to know without searching through the Supplementary Figures.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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