Membrane staining and phospholipid tracking in Pseudomonas aeruginosa PAO1 using the phosphatidylcholine mimic propargyl-choline
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The use of membrane-specific dyes for in vivo fluorescent microscopy is commonplace. However, most of these reagents are non-specific and cannot track specific lipid species movement, instead often acting as non-covalent lipid-associated probes or requiring the uptake of whole lipids and acyl tails into the membrane. This issue has been solved in eukaryotic cell biology by the use of click-chemistry-liable phospholipid headgroup pulse labels. Here, we describe a method for in vivo phospholipid labelling by fluorescent imaging in Pseudomonas aeruginosa using a phosphatidylcholine mimic, ‘propargyl-choline’ (PCho). This click-chemistry-liable headgroup mimic is visible by microscopy and allows the covalent labelling of lipids. Fluorescence of the cell membranes, visible in heterogeneous patches, is dependent on PCho concentration and is localized in the membrane fraction of cells, demonstrating that it is suitable for membrane labelling and cell imaging.
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Thank you very much for submitting your revised manuscript to Access Microbiology and addressing the reviewers' concerns. I am pleased to let you know that it has now been accepted for publication. Congratulations!
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The reviewers have highlighted minor concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
This manuscript describes a new membrane-labelling method in Pseudomonas aeruginosa. It is a straightforward paper that reads well and is mostly logical and clear. The authors show that this method for specific lipid labelling technically works well. I am less convinced by their data on labelling intensity on elongated cells, but the authors do show potential application of this new methods in tracking lipid movement in membranes. The authors compare this new labelling method with a more established method and discuss what are the areas of research where this new method may be more suitable than conventional methods. I have a list of comments below that the authors will need to address, which mostly relate to providing more clarity / details in the figures. Check legend of Fig. 1c - looks like 'of' …
Comments to Author
This manuscript describes a new membrane-labelling method in Pseudomonas aeruginosa. It is a straightforward paper that reads well and is mostly logical and clear. The authors show that this method for specific lipid labelling technically works well. I am less convinced by their data on labelling intensity on elongated cells, but the authors do show potential application of this new methods in tracking lipid movement in membranes. The authors compare this new labelling method with a more established method and discuss what are the areas of research where this new method may be more suitable than conventional methods. I have a list of comments below that the authors will need to address, which mostly relate to providing more clarity / details in the figures. Check legend of Fig. 1c - looks like 'of' is missing between 'maxima' and '1113' (or else the meaning of the sentence is unclear to me, in which case please clarify). Fig. 1c: more detail is needed to interpret this plot - I see various shades of green/yellow everywhere but don't know how they relate to fluorescence intensity. I guess a legend indicating how colours match fluorescence intensity could help (same comment for the BACTmap plots of Fig. 4). Fig. 3: I'm unclear about the 'banding patterns' and 'areas of higher intensity' that the authors claim they see in these cells - to me these elongated cells look more or less equally fluorescent across their membrane (but some cells clearly less intense than others). Please indicate (with arrows?) what banding/intensity the authors refer to. Also, please indicate how this image was selected - is it representative of a collection of pictures? Currently no information is given on this. Finally, the control of untreated cells is not shown here, even though it's referred to in the text. Is this an omission, or perhaps the authors wished to make a 'data no shown'-like statement? Please clarify. Fig. 4: not clear what is shown exactly in the (iii) and (iv) plots - I would have expected one plot per treatment, representing the average of the cells measured. Instead it shows 5 plots in total, please clarify (this fig has such low resolution that it is also impossible for me to read the axes so I just had to assume it was the same as for the plot in Fig. 3). Line 183-184: what do you mean with '..population of potential speeds over time..'. Please rephrase. Line 191: please write out TIRF Fig. 5: legend of this figure has gone wrong, please check. Lines 225-227: please elaborate how the use of PCho labelling in studying lipid movement relates to other labelling methods; i.e. do the authors think PCho is uniquely suitable for this application?
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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Comments to Author
The manuscript titled "Membrane staining and phospholipid tracking in Pseudomonas aeruginosa PAO1 using the phosphatidylcholine mimic propargyl-choline" describes a novel and interesting to method to label phospholipids in the bacterial membrane. The method has potential applications in studying membrane biology, as an alternative to other lipophilic fluorophores, such as the FM dyes. Sub-lethal amounts of the pCho reagent are used, and indeed the Cy3 fluorescence does label the bacterial membrane. The figures need some clarification. Comments. In Figure 1A, it appears that the propargyl-choline (pCho) binds to the phosphate in PC, but the structure of PC contains choline, not depicted in Figure 1A. It is not clear how pCho binds specifically to PC, and why "newly inserted PC lipids" are …
Comments to Author
The manuscript titled "Membrane staining and phospholipid tracking in Pseudomonas aeruginosa PAO1 using the phosphatidylcholine mimic propargyl-choline" describes a novel and interesting to method to label phospholipids in the bacterial membrane. The method has potential applications in studying membrane biology, as an alternative to other lipophilic fluorophores, such as the FM dyes. Sub-lethal amounts of the pCho reagent are used, and indeed the Cy3 fluorescence does label the bacterial membrane. The figures need some clarification. Comments. In Figure 1A, it appears that the propargyl-choline (pCho) binds to the phosphate in PC, but the structure of PC contains choline, not depicted in Figure 1A. It is not clear how pCho binds specifically to PC, and why "newly inserted PC lipids" are specifically labelled. Please clarify and elaborate. Is FM4-64X different than FM4-64? Can the pCho-Cy3 labelling be done in live cells without fixation? Cells were not actually 'fixed' but adhered or immobilized on an agarose bed, so this is a live cell imaging method. Can other Cy labels be used to expand the colour range? Does this labelling method label the outer membrane (OM), inner membrane (IM) or both? A simple plasmolysis experiment where cells are treated with 0.5 M NaCl can be used to cause 'plasmolysis bays" where the IM shrinks and separates from the OM, due to the release of cytoplasmic contents due to the osmotic shock. In this way, the fluorescence can be monitored in the "bays" to determine if one or both membranes are labelled. Figure 2 is unclear. Is the short, black arrow labelled "pCho-Cy3" referring to just pCho-Cy3 or does it also include pCho-Cy3-Phosphatydylcholine? In the middle fluorescent panel, there is a red background but a white spot? Is that white spot what is being highlighted in the black arrow above, right side panel? I was expecting to see a red spot of PC that is labelled with pCho-Cy3. What are the two main lipid species on the TLC plates? Figure 3. The fluorescence in the filaments does not look membrane-localized, but rather is more diffuse and appears cytoplasmic. What is meant by "regions of higher fluorescence"? Is that regions of bright red within the red stained filaments? Or does this refer to the filaments not stained at all? What is a "banded region"? Figure 4. A high resolution image could not be downloaded and it is difficult to read the Figure legends, for parts iii and iv. Could not access the data in the google drive, unusual way to link data. Line 217. Could the authors briefly elaborate on how pCho could be used to visualize techoic acid, rather than asking the reader to go to the reference (but include the reference).
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
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