In vitro peptidoglycan degradation assay using FITC labelled sacculi

  1. Dema Alodaini
  2. Adam Hart
  3. Patrick J. Moynihan
  4. Manuel Banzhaf

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Abstract

We describe a method using fluorescein-5-Isothiocyanate (FITC) labeled peptidoglycan sacculi to characterize peptidoglycan hydrolases in vitro. In this protocol soluble hydrolytic products released by peptidoglycan hydrolases are separated from the insoluble FITC-sacculi, quantified and set in relation to the overall available substrate. This enables the study of hydrolase activity in end-point or time- course experiments.

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  1. Access Microbiology

    Dear Authors, thank you for submitting your manuscript to Access Microbiology. It has now been reviewed by two experts in the field, whose comments are attached at the bottom of this email. Both reviewers noted that your assay could be replicated, and I believe it would be a valuable addition to the field. However, I welcome you to revise your manuscript in line with the reviewer comments.

  2. Access Microbiology

    Comments to Author

    The instructions provided in the methodology description are clear and can be followed. The pH of the suspension in point 2.3.1 is missing To enhance the validity of the procedure, it would be useful if you compare the results obtained with other protocols you mention in the introduction (Glauner. 1988) Several methods have been described in the literature since 1988. A comparison with them and the limitations they have, compared to the advantage of uours, would be useful and would higlight your contribution.

    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

    Poor

    Please rate the quality of the presentation and structure of the manuscript

    Poor

    To what extent are the conclusions supported by the data?

    Not at all

    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?

    No: there is no animal or human work involved

  3. Access Microbiology

    Comments to Author

    The authors described an interesting protocol to measure PG degradation using FITC labelled sacculi. The protocol is clear and I only have some minor comments: 1. To clarify how the assay works it would be nice to have a supportive graphic. 2. Line 101: I would replace `for no less` by `at least` 3. Line 116: Please add `PG `before `reaction buffer` (to ensure consistency)

    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

    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

  4. Version published to 10.1099/acmi.0.001178.v1 on Access Microbiology