Design and validation of a PCR screen for γ-butyrolactone-like regulatory systems in Streptomyces
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γ-butyrolactone and related signalling systems are found in Streptomyces and other actinobacteria where they control the production of secondary or specialized metabolites such as antibiotics. Genetic manipulation of these regulatory systems therefore leads to changes in the secondary metabolite profile of a strain and has been used to activate previously silent secondary metabolite gene clusters. However, there is no easy way to assess the presence of γ-butyrolactone-like systems in Streptomyces strains without whole-genome sequencing. We have therefore developed and tested a PCR screen that is able to detect homologues of the commonly co-located butenolide synthase and γ-butyrolactone receptor genes. This PCR screen could be employed for the screening of strain libraries to detect signalling systems without the necessity for whole-genome sequencing.
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The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. Thank you for you resubmission, with the changes made, i am happy to accept the manuscript. Congratulations and we welcome future submissions to Access!
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Comments to Author
The article in question concerns the development and testing of a PCR screen to detect homologs of co-located butanolide synthase and γ-butyrolactone receptor genes in Streptomyces strains. γ-butyrolactone is utilised by Streptomyces, along with other actinobacteria to control the production of secondary metabolites, including antibiotics. 1558 occurrences of ScbA homologs were found across 1020 assemblies, with 1092 of those occurrences within an intergenic distance of 600bp. 751 of the 1092 co-occurances were in a divergent position for the two genes, resulting in this orientation being used going forward for primer design. To estimate primer efficiency, the authors analysed the composition of the chosen binding sites in the 751 divergently oriented ScbA/ScbR homologs. When testing the primers …
Comments to Author
The article in question concerns the development and testing of a PCR screen to detect homologs of co-located butanolide synthase and γ-butyrolactone receptor genes in Streptomyces strains. γ-butyrolactone is utilised by Streptomyces, along with other actinobacteria to control the production of secondary metabolites, including antibiotics. 1558 occurrences of ScbA homologs were found across 1020 assemblies, with 1092 of those occurrences within an intergenic distance of 600bp. 751 of the 1092 co-occurances were in a divergent position for the two genes, resulting in this orientation being used going forward for primer design. To estimate primer efficiency, the authors analysed the composition of the chosen binding sites in the 751 divergently oriented ScbA/ScbR homologs. When testing the primers against extracted Streptomyces DNA, it was found that certain isolates possessed rare amino acid substitutions in the first or second N-terminal positions of the primer binding motif, resulting in no primer binding. However, in instances where the expected motif was bound, or where the motif possessed a substitution after the second N-terminal position, expected band lengths were seen. The paper illustrates the journey from concept of a PCR screen for γ-butyrolactone-like receptor genes to designing and trialling primers for the aforementioned screen in a concise and detailed manner and is easy to follow. While from reading it is clear that the PCR screen is not perfect (owed to its inability to bind to motifs with substitutions in the first and second N-terminal position), it is still a fantastic step in developing a novel screen for determining the presence of γ-butyrolactone-like systems in Streptomyces. Future work to consider includes widening the screen size of Streptomyces species, development of alternative primers for different motifs or different orientations, and application of this system in other receptor genes to determine efficacy with other genes. Overall, I approve of this paper's submission to Access Microbiology. Minor Revisions: Line 79: Include the number of PCR cycles Line 181: scbA and scbR should be italicised
Please rate the manuscript for methodological rigour
Very 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
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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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Thank you for your submission. Following reviewers comments i've selected minor corrections. Please address each point for resubmission.
