Microplate-Based Quantification of Poly-γ-Glutamic Acid (γ-PGA) levels in Biofilm Samples
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Poly-γ-glutamic acid (PGA) is a commercially useful biopolymer produced by many Bacillus species. PGA has a diverse range of applications across medicine and industry generating significant interest in optimising PGA production and enhancing yields. One approach to improve PGA recovery involves identification of high-yield PGA producer strains and determining optimal production conditions, both of which require an appropriate screening method. Here we present a sensitive and reproducible assay for quantifying PGA from B. subtilis biofilms, whereby the spectral profile of methylene blue changes when bound to PGA. PGA was purified and lyophilised from NCIB 3610 ΔtasA liquid cultures grown at 50°C, allowing production of protein-free PGA for use as a standard at known concentrations. Standard curves were generated from methylene blue absorbance readings at 564 nm and 664 nm, enabling subsequent quantification of PGA from biofilm extracts. We validated the quantification protocol and determined the treatment steps required to minimise interference. The assay has a 96-well plate format, enabling quantification of many samples at low sample volume, while minimising waste of laboratory consumables. Overall, our method offers a sensitive, reproducible approach for PGA quantification in biofilm research and should facilitate comparative analyses across strains, treatments, or environmental conditions.
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Thank you for submitting your manuscript to Access Microbiology addressing te reviewers' suggestions. I am pleased to inform you that your manuscript is now accepted for publication. Congratulations!
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Comments to Author
The work : "Microplate-Based Quantification of Poly-γ-Glutamic Acid (γ-PGA) levels in Biofilm Samples" by Steneson et al., is a valuable contribution to the field. I noticed the absence of high throughput screening method for PGA years ago, therefore the described method is welcome. However, there are a few points, where I believe clarification or additional explanation will make the presenting method more convincing and useful. 1. Terminology and text clarity: authors frequently use "normalize" in different context. Although it is never fully wrongly applied, it is often confusing. In analytical chemistry, "normalized" typically means division of a (measured) value by a reference or scaling to 1, often leading to the expression of relative values. In the proposed article, "normalized" appears in some …
Comments to Author
The work : "Microplate-Based Quantification of Poly-γ-Glutamic Acid (γ-PGA) levels in Biofilm Samples" by Steneson et al., is a valuable contribution to the field. I noticed the absence of high throughput screening method for PGA years ago, therefore the described method is welcome. However, there are a few points, where I believe clarification or additional explanation will make the presenting method more convincing and useful. 1. Terminology and text clarity: authors frequently use "normalize" in different context. Although it is never fully wrongly applied, it is often confusing. In analytical chemistry, "normalized" typically means division of a (measured) value by a reference or scaling to 1, often leading to the expression of relative values. In the proposed article, "normalized" appears in some instances to mean " to adjust by procedure" as , for example in the case: "Biofilm extract samples should be normalised to 5-10 μg protein per sample/well depending on sample type." Probably it was meant: "Biofilm extract samples should be adjusted to contain 5-10 μg protein per sample/well depending on sample type." In other instances authors use normalise to say that the samples were background corrected. Indeed, in analytical chemistry, there are in some methods procedures where normalization can reduce background, but in the present case, the background absorbance was subtracted from the sample absorbance- I believe the division by a factor or similar was not done. On the other hand, in some instances "normalised" appears to be used correctly, i.e. meaning division to obtain relative values as in the case: "Protein values were used to normalise samples for SDS-PAGE and PGA quantification." Authors should take care that they use "normalize" properly. The use of symbol " / " : in some instances it is not clear if it means "or" or "per" or it is just redundant. Yes, often it can be inferred from the context, but it makes it more difficult to read. For example: "In a spreadsheet normalise/blank correct the raw data…" or "The greatest/maximum peaks formed (when PGA binds methylene blue) should be around 564 nm and 664 nm." or "Methylene blue staining/assay solutions were prepared…" or "Spectral profile of methylene blue incubated with 10 μg (black dotted line), 1 μg (green dotted line), and 0.1 μg (blue dotted line) of protein/biofilm extract from NCIB 3610 capB in BugBuster." Authors should use "/" in an unambiguous way or omit it throughout the text. It is not well presented why BugBuster® Master Mix was used. At first mention in line 84-85 it says : "BugBuster® Master Mix (Merck Millipore Novagen, 71456-4) was used for biomass disruption and extraction." Extraction of what? One would think, it is for PGA- but this reagent is used lo lyse the cells, so one could get intracellular content. This appears again contra intuitive as PGA is extracellular. It appears one would contaminate the PGA by additional intracellular proteins and DNA, RNA, which needs to be removed by DNase and Proteinase in later steps as given by the authors. After further reading of the manuscript it becomes clear to me that authors wanted to normalize the amount of PGA per unit protein in the biofilm. This information is important and should be better presented or justified to the reader. On the other hand, wouldn't be better to divide the sample of the biofilm into two parts: 1. One only to extract PGA ( no BugBuster) 2. and one two obtain total proteins (with BugBuster) and protein analysis? Perhaps this suggestion is discussed as an option or alternative to the proposed method. Also, considering the method for protein quantification, i.e. BCA. In a given case it might be OK, but according to my experience, whenever there are reducing sugars present in the sample, BCA will detected them as well. I have used this method to observe polysaccharide degradation. I believe authors should mention this potential disadvantage of BCA that can occur, especially if the growth