Technical note: Flow cytometry assays for the detection, counting and cell-sorting of polyphosphate-accumulating bacteria

Bouquet, Clémentin; Billard, Hermine; Bidaud, Cécile; Colombet, Jonathan; Chang, Young-Tae; Benzerara, Karim; Skouri-Panet, Fériel; Duprat, Elodie; Lehours, Anne-Catherine

doi:https://doi.org/10.5194/egusphere-2024-1085

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https://doi.org/10.5194/egusphere-2024-1085

Preprints

02 May 2024

| 02 May 2024

Technical note: Flow cytometry assays for the detection, counting and cell-sorting of polyphosphate-accumulating bacteria

Clémentin Bouquet, Hermine Billard, Cécile Bidaud, Jonathan Colombet, Young-Tae Chang, Karim Benzerara, Fériel Skouri-Panet, Elodie Duprat, and Anne-Catherine Lehours

Abstract. In the context of the ecological sustainability of phosphorus (P), the emerging evidence of the ubiquitous presence of polyphosphate-accumulating bacteria (PAB) in natural environments invites efforts to reveal their unknown functions and roles in the biogeochemical cycle of P. This requires high-throughput methods to characterise PAB structure and dynamics in the environment. A promising strategy is to combine efficient staining of intracellular polyphosphate (polyP) granules in PAB and their subsequent detection by flow cytometry, enabling rapid data acquisition and multiparametric analysis. In this study, we provide a generic protocol for the detection, quantification, and cell sorting of PAB by flow cytometry using the dye 4’6-diamidino-2-phenylindole (DAPI). The assays were performed using Tetrasphaera elongata, which represent a large part of the microbial biomass in enhanced biological phosphate removal systems for wastewater treatment. We also included, as a negative control, a bacterial strain characterized by very low quantities of cellular polyP and carried out tests on water and lake sediment samples. We also show that the synthetic fluorochrome JC-D7, a new selective fluorescent dye used for the specific labeling of endogenous polyP in living cells, is promising for achieving these purposes, particularly in complex environmental samples.

Received: 10 Apr 2024 – Discussion started: 02 May 2024

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07 Apr 2025

Technical note: Flow cytometry assays for the detection, counting and cell sorting of polyphosphate-accumulating bacteria

Clémentin Bouquet, Hermine Billard, Cécile C. Bidaud, Jonathan Colombet, Young-Tae Chang, Joan Artigas, Isabelle Batisson, Karim Benzerara, Fériel Skouri-Panet, Elodie Duprat, and Anne-Catherine Lehours

Biogeosciences, 22, 1729–1744, https://doi.org/10.5194/bg-22-1729-2025,https://doi.org/10.5194/bg-22-1729-2025, 2025

Short summary

Clémentin Bouquet, Hermine Billard, Cécile Bidaud, Jonathan Colombet, Young-Tae Chang, Karim Benzerara, Fériel Skouri-Panet, Elodie Duprat, and Anne-Catherine Lehours

Interactive discussion

Status: closed

RC1:
'Comment on egusphere-2024-1085', Anonymous Referee #1, 03 May 2024

The preprint of Bouquet et al. aims to establish a FACS approach for detection, sorting, and quantification of polyphosphate-accumulating bacteria in environmental samples. PolyP is an interesting energy-rich substance accumulated intracellularly by many organisms. However, we still lack a clear understanding of which taxa accumulate PolyP and under what environmental circumstances. Consequently, I agree with the authors that such an approach, and especially a relatively high-throughput approach via FACS, would be very helpful for future research.
General comments:
The authors grew a known PolyP-accumulating strain (T. elongata) and used a second strain as a control (their own isolate), that still showed “low” levels of PolyP accumulation. This is one of the first flaws of the experimental approach, as the control strain did in fact also accumulate polyphosphates. The authors tested a multitude of conditions for staining and storage of cells, although it does not become clear why all these conditions were tested in the first place. More severely, most of the resulting data is either not shown or not conclusive (see comments Figure 2 and 3). Following their tests, the authors applied their FACS approach to environmental samples and find that the FACS detection highly overestimates the cell numbers/proportions as compared to microscopy (which is the benchmarking tool). Surprisingly, the authors conclude that their approach is robust. I strongly disagree with this conclusion. Also, I fail to understand how the authors classified JC-D7 as a useful PolyP-stain when it failed to show similar numbers to DAPI (which is also has a questionable PolyP-specificity). The methods section is not detailed enough to allow for reproduction of the presented approach and tests. Many data are not shown despite the possibility to add data to a supplementary file. I ask the authors to resubmit a carefully revised version where all necessary data is shown, where the specificity of DAPI as a PolyP stain is critically discussed which will then also allow to critically interpret the data produced in this study. Currently, I cannot support the notion that the presented FACS-approach is a robust method to quantify and sort PolyP-accumulating cells from environmental samples. Please also include more literature including (https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1046/j.1462-2920.2001.00164.x, https://journals.asm.org/doi/10.1128/aem.02592-12, https://doi.org/10.1038/s41396-019-0399-7)