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Comments to Author
Waschulin et al. report a PCR method for identifying scbR-scbA operons in Streptomyces species. The authors' identified 1091 putative scbR-scbA operons from genomic analysis. The majority these operons (751 operons) were divergently encoded, for which the authors then identified conserved amino acid motifs for primer design. The primers, overall, worked reasonably well, but did not detect all operons, because of nucleotide mismatches between template and the 3' end of primer(s). The authors suggested ways the PCR method could be improved and noted a clever and facile way of applying their method would be to design a standardised CRISPR-Cas9 vector targeting a considered ~20nt region to knockout the locus. Minor comments only: Abstract/Introduction - Be aware that the use of specialised vs secondary …
Comments to Author
Waschulin et al. report a PCR method for identifying scbR-scbA operons in Streptomyces species. The authors' identified 1091 putative scbR-scbA operons from genomic analysis. The majority these operons (751 operons) were divergently encoded, for which the authors then identified conserved amino acid motifs for primer design. The primers, overall, worked reasonably well, but did not detect all operons, because of nucleotide mismatches between template and the 3' end of primer(s). The authors suggested ways the PCR method could be improved and noted a clever and facile way of applying their method would be to design a standardised CRISPR-Cas9 vector targeting a considered ~20nt region to knockout the locus. Minor comments only: Abstract/Introduction - Be aware that the use of specialised vs secondary metabolite is controversial. It is up to you whether to continue to use 'specialised', however the following article would suggest (and I agree) that its use should be restricted to cases where the in situ function of a metabolite has been established https://pubs.rsc.org/en/content/articlelanding/2020/np/c9np00048h Line 43 - It is probably better to use '…manipulate components…' instead of '…manipulate elements…' just to avoid confusion with AREs. Line 61 - Please give a little more detail, for example, how was R used (what package(s)?) to assess gene orientation. Line 181 - scbR and scbA should be in italics.
Please rate the manuscript for methodological rigour
Very 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
-
Comments to Author
Waschulin and colleagues have designed degenerate oligonucleotides for screening Streptomyces strains for the presence of gamma-butyrolactone-like regulatory systems. These systems have been demonstrated to be important in the regulation of specialised metabolism in several strains already and therefore represent a useful target for manipulation to effect production of specialised metabolites. I support the publication of this manuscript in Access Microbiology and I think the content of the manuscript has been completed to a high level. However, the screen designed is only for the detection of the two target genes when in an opposite orientation (~69% of co-occurrences are in divergent orientations). The successful identification of divergent gene pairs is believed to be ~72%. This means ~50% of gene …
Comments to Author
Waschulin and colleagues have designed degenerate oligonucleotides for screening Streptomyces strains for the presence of gamma-butyrolactone-like regulatory systems. These systems have been demonstrated to be important in the regulation of specialised metabolism in several strains already and therefore represent a useful target for manipulation to effect production of specialised metabolites. I support the publication of this manuscript in Access Microbiology and I think the content of the manuscript has been completed to a high level. However, the screen designed is only for the detection of the two target genes when in an opposite orientation (~69% of co-occurrences are in divergent orientations). The successful identification of divergent gene pairs is believed to be ~72%. This means ~50% of gene pairs are possible to identify. Although I sympathise with the authors about the cases where the degenerate oligonucleotides don't work in the divergent orientation, I don't understand why the authors didn't make additional oligonucleotides to screen when the two genes are in other orientations (the remaining 31% of loci). I recommend the authors improve the manuscript by designing and testing these additional oligonucleotides. This will ultimately provide the field with a more definitive screen for gamma-butyrolactone-like regulatory systems. Minor revisions include; -Line 79, include the number of PCR cycles used. -Line 103, the figure reference should be Figure 1B. -Line 104, the figure reference should be Figure 1C. -Line 137, the figure reference should be Figure 1D. -Table 3, include a column to indicate if the PCR product has been confirmed to be the correct intergenic region. -Line 183; without genome sequencing I am curious about the application of CRISPR-Cas9 as target guide RNA specificity cannot be predicted, repair templates can't be designed, etc. It could be possible without verifying the specificity of the guide RNA's to modify a single nucleotide using CRISPR-BEST but you can only modify a base at a time and I think you would probably need to change a few bases in the ARE sequence, and without the specificity of the guide RNA it would be taking a rather large leap of faith.
Please rate the manuscript for methodological rigour
Very 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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