eDNA, Amyloid Fibers and Membrane Vesicles Identified in Pseudomonas fluorescens SBW25 Biofilms
, Ianina Pokholenko 1, Olga Iungin 1,2, Geert Potters 3,4,* and Andrew J. Spiers 5Round 1
Reviewer 1 Report
This is an interesting and carefully done paper. It adds considerably to our knowledge of the biofilm made by the wrinkly spreader mutant of SBW 25 which has been used as a model system.
I have two minor corrections:
Figure 2 some of the bars lack letters above them.
Lines 360-2 The reasoning here is not clear to me. Please expand this.
Author Response
This is an interesting and carefully done paper. It adds considerably to our knowledge of the biofilm made by the wrinkly spreader mutant of SBW 25 which has been used as a model system.
I have two minor corrections:
Figure 2 some of the bars lack letters above them.
This was apparently an issue of converting the original figure to a pdf, and we will send the original pptx files to the Editor when the paper will be accepted. Suffice to say that all bars should have the text “10 µm” above them.
Lines 360-2 The reasoning here is not clear to me. Please expand this.
We propose to change this sentence to: "Since a cellulose-deficient Wrinkly Spreader mutant does not produce a biofilm in experimental microcosms [24], we can conclude that eDNA is not the only critical component of biofilm formation."
Reviewer 2 Report
The authors investigated “eDNA, amyloid fibers and membrane vesicles identified in Pseudomonas fluorescens SBW25 biofilms”. The manuscript falls into the scope of the journal. The manuscript is well-written and contains valuable information. The content is meaningful and understandable. The structure of the MS is considerably organized. The authors present adequate information in the methodology to allow the reproduction of the estimated methods properly. However, the findings are partially novel. I have just a few questions/comments.
1) Bacterial biofilm-EPS contains curli fimbriae, proteins, nanocellulose rich polysaccharides, nucleic acids, lipids, and amide A of peptidoglycan and amide B of peptidoglycan (https://0-doi-org.brum.beds.ac.uk/10.3389/fmicb.2020.542053; https://0-doi-org.brum.beds.ac.uk/10.1016/j.envpol.2022.12023; https://0-doi-org.brum.beds.ac.uk/10.1038/s41598-022-09519-9). Therefore, it is suggested to improve the introduction section with more recent citations.
2) Line # 37-41: Please simplify the statement, also delete “see”, and write [8-10].
3) Line # 52: Delete “reviewed by”. A full stop is needed after [18,19), and delete the first bracket.
4) Line # 55: Delete the first bracket.
5) Line # 61-62: A full stop is needed after [21), and delete the first bracket.
6) Line # 69-73: Please simplify the statement.
7)Line # 77-78: Include the reference of https://0-doi-org.brum.beds.ac.uk/10.3389/fmicb.2020.542053. In this article, the authors shown that eight Pseudomonas species forms air-liquid biofilm isolated from rhizosphere soil of tomato plants grown in drought prone ecosystem.
8) Nucleic acid (i.e., eDNA and eRNA) is one of the major components of biofilm-EPS. Is it possible to identify the eDNA using Furrier Transform Infrared Spectroscopy?
Author Response
1) Bacterial biofilm-EPS contains curli fimbriae, proteins, nanocellulose rich polysaccharides, nucleic acids, lipids, and amide A of peptidoglycan and amide B of peptidoglycan (https://0-doi-org.brum.beds.ac.uk/10.3389/fmicb.2020.542053; https://0-doi-org.brum.beds.ac.uk/10.1016/j.envpol.2022.12023; https://0-doi-org.brum.beds.ac.uk/10.1038/s41598-022-09519-9). Therefore, it is suggested to improve the introduction section with more recent citations.
6) Line # 69-73: Please simplify the statement.
7) Line # 77-78: Include the reference of https://0-doi-org.brum.beds.ac.uk/10.3389/fmicb.2020.542053. In this article, the authors shown that eight Pseudomonas species forms air-liquid biofilm isolated from rhizosphere soil of tomato plants grown in drought prone ecosystem.
We propose to answer comments 1, 6 and 7 by changing lines 69-74 as follows: “Several other pseudomonad species are known to produce eDNA, such as P. chlororaphis 30-84 [33] and P. putida KT2440 [34] in A-L interface biofilms. P. putida KT2440 also produces cellulose matrix-based biofilms and Wrinkly Spreader-like mutants [35-39], as do Pseudomonas sp. FW1 [40] in a S-L interface biofilm, and P. fluorescens B/S1000 in a slime [41]. Fourier-transform infrared spectroscopy spectra suggest eDNA, besides nanocellulose, may be in other pseudomonad A-L interface biofilms, e.g. [XX].” and add the suggested references at the site of the “XX”.
2) Line # 37-41: Please simplify the statement, also delete “see”, and write [8-10].
3) Line # 52: Delete “reviewed by”. A full stop is needed after [18,19), and delete the first bracket.
4) Line # 55: Delete the first bracket.
5) Line # 61-62: A full stop is needed after [21), and delete the first bracket.
The changes suggested in comments 2-5 have been introduced.
8) Nucleic acid (i.e., eDNA and eRNA) is one of the major components of biofilm-EPS. Is it possible to identify the eDNA using Furrier Transform Infrared Spectroscopy?
Bonds characteristic of nucleic acids can be identified by FTIR (eg. the two references of Haque et al. provided by Reviewer 2 themselves), but in complex mixtures such as a biofilm, some of these may be difficult to identify. Furthermore, FTIR cannot differentiate between inter- and intracellular nucleic acids, so to identify eDNA/eRNA, these analyses would need to be conducted on processed material (e.g., comparing DNase I treated samples – not done by Haque et al.). A sentence linking to FTIR has been added to lines 69-74 as indicated above.
