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The original Figure 1 was deleted and a few words added to the legend of the original Figure 2, now the new Figure 1.
| 2 | 1 |
Figure 1 is a bit redundant. A short comment about the inclusion of that particular sequence into the Thermoplasmatales can be added into the legend of Figure 2.
| 1 | 2 |
life5010949_makarova
| 1 |
Judging by the cluster labeled as Euryarchaeote{0+3} in Figure 2 Methanomassiliicoccus was not reclassified into Thermoplasmataceae but to an yet un-specified class.
| 2 | 1 |
However, the reclassification of Methanomassiliicoccus into Thermoplasmataceae needs more explanation. To be objective, authors should address the following question, why in the same family and not in another new family?
| 1 | 2 |
life5010949_makarova
| 1 |
The sentence has been moved to the legend of Fig. 1 and slightly rephrased.
| 2 | 1 |
L106–107: maybe a bit inappropriate on that position. I would suggest to add a comment on the figure legend instead.
| 1 | 2 |
life5010949_makarova
| 1 |
Done.
| 2 | 1 |
Part 3.2 L109–118: Summarize and move to introduction. Those sentences are of general importance for the topic and not specific to part 3.2.
| 1 | 2 |
life5010949_makarova
| 1 |
the K issue is discussed in the newly added “Material and Method” section; so scattered mentioning of K has been deleted from the rest of text.
| 2 | 1 |
L119–123: if the K issue is relevant to understand the text then please add a proper explanation. If not, then keep it simple and avoid entering into the K issue (L122–123, L132, L135, L139, 183, and also remove this K = 6 from Figure 3.)
| 1 | 2 |
life5010949_makarova
| 1 |
Done in the caption of Figure 2.
| 2 | 1 |
Add more explanations in legend of Figure3.
| 1 | 2 |
life5010949_makarova
| 1 |
We have rewritten the paragraph as: “The new phylum Korarchaeota violates the monophyly of the phylum Crenarchaeota by drawing to itself the family Thermofilaceae. However, in an on-going study of ours (not published yet) using a much larger data set, this violation no longer shows up; both R8 Korarchaeota and Crenarchaeota restore their phylum status. Taking into account the fact that both Korarchaeota and Thermofilaceae are represented by single species for the time being, their placement certainly requires further study with broader sampling of genomes.”
| 2 | 1 |
L143: Sounds clearer if you avoid mixing class and phylum, for example: “The placement of phylum Korarchaeota, as a closest neighbor of family Thermofilaceae, violates the monophyly of phylum Crenarchaeota.” Response:
| 1 | 2 |
life5010949_makarova
| 1 |
From the original Figures 3 and 4 only one has been kept and the legend rewritten. In fact, the whole paragraph changed to: “The newly proposed phylum Thaumarchaeota appears to be non-monophyletic as an outlying strain Candidatus Caldiarchaeum subterranum was assigned to this phylum according to the NCBI taxonomy. The NCBI assignment might reflect its position in some phylogenetic tree based on concatenated proteins, e.g., Figure 2 in […]. However, in the original paper reporting the discovery of this strain […] and in recent 16S rRNA studies, e.g., […], Candidatus Caldiarchaeum subterranum was proposed to make a new phylum Aigarchaeota. CVTrees support the introduction of this new phylum. A lineage modification of Candidatus Caldiarchaeum subterranum from Thaumarchaeota to Aigarchaeota would lead to a monophyletic Thaumarchaeota{7}.”
| 2 | 1 |
L146: No need to explain the 6+2 if it is properly explained in the figure legend. Figure 3 is redundant. I would recommend to avoid presenting different versions of the same tree; just the final tree is OK (use final/valid labels) and all important explanations in the text or legend. Perhaps the whole reasoning in Lines 142–180 is not so relevant for the current objective of comparing CVT, LTP, Bergeys? Or, perhaps, define this objective more clearly.
| 1 | 2 |
life5010949_makarova
| 1 |
The problem of taxonomic placement of Aciduliprofundum is a good example to demonstrate how one extract information from CVTrees. In the Reysenbach et al. Nature 2006 paper it was taken as the first cultivated member of the DHEV2 (deep-sea hydrothermal euryarchaeate 2) clade based on a maximum-likelihood 16S rRNA tree. Unfortunately, all other 13 members of this clade were represented by 16S rRNA sequences only and no genome data are available so far. The NCBI taxonomy gave an incomplete lineage: Archaea; Euryrchaeota; unclassified Euryarchaeota; missing taxonomic assignment at the rank class and below. In order to make use of CVTree we must touch on the K-issue a little more. The alignment-free comparison of genomes in CVTree is implemented by counting the number of K-peptides in the protein products encoded in a genome followed by subtraction of a random background caused by neutral mutations. The peptide length K looks like a parameter, but it is actually not a parameter. Using a longer K emphasizes species-specificity, while a shorter K takes into account more common features with neighboring species. However, we never adjust K: a fixed K is used for all genomes to construct a tree, but one may construct a series of trees for K = 3, 4, 5, 6, 7, 8, … We have shown repeatedly that R9 K = 5 and 6 lead to best results in the sense of agreement with taxonomy, so usually only a K = 6 tree is given in publications. Let us look at a subtree, i.e., part of a tree, containing the organisms of interest. If the branching order in all trees built for different Ks turns out to be same, it would be a strong support to the branching order. In most cases the branching order varies with K: K = 3 and 4 make sense, K = 5 and 6 yield the best, K = 7 and 8 become slightly worse, etc. For too big a K, even if the closest strains remain grouped together the whole tree may tend to become a star-tree, i.e., every small clade stands in its own and their mutual placements become less meaningful. Therefore, inspection of trees for a range of K-values provides an additional dimension to