Specific comments:
Please do not use abbreviations in the abstract
It seems that JC-D7 and DAPI could be applied together as their spectrum is different from each other. This would also allow to see how specific both stains are.
L65f: add some results please
L77: I guess it should be MgSO4 x 7 H2O?
L901. 1500 x g does not sound much? How were the authors sure this sample did not contain particulate organic matter or non-microbial particles that could cause autofluorescence in the FACS?
L94: remove “The” at the beginning of the sentence please
L97f: please remove the ‘.’ Between µg or mg and ml-1. It should be µm ml-1. This appears throughout the manuscript
L100: what was the solvent used for DAPI stocks and why were they stored at -20°C?
L103: please choose to use either concentration or molarity throughout the methods section
113: what does qsp stand for?
L120: µm not µM
L125: Darmstadt
L126: how were the cells stored at these different temperatures? This is critical information missing
L131: again, please remove the dot ‘.’ between cell and s-1
L129 and L140: is it correct that two different machines were used for the FACS analysis (counting and sorting)?
L150f: it sounds as if the cells were filtered onto a black membrane, DAPI-stained, washed and DAPI-stained again. Is this correct? If so, why were the cells stained twice?
L153: Did the authors not use a mounting medium between filters and coverslips (e.g. Citifluor or Vectashield)?
L165 and also before: please indicate what solution was used to prepare the formaldehyde fixative. Water, 1 x PBS, or something else? Same applies for all stains (e.g. JC-D7) and solutions used. Please be more diligent in adding information
L181: it does not become clear what independent staining refers to and how that helped to establish thresholds. Please elaborate.
L185f: what software was used to perform statistical analyses?
Figure 1: Please try to use another micrograph for Figure 1B where the cells are magnified in a similar way as in Figure 1C.
L246: It does not become clear what the authors mean with “population structure”. Why would the staining buffer interfere with population structure in the first place?
L254: I am convinced that in the era of almost unlimited online space there is no need for “data not shown” anymore. I would like to ask the authors to show these data as well.
L288: why is this reference showing up in the brackets again? It is clear from the methods how the labelling was performed.
L290: Again, it is pretty unacceptable to not show these results. Please add them to the note or to a supplementary file so the readers can judge your conclusions.
L290: How did the authors decide if there was a strong or a weak fluorescent signal. This needs to be clarified in order to judge the results, e.g. the data shown in Figure 2C (comparison of HEPES and PBS).
L294f and Figure 1C: The interpretation of these results is pretty unclear to me. The FCM detection of DAPI-stained PolyP signal in the strain that supposedly did accumulate low values of PolyP in HEPES buffer showed a PolyP signal for 99% of the cells. This is surprising. The authors then state that in PBS, the number of cells with a PolyP signal was around 1%. Given the size of the bar in Figure 1C, this value should be around 5-10%. Please clarify. The authors then state that under PBS and microscopy a “correct” ratio was observed which is why they excluded HEPES buffer from further experimentation. First of all, how do the authors know what the correct value is if their “control” strain accumulated PolyP as well? Also, HEPES buffer without any P should in principle be more suited for PolyP detection than PBS, which introduces P to the cells. In addition, why does the microscopy data and the FCM data not agree more with each other? Microscopy should be the control here so this means that FCM highly overestimates the PolyP-detection?
Also I would like to ask the authors to also show the data of Figure 2C with TE cells. What are the proportions here? How did HEPES buffer fare in comparison to PBS?
L304: The data referred to here is not shown
Figure 3B shows the proportion of PolyP detected in cells RX (low-accumulators) for different fixation treatments (8-14%). These proportions are similar to the values shown in Figure 2C where they were referred to as 1%. Again, the data does not really add up and disagrees with the microscopy data shown in Figure 2C. In addition, I want to see the proportion of TE cells according to these tests as they were also counted (see Figure 3A). Why were they not shown in the first place?
L332: I am highly surprised to see that the storage at -80°C did not affect cell morphology, and thus detectability of PolyP. But again the authors chose to not show these data.
L334: How did the authors prepare the 50/50 mixture relative abundance for these tests?
L383f: The overestimation was already visible in Figures 2 and 3 which indicates that FCM as established in this study is not very useful for the quantitative detection of PolyP accumulating strains
L424: I strongly disagree with the conclusion that the “present study established the basis of a robust protocol for the detection and enrichment of PAB by flow cytometry”. The current study, in my opinion, fails to establish the FACS analysis in a comparable manner to fluorescence microscopy which serves as the benchmark.

Citation: https://doi.org/10.5194/egusphere-2024-1085-RC1
- AC1: 'Reply on RC1', Anne-Catherine Lehours, 02 Jul 2024
  
  The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2024/egusphere-2024-1085/egusphere-2024-1085-AC1-supplement.pdf
  
  Citation: https://doi.org/10.5194/egusphere-2024-1085-AC1
RC2:
'Comment on egusphere-2024-1085', Anonymous Referee #2, 06 May 2024
The paper submitted by Bouquet et al. tries to establish a protocol to efficiently stain, detect and and sort polyphosphate accumulating bacteria using flow cytometry. Considering the importance of poly-P in wastewater treatment systems and the limited knowledge on poly-P cycling in natural environments such a protocol would be very useful.
General comments:
I have three major issues with this submission.

