Identification of NpdA as the protein forming the surface layer in Paracidovorax citrulli and evidence of its occurrence as a surface layer protein in diverse genera of the Betaproteobacteria and Gammaproteobacteria
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Stylized cryo-electron image of two bacterial cells and an extracellular vesicle highlighting the presence of a surface layer (gold-colored structure).
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Thank you very much for submitting your revised manuscript to Access Microbiology. We really appreciate your diligence in addressing the reviewer concerns and applying their suggestions in this new version. After addressing these changes, the manuscript will be accepted for publication. Congratulations to all authors!
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Than you very much for submitting your manuscript to Access Microbiology. It has now been reviewed by two experts in the field, with whom I agree that the manuscript is well-written and clear, and the results are a valuable contribution to the field. However, the reviewers point out some concerns that need correction. Please pay special attention to the corrections involving the current nomenclature of the model species used in the study, the addition of further information on certain methodologies and the addition of the virulence results as a bar chart. With respect to image edition, I will kindly request to present the figures keeping the image edition as minimalist as possible, and in any case, providing the original, non-edited image as a supplementary figure. I also agree with the reviewers that some figures can be provided as …
Than you very much for submitting your manuscript to Access Microbiology. It has now been reviewed by two experts in the field, with whom I agree that the manuscript is well-written and clear, and the results are a valuable contribution to the field. However, the reviewers point out some concerns that need correction. Please pay special attention to the corrections involving the current nomenclature of the model species used in the study, the addition of further information on certain methodologies and the addition of the virulence results as a bar chart. With respect to image edition, I will kindly request to present the figures keeping the image edition as minimalist as possible, and in any case, providing the original, non-edited image as a supplementary figure. I also agree with the reviewers that some figures can be provided as supporting material rather than in the main text to improve the readability. Reviewer 2 suggests additional experiments to further increase the significance of the work. Please consider this suggestion, and in case it resulted unfeasible, discuss these aspects in the manuscript.
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Comments to Author
The authors have presented the role of NpdA in S-Layer formation in AAC00-1, as well as demonstrating the importance of this in biofilm formation. Although differences were seen in biofilm forming capabilities, when the authors tested the effects of npdA deletion strain in watermelon fruit, they did not see a link to "virulence". Main comments: 1) Is the strain Acidovorax citrulli (strain AAC00-1) renamed to be Paracidovorax citrulii? If not, could you make sure you refer to this strain as Acidovorax citrulli? 2) For the Methodological rigour, reproducibility and availability of underlying data: - Fiji (Image J) and Prism (GraphPad) softwares were used. Can the authors list which version of these software they used for their work? - Methods are explained in detail, except for Section 3.6 - this …
Comments to Author
The authors have presented the role of NpdA in S-Layer formation in AAC00-1, as well as demonstrating the importance of this in biofilm formation. Although differences were seen in biofilm forming capabilities, when the authors tested the effects of npdA deletion strain in watermelon fruit, they did not see a link to "virulence". Main comments: 1) Is the strain Acidovorax citrulli (strain AAC00-1) renamed to be Paracidovorax citrulii? If not, could you make sure you refer to this strain as Acidovorax citrulli? 2) For the Methodological rigour, reproducibility and availability of underlying data: - Fiji (Image J) and Prism (GraphPad) softwares were used. Can the authors list which version of these software they used for their work? - Methods are explained in detail, except for Section 3.6 - this methodology should be expanded to include more information on how the batch experiments were done. - Could all Cryo-EM / TEM data be deposited somewhere? 3) Presentation of results: - Figure 1, Panel A looks excessively contrasted. Could you please provide the original gel image, without bleaching it? Are these two lanes run on the same gel? Authors should include a non-altered version of this image. - Figure 3, Could you please add a few more of the "detailed view" Cryo-EM images to this figure? The close-up detailed view for Panel B is lacking the clarity of the panel A detailed view. Additional images would help. - Biofilm and virulence on watermelon fruit results could be shown in bar graphs for visual help to the readers, rather than in-text mention alone (Line 360). - Figure 4 and 5 - Were the "nanopods" also checked/seen in npdA mutants? This would add to the findings presented in the manuscript. At the moment, this section (4.5) is in between npdA results, and does not read well. - Figure 6 - When zoomed in, the legends are un-readable and blurry. Could a higher resolution version of this figure be imported so can be read when zoomed in? 4) Suggested experiments: - Can the authors check the cell surface charge of the npdA mutant vs WT cells? This would add to the significance of the main findings of this work. - If the mutant vs WT is not injected, but is inoculated to the surface of an intact watermelon, would there be differences in their infection capability? 5) For the style and organization of the paper for communicating key findinfs: - Figure 1, Figure 2, and Figure 7 can all be supplemental figures, rather than main figures. Please re-arrange main figures accordingly. 6) Other comments: I really like the graphical abstract. Literature analysis and discussion reads well. Line 58: didn't -> did not. Line 238: pipet -> pipette. 489: surface energy -> surface charge? Line 506: it's -> it is Line 641: it's -> it is
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
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?
Yes: Figure 1 - Panel A: Are these two lanes run on the same gel? Background is bleached out a lot / contrast settings played with?... Should use non-altered version of this image.
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
In this study, the authors investigated the surface layer protein NpdA in the phytopathogen Paracidovorax citrulli. The authors demonstrated the presence of an NpdA-dependent S-layer using electron microscopy. Using a phylogenetic approach, the authors surveyed betaproteobacteria and gammaproteobacteria for the existence of NpdA orthologs. The presence of S-layer and cell envelope structures was further examined in select species through electron microscopy. Notably, in P. citrulli, the data suggest that NpdA production leads to a deficiency in biofilm formation. The paper is well-written and clear. My main concern pertains to the nanopod experiments. While the authors beautifully showed the existence of extracellular structures, the structures were quite different in planktonic and biofilm culture. …
Comments to Author
In this study, the authors investigated the surface layer protein NpdA in the phytopathogen Paracidovorax citrulli. The authors demonstrated the presence of an NpdA-dependent S-layer using electron microscopy. Using a phylogenetic approach, the authors surveyed betaproteobacteria and gammaproteobacteria for the existence of NpdA orthologs. The presence of S-layer and cell envelope structures was further examined in select species through electron microscopy. Notably, in P. citrulli, the data suggest that NpdA production leads to a deficiency in biofilm formation. The paper is well-written and clear. My main concern pertains to the nanopod experiments. While the authors beautifully showed the existence of extracellular structures, the structures were quite different in planktonic and biofilm culture. Does the formation of both types of "nanopods" requires NpdA in P. citrulli? Without the evidence for NpdA, this section, together with figures 4 and 5, appears somewhat disjointed from the rest of the manuscript. Additional comments: 1. In Section 4.4, it could be nice if the authors plotted the data from the crystal violet assay and the virulence test into a figure. 2. In tables 2 and 3, a number of species names are listed as "species." Are these species unidentified or omitted due to some other reason?
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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