media containing sugars is used. For PGA detection and quantification two method are presented SDS-PAGE, which authors say is less sensitive and therefore preffer VIS absorbance spectroscopy for quantification of PGA. SDS-PAGE: Fig. 1. This gel is convincing me that ∆tas dosen't produce TasA, but reader is left to simply trust that a cloud in the beginning of the gel is PGA. Here, I believe the author should give a reference or add a gel line showing that in ∆capB no cloud appears in the beginning. Fig. 2. Here, when I first looked at gels I was not sure if there is protein or PGA changing or both…also, which I think is a slight problem in the manuscript, the biofilm extract and proteins are used almost interchangeably and this is confusing. I believe, the authors use always biofilm extracts that contain proteins and protein content in the biofilm extract is manipulated only by diluting the biofilm extracts. So, PGA (ul) + protein , PGA alone label above the gel should be: biofilm extract + PGA (uL), PGA alone (uL). In figure caption "in (B)" in first line is redundant. VIS spectroscopy: Fig 3. This figure was supposed to convince the reader that the method with methylene blue can be tweaked to be specific for detection of PGA. But, it actually gave the opposite impression. Perhaps something is not clearly labelled or I don't understand it. I will describe how I see it, so authors can improve it accordingly. From Fig. 3AB it follows- ∆capB, which doesn't have PGA produces the expected peak shift for PGA ( at 564 nm increased absorbance and decreased absorbance at 664 nm relative to Methyleneblue only solution). This suggest strong interference with non-PGA substances. Here, a comparison of the spectrum of wt strain would be welcome. Now, in 3C the authors reduced the spectrum intensity of interfering substances, by deploying proteinase and DNase, by something like 30-40 %. The rest is still there- perhaps this is from EpsA-O that was recently shown to contain negatively charged pyruvyl residues (https://doi.org/10.1038/s41522-024-00555-z) that could potentially bind positively charged methylene blue. Then in Fig. 3D we see spectra of protein extracts that are probably biofilm extracts diluted to contain the shown protein concentrations. Well, the absorbance decreases with the dilution of the biofilm extract, but I do not see how this contributes to the specificity of the methylene method for PGA. 3E then shows what happens when you add PGA to most diluted biofilm extract. The background corrected spectrum appears the same as a green spectrum in Fig 3D where no PGA is present. I guess, authors will understand from my comments where was the problem. Now, even if the method has interference drawback that cannot be fully accounted for, the method might still be useful. One can still run a large survey of many potential PGA producers and find out those that produce best quantities according to the methylene blue method described. way And the isolated PGA from a few strains (best producers) can then be quantified and confirmed by some more specific method as for example, HPLC, as we have done in the past in our lab. Authors should discuss this scenario, if their method lacks specificity. A huge time saving can be obtained in this way. Fig 4: In Fig. caption 0.25 mg/mL is probably 0.025 mg/mL In the section Procedure: Analysis of PGA by electrophoresis: maybe describe what the user should see in the end of electrophoresis. Is cooking samples at exactly 99 °C crucial or why not saying 100 °C? Quantification of PGA: Static? It was likely meant static electricity.
Please confirm that no generative AI tools or large language models have been used to generate this peer review report or to assist with any part of the peer review process.
I confirm no generative AI tools were used in preparation of this review.
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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Thank you for submitting your manuscript to Access Microbiology. It has now been reviewed by two experts in the filed, whose comments are attached here. They were pleased to see an optimised protocol for the quantification of PGA in biofilms, and they have provided some suggestions and feedback to further strengthen the manuscript. Based on their recommendations, please provide a revised manuscript (including a tracked-changes document) along with a point-by-point response to the reviewers' comments within 2 months.
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Comments to Author
Overall, this is a really nice paper outlining a methodology for quantifying the amount of poly-g-glutamate secreted by Bacillus subtilis, a technique the field currently lacks and would benefit greatly from. Fig 2B. The lower majority of the gel is dark. Is this a problem with the image or conversion to pdf? Figure legend 3. I think the figure legend title could be made more specific "Protease and DNase treatment reduces background interference from biofilm extracts." Line 219. Confirm and/or clarify the citation to Fig 3D. Fig 3D looks like a titration of extract rather than an experiment testing the effect of DNase/protease treatment. The authors eliminate the contribution of DNA and protein to the absorbance measured in the assay but what about the biofilm EPS? Even if the EPS …
Comments to Author
Overall, this is a really nice paper outlining a methodology for quantifying the amount of poly-g-glutamate secreted by Bacillus subtilis, a technique the field currently lacks and would benefit greatly from. Fig 2B. The lower majority of the gel is dark. Is this a problem with the image or conversion to pdf? Figure legend 3. I think the figure legend title could be made more specific "Protease and DNase treatment reduces background interference from biofilm extracts." Line 219. Confirm and/or clarify the citation to Fig 3D. Fig 3D looks like a titration of extract rather than an experiment testing the effect of DNase/protease treatment. The authors eliminate the contribution of DNA and protein to the absorbance measured in the assay but what about the biofilm EPS? Even if the EPS doesn't react with methylene blue, wouldn't it add mass to the standard curve made from purified PGA? I'm not sure it is a problem per se, but the authors to use a tasA eps double mutant to check.
Please confirm that no generative AI tools or large language models have been used to generate this peer review report or to assist with any part of the peer review process.
I confirm no generative AI tools were used in preparation of this review.
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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