The experimental set-up; using T. elongata as a positive control is excellent and the use of environmental samples is good and necessary since a high-throughput method that is able to detect poly-P in natural samples could potentially give much more insights into the spatial and temporal dynamics of polyphosphate cycling. However, no real negative control has been used. A strain named “RX” was used as a negative control, even though it was shown to accumulate poly-P. Why not use an actual negative control? The data that has been collected for the “RX” strain could be an excellent addition to show how the protocol functions for species with minor amounts of poly-P, which might be relevant in natural settings.

A lot of the data is not shown. Add this data, either to the main results or the supplement.

The authors claim that this is a robust protocol whereas the data tells a different story. The observed differences between the proportion of poly-P cells detected by either FCM or Epifluorescence microscopy in sediment samples (Fig 2C, Fig 4BEF, Fig 5C and Fig 6C) shows that this is not a robust protocol and that FCM grossly overestimates the amount of cell accumulating poly-P.

Overall, I would conclude that the combination of DAPI staining and FCM would not work and that this method should not be used to detect and quantify polyP. However, since DAPI staining combined with fluorescence microscopy is often used to detect polyP, it is still important to get the knowledge out there to show that it does not work when combining with FCM. The data shown here where the dye FC-D7 was used seems to be more promising but requires more data (i.e., fluorescence microscopy to show that it works in prokaryotes) and a similar validation protocol as presented here for DAPI.
Specific comments:
Introduction
I think the introduction could elaborate a bit more or the functions and ubiquitousness of polyphosphate and put it in an environmental context. Almost all bacteria have the enzymatic potential for poly-P built-up and breakdown and besides being used as a storage compounds/energy reserve, many functions are known and none of them are mentioned in the introduction here. The following papers are excellent references:

Brown, M. R. W., and Kornberg, A. (2004). Inorganic polyphosphate in the origin and survival of species. Proc. Natl. Acad. Sci. 101, 16085–16087. doi: 10.1073/ pnas.0406909101

Kornberg, A. (1995). Inorganic polyphosphate - toward making a forgotten polymer unforgettable. J. Bacteriol. 177, 491–496. doi: 10.1128/jb.177.3.491496.1995

Kornberg, A., Rao, N. N., and Ault-riché, D. (1999). Inorganic polyphosphate: a molecule of many functions. Annu. Rev. Biochem. 68, 89–125. doi: 10.1146/ annurev.biochem.68.1.89

Rao, N. N., Gómez-García, M. R., and Kornberg, A. (2009). Inorganic polyphosphate: essential for growth and survival. Annu. Rev. Biochem. 78, 605–647. doi: 10.1146/annurev.biochem.77.083007.093039

Material and methods
L75: What are “large amounts” of polyP? Is it a large fraction of the cell volume? And if so, how much? Do most cells accumulate poly-P? And if so, how much?
L78: What is “a very small amount of intracellular polyP? Only few cells or does polyP only take up a small amount of the cell volume?
Results
Figure 1A: Beautiful TEM image, the A is on a weird spot in the figure. I would like to see false quantitative color scales for the EDX analyses if that is possible.
Figure 1BC: Make the figures bigger.
L247: What is meant by “population structure”?
L254 & 255: Show the data somewhere.
L290: Show the data.
L292 and Figure 2C: There are significant differences between the proportion of polyP+ cells observed by labeling with DAPI in PBS using the FCM or epifluorescence microscopy. Why is this? Isn’t this already a first insight the DAPI in combination with FCM is not an appropriate method to detect polyP accumulating bacteria because there are too many false positives?
Also, do you have any idea why the use of HEPES would give you so many polyP+ cells? Please elaborate.
Figure 3: Both staining periods, DAPI concentrations, and fixation with formaldehyde seem to significantly affect the detection of polyP+ cells with FCM. How was this with epifluorescence microscopy? Is the detection of polyP+ cells using fluorescence microscopy also affected by the staining period and DAPI concentration? This seems especially relevant since the differences in detection by epifluorescence microscopy when compared to FCM all seem to be significant according to the data presented in figure 1C, and later in Figure 4B, E, F, Figure 5B and Figure 6C. Also, even though there are no significant differences observed between fixed and unfixed polyP+ cells for T. elongata (L307), still show the data.
L332: Show the data. Especially because storage is highly relevant for natural samples.
Figure 4B, 4E, 4F: Where are the statistics between FCM and Epifluorescence? By eyeballing it, the differences seem significant and FCM appears to overestimate the amount of polyP+ cells before sorting and underestimates the amount op polyP+ cells after sorting.
L337: What is significant here? Which values are compared? FCM before sorting to FCM after sorting?
L339: PAB represented less than 10% of the polyP- fraction according to Epifluorescence but the proportion found by FCM is much lower. Why is this?
L378: I do not think that the method has been validated. There are significant differences observed between the FCM and Epifluorescence microscopy in almost every figure shown. The only thing that is validated is that this protocol can be used to enrich polyP+ cells (Figure 4 and Figure 5A).
Figure 5B: This really shows that this is not a robust method to be used for environmental samples.
L399-400: Show the data. Was this fluorescence intensity measured with FCM or epifluorescence microscopy.
Figure 6: Why was there no (epi)fluorescence microsocopy performed with JC-D7 labeling? And what is the significance between JC-D7 and DAPI-epifluo in the sediment? Again, the enormous difference between DAPI stained poly+ cells observed via FCM and Epifluorescence show that this is not a robust method.
The focus of the paper lies on the use of DAPI but looking at Figure 6, the dye JC-D7 looks much more promising for the use in natural samples (or at least for sediment samples). I think validating JC-D7 as a polyP specific dye in combination with epifluorescence microscopy that can also be used in prokaryotes would be a big step forward.
Discussion
L423: This line does not seem to be relevant here.
L458-462: I think this is an overestimation of what this protocol can do. In almost all the data shown, the observations with FCM and epifluorescence are significant, and especially in the natural samples, the differences are striking (Figure 5B and Figure 6C). The only thing that is shown is that polyP+ cells can be enriched. I would not recommend DAPI staining in combination with FCM to quantify the amount of polyP accumulating bacteria in a natural sample based on this data.
L469-472: This conclusion cannot be made without comparing the different staining periods and DAPI concentrations with epifluorescence microscopy.
L481: Show the results. The publication of negative results is important so other researchers do not have to try it for themselves.
L484-485: The data obtained shows promising results but it does not show the specific nature of PAB labeling. This would require fluorescence microscopy to visualize the JC-D7 dye in combination with for instance SEM-EDS so co-localization analysis can be performed
Citation: https://doi.org/10.5194/egusphere-2024-1085-RC2
- AC2: 'Reply on RC2', Anne-Catherine Lehours, 02 Jul 2024
  
  The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2024/egusphere-2024-1085/egusphere-2024-1085-AC2-supplement.pdf
  
  Citation: https://doi.org/10.5194/egusphere-2024-1085-AC2

Interactive discussion

Status: closed

RC1:
'Comment on egusphere-2024-1085', Anonymous Referee #1, 03 May 2024

The preprint of Bouquet et al. aims to establish a FACS approach for detection, sorting, and quantification of polyphosphate-accumulating bacteria in environmental samples. PolyP is an interesting energy-rich substance accumulated intracellularly by many organisms. However, we still lack a clear understanding of which taxa accumulate PolyP and under what environmental circumstances. Consequently, I agree with the authors that such an approach, and especially a relatively high-throughput approach via FACS, would be very helpful for future research.
General comments:
The authors grew a known PolyP-accumulating strain (T. elongata) and used a second strain as a control (their own isolate), that still showed “low” levels of PolyP accumulation. This is one of the first flaws of the experimental approach, as the control strain did in fact also accumulate polyphosphates. The authors tested a multitude of conditions for staining and storage of cells, although it does not become clear why all these conditions were tested in the first place. More severely, most of the resulting data is either not shown or not conclusive (see comments Figure 2 and 3). Following their tests, the authors applied their FACS approach to environmental samples and find that the FACS detection highly overestimates the cell numbers/proportions as compared to microscopy (which is the benchmarking tool). Surprisingly, the authors conclude that their approach is robust. I strongly disagree with this conclusion. Also, I fail to understand how the authors classified JC-D7 as a useful PolyP-stain when it failed to show similar numbers to DAPI (which is also has a questionable PolyP-specificity). The methods section is not detailed enough to allow for reproduction of the presented approach and tests. Many data are not shown despite the possibility to add data to a supplementary file. I ask the authors to resubmit a carefully revised version where all necessary data is shown, where the specificity of DAPI as a PolyP stain is critically discussed which will then also allow to critically interpret the data produced in this study. Currently, I cannot support the notion that the presented FACS-approach is a robust method to quantify and sort PolyP-accumulating cells from environmental samples. Please also include more literature including (https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1046/j.1462-2920.2001.00164.x, https://journals.asm.org/doi/10.1128/aem.02592-12, https://doi.org/10.1038/s41396-019-0399-7)

Specific comments:
Please do not use abbreviations in the abstract
It seems that JC-D7 and DAPI could be applied together as their spectrum is different from each other. This would also allow to see how specific both stains are.
L65f: add some results please
L77: I guess it should be MgSO4 x 7 H2O?
L901. 1500 x g does not sound much? How were the authors sure this sample did not contain particulate organic matter or non-microbial particles that could cause autofluorescence in the FACS?
L94: remove “The” at the beginning of the sentence please
L97f: please remove the ‘.’ Between µg or mg and ml-1. It should be µm ml-1. This appears throughout the manuscript
L100: what was the solvent used for DAPI stocks and why were they stored at -20°C?
L103: please choose to use either concentration or molarity throughout the methods section
113: what does qsp stand for?
L120: µm not µM
L125: Darmstadt
L126: how were the cells stored at these different temperatures? This is critical information missing
L131: again, please remove the dot ‘.’ between cell and s-1
L129 and L140: is it correct that two different machines were used for the FACS analysis (counting and sorting)?
L150f: it sounds as if the cells were filtered onto a black membrane, DAPI-stained, washed and DAPI-stained again. Is this correct? If so, why were the cells stained twice?
L153: Did the authors not use a mounting medium between filters and coverslips (e.g. Citifluor or Vectashield)?
L165 and also before: please indicate what solution was used to prepare the formaldehyde fixative. Water, 1 x PBS, or something else? Same applies for all stains (e.g. JC-D7) and solutions used. Please be more diligent in adding information
L181: it does not become clear what independent staining refers to and how that helped to establish thresholds. Please elaborate.
L185f: what software was used to perform statistical analyses?
Figure 1: Please try to use another micrograph for Figure 1B where the cells are magnified in a similar way as in Figure 1C.
L246: It does not become clear what the authors mean with “population structure”. Why would the staining buffer interfere with population structure in the first place?
L254: I am convinced that in the era of almost unlimited online space there is no need for “data not shown” anymore. I would like to ask the authors to show these data as well.
L288: why is this reference showing up in the brackets again? It is clear from the methods how the labelling was performed.
L290: Again, it is pretty unacceptable to not show these results. Please add them to the note or to a supplementary file so the readers can judge your conclusions.
L290: How did the authors decide if there was a strong or a weak fluorescent signal. This needs to be clarified in order to judge the results, e.g. the data shown in Figure 2C (comparison of HEPES and PBS).
L294f and Figure 1C: The interpretation of these results is pretty unclear to me. The FCM detection of DAPI-stained PolyP signal in the strain that supposedly did accumulate low values of PolyP in HEPES buffer showed a PolyP signal for 99% of the cells. This is surprising. The authors then state that in PBS, the number of cells with a PolyP signal was around 1%. Given the size of the bar in Figure 1C, this value should be around 5-10%. Please clarify. The authors then state that under PBS and microscopy a “correct” ratio was observed which is why they excluded HEPES buffer from further experimentation. First of all, how do the authors know what the correct value is if their “control” strain accumulated PolyP as well? Also, HEPES buffer without any P should in principle be more suited for PolyP detection than PBS, which introduces P to the cells. In addition, why does the microscopy data and the FCM data not agree more with each other? Microscopy should be the control here so this means that FCM highly overestimates the PolyP-detection?
Also I would like to ask the authors to also show the data of Figure 2C with TE cells. What are the proportions here? How did HEPES buffer fare in comparison to PBS?
L304: The data referred to here is not shown
Figure 3B shows the proportion of PolyP detected in cells RX (low-accumulators) for different fixation treatments (8-14%). These proportions are similar to the values shown in Figure 2C where they were referred to as 1%. Again, the data does not really add up and disagrees with the microscopy data shown in Figure 2C. In addition, I want to see the proportion of TE cells according to these tests as they were also counted (see Figure 3A). Why were they not shown in the first place?
L332: I am highly surprised to see that the storage at -80°C did not affect cell morphology, and thus detectability of PolyP. But again the authors chose to not show these data.
L334: How did the authors prepare the 50/50 mixture relative abundance for these tests?
L383f: The overestimation was already visible in Figures 2 and 3 which indicates that FCM as established in this study is not very useful for the quantitative detection of PolyP accumulating strains
L424: I strongly disagree with the conclusion that the “present study established the basis of a robust protocol for the detection and enrichment of PAB by flow cytometry”. The current study, in my opinion, fails to establish the FACS analysis in a comparable manner to fluorescence microscopy which serves as the benchmark.

Citation: https://doi.org/10.5194/egusphere-2024-1085-RC1
- AC1: 'Reply on RC1', Anne-Catherine Lehours, 02 Jul 2024
  
  The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2024/egusphere-2024-1085/egusphere-2024-1085-AC1-supplement.pdf
  
  Citation: https://doi.org/10.5194/egusphere-2024-1085-AC1
RC2:
'Comment on egusphere-2024-1085', Anonymous Referee #2, 06 May 2024
The paper submitted by Bouquet et al. tries to establish a protocol to efficiently stain, detect and and sort polyphosphate accumulating bacteria using flow cytometry. Considering the importance of poly-P in wastewater treatment systems and the limited knowledge on poly-P cycling in natural environments such a protocol would be very useful.
General comments:
I have three major issues with this submission.

The experimental set-up; using T. elongata as a positive control is excellent and the use of environmental samples is good and necessary since a high-throughput method that is able to detect poly-P in natural samples could potentially give much more insights into the spatial and temporal dynamics of polyphosphate cycling. However, no real negative control has been used. A strain named “RX” was used as a negative control, even though it was shown to accumulate poly-P. Why not use an actual negative control? The data that has been collected for the “RX” strain could be an excellent addition to show how the protocol functions for species with minor amounts of poly-P, which might be relevant in natural settings.

A lot of the data is not shown. Add this data, either to the main results or the supplement.

The authors claim that this is a robust protocol whereas the data tells a different story. The observed differences between the proportion of poly-P cells detected by either FCM or Epifluorescence microscopy in sediment samples (Fig 2C, Fig 4BEF, Fig 5C and Fig 6C) shows that this is not a robust protocol and that FCM grossly overestimates the amount of cell accumulating poly-P.

Overall, I would conclude that the combination of DAPI staining and FCM would not work and that this method should not be used to detect and quantify polyP. However, since DAPI staining combined with fluorescence microscopy is often used to detect polyP, it is still important to get the knowledge out there to show that it does not work when combining with FCM. The data shown here where the dye FC-D7 was used seems to be more promising but requires more data (i.e., fluorescence microscopy to show that it works in prokaryotes) and a similar validation protocol as presented here for DAPI.
Specific comments:
Introduction
I think the introduction could elaborate a bit more or the functions and ubiquitousness of polyphosphate and put it in an environmental context. Almost all bacteria have the enzymatic potential for poly-P built-up and breakdown and besides being used as a storage compounds/energy reserve, many functions are known and none of them are mentioned in the introduction here. The following papers are excellent references:

Brown, M. R. W., and Kornberg, A. (2004). Inorganic polyphosphate in the origin and survival of species. Proc. Natl. Acad. Sci. 101, 16085–16087. doi: 10.1073/ pnas.0406909101

Kornberg, A. (1995). Inorganic polyphosphate - toward making a forgotten polymer unforgettable. J. Bacteriol. 177, 491–496. doi: 10.1128/jb.177.3.491496.1995

Kornberg, A., Rao, N. N., and Ault-riché, D. (1999). Inorganic polyphosphate: a molecule of many functions. Annu. Rev. Biochem. 68, 89–125. doi: 10.1146/ annurev.biochem.68.1.89

Rao, N. N., Gómez-García, M. R., and Kornberg, A. (2009). Inorganic polyphosphate: essential for growth and survival. Annu. Rev. Biochem. 78, 605–647. doi: 10.1146/annurev.biochem.77.083007.093039

Material and methods
L75: What are “large amounts” of polyP? Is it a large fraction of the cell volume? And if so, how much? Do most cells accumulate poly-P? And if so, how much?
L78: What is “a very small amount of intracellular polyP? Only few cells or does polyP only take up a small amount of the cell volume?
Results
Figure 1A: Beautiful TEM image, the A is on a weird spot in the figure. I would like to see false quantitative color scales for the EDX analyses if that is possible.
Figure 1BC: Make the figures bigger.
L247: What is meant by “population structure”?
L254 & 255: Show the data somewhere.
L290: Show the data.
L292 and Figure 2C: There are significant differences between the proportion of polyP+ cells observed by labeling with DAPI in PBS using the FCM or epifluorescence microscopy. Why is this? Isn’t this already a first insight the DAPI in combination with FCM is not an appropriate method to detect polyP accumulating bacteria because there are too many false positives?
Also, do you have any idea why the use of HEPES would give you so many polyP+ cells? Please elaborate.
Figure 3: Both staining periods, DAPI concentrations, and fixation with formaldehyde seem to significantly affect the detection of polyP+ cells with FCM. How was this with epifluorescence microscopy? Is the detection of polyP+ cells using fluorescence microscopy also affected by the staining period and DAPI concentration? This seems especially relevant since the differences in detection by epifluorescence microscopy when compared to FCM all seem to be significant according to the data presented in figure 1C, and later in Figure 4B, E, F, Figure 5B and Figure 6C. Also, even though there are no significant differences observed between fixed and unfixed polyP+ cells for T. elongata (L307), still show the data.
L332: Show the data. Especially because storage is highly relevant for natural samples.
Figure 4B, 4E, 4F: Where are the statistics between FCM and Epifluorescence? By eyeballing it, the differences seem significant and FCM appears to overestimate the amount of polyP+ cells before sorting and underestimates the amount op polyP+ cells after sorting.
L337: What is significant here? Which values are compared? FCM before sorting to FCM after sorting?
L339: PAB represented less than 10% of the polyP- fraction according to Epifluorescence but the proportion found by FCM is much lower. Why is this?
L378: I do not think that the method has been validated. There are significant differences observed between the FCM and Epifluorescence microscopy in almost every figure shown. The only thing that is validated is that this protocol can be used to enrich polyP+ cells (Figure 4 and Figure 5A).
Figure 5B: This really shows that this is not a robust method to be used for environmental samples.
L399-400: Show the data. Was this fluorescence intensity measured with FCM or epifluorescence microscopy.
Figure 6: Why was there no (epi)fluorescence microsocopy performed with JC-D7 labeling? And what is the significance between JC-D7 and DAPI-epifluo in the sediment? Again, the enormous difference between DAPI stained poly+ cells observed via FCM and Epifluorescence show that this is not a robust method.
The focus of the paper lies on the use of DAPI but looking at Figure 6, the dye JC-D7 looks much more promising for the use in natural samples (or at least for sediment samples). I think validating JC-D7 as a polyP specific dye in combination with epifluorescence microscopy that can also be used in prokaryotes would be a big step forward.
Discussion
L423: This line does not seem to be relevant here.
L458-462: I think this is an overestimation of what this protocol can do. In almost all the data shown, the observations with FCM and epifluorescence are significant, and especially in the natural samples, the differences are striking (Figure 5B and Figure 6C). The only thing that is shown is that polyP+ cells can be enriched. I would not recommend DAPI staining in combination with FCM to quantify the amount of polyP accumulating bacteria in a natural sample based on this data.
L469-472: This conclusion cannot be made without comparing the different staining periods and DAPI concentrations with epifluorescence microscopy.
L481: Show the results. The publication of negative results is important so other researchers do not have to try it for themselves.
L484-485: The data obtained shows promising results but it does not show the specific nature of PAB labeling. This would require fluorescence microscopy to visualize the JC-D7 dye in combination with for instance SEM-EDS so co-localization analysis can be performed
Citation: https://doi.org/10.5194/egusphere-2024-1085-RC2
- AC2: 'Reply on RC2', Anne-Catherine Lehours, 02 Jul 2024
  
  The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2024/egusphere-2024-1085/egusphere-2024-1085-AC2-supplement.pdf
  
  Citation: https://doi.org/10.5194/egusphere-2024-1085-AC2

Peer review completion

AR: Author's response | RR: Referee report | ED: Editor decision | EF: Editorial file upload

ED: Reconsider after major revisions (09 Jul 2024) by Lucia Fuchslueger

AR by Anne-Catherine Lehours on behalf of the Authors (24 Jul 2024) Author's response Author's tracked changes Manuscript

ED: Referee Nomination & Report Request started (01 Aug 2024) by Lucia Fuchslueger

RR by Anonymous Referee #1 (05 Aug 2024)

Suggestions for revision or reasons for rejection

The revised version of the technical note represents a highly improved manuscript as compared to the first version. I would like to thank the authors for addressing many of my previous points.

The study revolves around the comparison of PolyP/DNA labelling methods and downstream quantification/sorting via microscopy and cell sorting and counting. The methods are mainly tested on two pure cultures. One of them (T. elongata) is a well-documented PolyP-accumulating strain. All sorting and counting efforts worked very well with T. elongata in this study. On the other side there is strain “RX”, that does not seem to comply with the methods at all. Unfortunately, it remains difficult to speculate what could be the issue with this strain as it remains unclear which group of bacteria it belongs to. I ask the authors to detail the taxonomy of this strain as they have certainly performed sanger sequencing if it was taken from a ‘library of bacterial strains’ located at the LMGE. This will also allow that others can repeat similar experiments. In general, the low number of the strains tested together with the differing results does not allow to draw conclusions on how well the approach actually works for a mixed community. In fact, the mixed data presented in Figure 4 is everything but convincing. The application of JC-D7 looks promising, especially in the sediment samples. I would enjoy to see more data/testing on the usage of JC-D7 with potentially some downstream analysis of PolyP or sequencing.

Specific questions:
Why was JC-D7 diluted in HEPES buffer when it was shown to cause problems with strain RX?

L204: I still do not understand why there was the need to stain cells twice. First cells were stained with a high concentration (different conc in the manuscript as given in the response to the reviewers), washed and then stained with a low concentration. The authors argue that the second staining was done to stain the DNA of the cells but that was already achieved during the high concentration staining in my opinion. DAPI does not bind specific to a target based on the concentration. It does not create a problem for the analysis, but this could be unnecessary and introduce bias.

Figure 2: where could the artifact in the McIlvaine buffer come from? The buffer only contains NaH2PO4, citric acid and water. Would it be possible to show the exact same panels for Tris-EDTA for PBS, HEPES, McIlvaine in the supplements? This way one could judge better.

Figure 3 and other data show that there cannot be a unified protocol for all bacteria when there is such a high difference between a high accumulator and low accumulator strain?

Figure 4: It is shown in Figures 3 and 5 that almost 100% of the T. Elongata cells are repeatedly counted/detected via FCM and epifluorescence microscopy. For this strain a good congruence between FCM and microscopy extist. After mixing the strains T.elongata and RX in equal cell number (50:50 abundance ratio) both methods only detect 36.5 or 12.6 % of PolyP+ cells when it should be approximately 50%. How do the authors explain this discrepancy? Either the cells were not mixed in equal abundance, or the counting does not work anymore as soon as a second bacterial strain is in the mixture which makes the approach not robust enough for any type of mixed communities.

Fig. 4: Why was only one sample counted with FCM? This makes it very difficult to judge the result, especially if the goal is to show that FCM is a robust way to count cells after sorting

Fig5: Here JC-D7 looks promising in the sediment, that would actually be something to extend on

Previosuly unanswered questions/points:

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RR by Anonymous Referee #2 (12 Aug 2024)

Suggestions for revision or reasons for rejection

Overall, I think the manuscript has improved and that it is much more realistic about the capacities of the tested methods. The authors have clearly taken the reviewer comments into accounts and I appreciate both the additional paragraphs (4.2 and 4.3) as well as the absence of “data not shown”. I agree with the response that this information needs to be published to avoid pitfalls for other researchers and to improve on the methodology and I would therefore recommend it for publication after another round of minor revisions.

I do think that the authors should be more careful with some of the wording and that they need to specify what other experiments need to be done in order to get a more comprehensive view of the methodology (see comments below).

L61: Change to: (< µm)
L86: “carried out out tests on microbial cells extracted from water and lake sediment samples”
L90: It also points out that..

L100: There is no natural negative control. However, it is possible to modify species to get a true negative control by knocking out the ppk gene (see e.g.,: https://www.pnas.org/doi/full/10.1073/pnas.060030097, https://www.pnas.org/doi/full/10.1073/pnas.170283397). I do not think the authors should do this and the reasoning behind using a low poly-P accumulator are valid, but be precise in the formulations.

L345 and Figure 2C, L 344-347, L463-465: While I agree with the authors that the HEPES buffer should not be used to analyze DAPI-stained cells with FCM, I would also argue that PBS is not great, at least for the low polyP accumulators and caution should be applied. I do not think that the proportion is in “a simlar range” (L344-L347) (7.2% is very different from 0.9% and accounts for a significant fraction of false positives) and the significant difference observed already shows that this method is problematic for low poly-P accumulating organisms. I would state that the PBS buffer is a better option that the HEPES buffer and therefore, one should not use HEPES but using PBS buffer is already a compromise and also results in artefactual labeling and false positives.

Figure 3A and 3C: I think it is good that the authors tested the different conditions of fixation, storage time and storage temperature for the RX strain. It is clear that this affects the propotion of polyP cells measured but it is very hard to give any advice on sample preparation if there is no “ground truth” present, i.e, if we do not know the amount of actual poly-P+ cells present (i.e., measured by epifluorescence microscopy). Is it in a similar range as the amount measured in Figure 1C (~0.9% poly-P+ cells)? If so, why does the proportion of polyP+ differ so much from the actual amount? And why does the proportion significantly decrease after fixation with PFA?

Furthermore, RX is a gram-negative strain and it is unclear whether the differences between the treatments are the result of the low polyP accumulation or from the difference in the membrane structure. Overall, it is not possible to draw conclusions about sample preservation on gram negative strains or low polyP accumulators from the data presented here since the testing was done on a low polyP gram-negative strain. To untangle the results, one would have to test the same protocolson a gram-negative strain that accumulates high amounts of poly-P and a gram-positive strain that accumulates very little poly-P.

Line 372: There is no figure 3D, it is only figure 3C.

L475-480: I agree with what the authors state here but specify that this only works for gram-positive high polyP+ accumulating organisms, it does not work for gram-negative low polyP accumulators and that further testing is needed for gram-positive low polyP accumulators and gram-negative high polyP accumulators.

L499-501: This would only work if the polyP accumulators accumulate high amounts of polyP
L510-515: It disagree with the statement that it shows the proportions of polyP+ cells in a congruent manner. To me, it did not show this in a congruent manner for flow cytometry in the RX strain. The differences are significant and a lot of false positives would be measured overestimating the amount of polyP+ cells present. More research would be needed to establish this. For now, the only application is ‘simple’ matrices in the case of gram-positive high polyP accumulators.

L516: remove the comma and the however, that way the sentence is much clearer.
L519: remove the very
L520-521: I would specify that you can effectively enrich and isolate PAB that accumulate large amount of polyP, as this method would not work for low polyP accumulators.

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ED: Reconsider after major revisions (06 Sep 2024) by Lucia Fuchslueger

AR by Anne-Catherine Lehours on behalf of the Authors (25 Nov 2024) Author's response Author's tracked changes Manuscript

ED: Referee Nomination & Report Request started (03 Dec 2024) by Lucia Fuchslueger

RR by Anonymous Referee #1 (16 Dec 2024)

RR by Nicole Geerlings (30 Dec 2024)

ED: Publish subject to technical corrections (22 Jan 2025) by Lucia Fuchslueger

AR by Anne-Catherine Lehours on behalf of the Authors (29 Jan 2025) Author's response Manuscript

Journal article(s) based on this preprint

07 Apr 2025

Technical note: Flow cytometry assays for the detection, counting and cell sorting of polyphosphate-accumulating bacteria

Biogeosciences, 22, 1729–1744, https://doi.org/10.5194/bg-22-1729-2025,https://doi.org/10.5194/bg-22-1729-2025, 2025

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Clémentin Bouquet, Hermine Billard, Cécile Bidaud, Jonathan Colombet, Young-Tae Chang, Karim Benzerara, Fériel Skouri-Panet, Elodie Duprat, and Anne-Catherine Lehours

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https://doi.org/10.5194/egusphere-2024-1085-supplement

Clémentin Bouquet, Hermine Billard, Cécile Bidaud, Jonathan Colombet, Young-Tae Chang, Karim Benzerara, Fériel Skouri-Panet, Elodie Duprat, and Anne-Catherine Lehours

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In the context of the ecological sustainability of phosphorus (P), the ubiquitous presence of polyphosphate-accumulating bacteria (PAB) in natural environments invites efforts to reveal their unknown roles in the biogeochemical cycle of P. In this study, we provide a generic protocol for the detection, quantification, and cell sorting of PAB by combining efficient staining of intracellular polyphosphate (polyP) granules in PAB and their subsequent detection by flow cytometry.


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