evaluate the results. For Aciduprofumdum we have a stable pair (<C>Thermococci{18}, <G>Aciduprofumdum{2}) at K=3, 5, 6, 7. At K = 4 we have (<C>Thermococci{18}, (<G>Staphylothermus{2}, <G>Aciduprofundum{2})) In all these cases Thermoplasmata stands farther away from the above pair. However, at K = 8 and 9, when the overall tree picture has been largely distorted, Aciduliprofundum does stand closer to Thermaplasmata. Putting together all the above results we tend to consider the pair (<C>Thermococci{18}, <G>Aciduliprofundum{2}) as reflecting a more probable relation. Confined to the available data for the time being one may assign <G>Aciduliprofundum to <C>Thermococci, e.g., to denote the pair as <C>Thermococci{20}=(<F>Thermococcaceae{18}, <G>Aciduliprofundum{2}) leaving its family unclassified or assign it to a new family. Without further phenotypic and chemotaxonomic evidence it is better not to introduce new taxon names if the present naming scheme is capable to accommodate the leaves without conflict. This was why we wrote “this modification would hold as long as no new facts challenge it”. Anyway, taxonomy has always been a work in progress. One has to be prepared for modifications when new data appear. To make a long story short, we have rewritten the paragraph as: “The Candidatus genus Aciduliprofundum is considered a member of the DHEV2 (deap-sea hydrothermal vent euryarchaeotic 2) phylogenetic cluster. No taxonomic information was given in the original papers [55,56]. The NCBI Taxonomy did not provide definite lineage information for this taxon at the class, order, and family ranks. According to [55] the whole DHEV2 cluster was located close to Thermoplamatales in a maximum-likelihood analysis of 16S rRNA sequences. A similar placement was seen in [54] where a Bayesian tree of the archaeal domain based on concatenation of 57 ribosomal proteins put a lonely Aciduliprofundum next to Thermoplasmata. However, in CVTrees, constructed for all K-values from 3 to 9, Aciduliprofundum juxstaposes with the class Thermococci{18}. An observation in [56] that this organism shares a rare lipid structure with a few species from Thermococcales may hint on its possible association with the latter. If we temporarily presume a lineage <C>Thermococci<O>Unclassified<F>Unclassified<G>Aciduliprofundum… R10 one might have a monophyletic class <C>Thermococci{20}. Since none of the 13 DHEV2 members listed in [55] has a sequenced genome so far, CVTree cannot tell the placement of the DHEV2 cluster as a whole for the time being. It remains an open problem as whether DHEV2 is close to Thermoplasmata or to Thermococci, or a new class is needed to accommodate DHEV2.”
| 2 | 1 |
L156–166. If I understood right, there was good support for Candidatus Aciduliprofundum as part of a clade called DHEV2, which is a sister clade of Thermoplasmatales. However in the present work the authors intend here to reclassify Aciduliprofundum into family Thermococcaceae of Thermococcales. This needs further explanation. Since the new affiliation is quite in disagreement with previous observations, and this is not properly justified in the results/discussion, the final statement “this modification would hold as long as no new facts challenge it” seems unacceptable. In addition, why should Aciduliprofundum be regarded as member of Thermococcaceae and not as another distinct family?
| 1 | 2 |
life5010949_makarova
| 1 |
No, we did not mean it.
| 2 | 1 |
L163: If I’m right the current observation actually does not support the previous work done by Brochier-Armanet.
| 1 | 2 |
life5010949_makarova
| 1 |
We should first explain how these inappropriate names appeared. We have insisted to use the directory name at the NCBI FTP site as genome name. However, in November 2013 NCBI announced that they would not release genomes of different strains of the same species as before. In a period thereafter NCBI sometimes put several genomes in a directory and we had to extract the data and to assign a name from the “Source” line of the GenBank file. This caused some confusion. For example, as of February 27, 2015, a directory name at NCBI remained “archaeon_Mx1201_uid196597” and we had to change it to: Candidatus_Methanomethylophilus_alvus_Mx1201_uid196597 Now all “wrong names” as pointed out by the Reviewer no longer appear in figures. In the text we tried to refer to their names as complete as possible.
| 2 | 1 |
L167–173: The names are wrongly written (please check the original submission). Authors have to explain with more clarity, why is this clade of rank class. If that is the case, is it a single-order class? A single family order?
| 1 | 2 |
life5010949_makarova
| 1 |
The genome name at NCBI FTP site is “halophilic_archaeon_DL31_uid72619”. The uid number was dropped when mentioned in the text. We put it back and capitalized the first letter to “Halophilic”, still an illegal genus name.
| 2 | 1 |
L174–180: I don’t understand the reasoning along this paragraph. In addition “haolphiic_archaeon_DL31” is not well written, please be careful when copying names from other source. Also, a similar question about the objectiveness for detecting high taxa: why not to create new family?
| 1 | 2 |
life5010949_makarova
| 1 |
It is Figure 2 in the revised manuscript. We discussed it at some length.
| 2 | 1 |
Figure 4: the reclassifications must be clearly justified in the text.
| 1 | 2 |
life5010949_makarova
| 1 |
No, organism cannot be published. Thanks for correcting our mistake.
| 2 | 1 |
L191: organisms can't be validly published, but their names.
| 1 | 2 |
life5010949_makarova
| 1 |
The latter can be assigned only after comparison with a reference taxonomy which is not a rigid framework but a modifiable system. Though a dissimilarity measure figures in the CVTree algorithm, it is not realistic to delineate taxa by using this measure at least for the time being. Even if defined in the future, it must be lineage-dependent. For example, it cannot be expected that the same degree of dissimilarity may be used to delineate classes in all phyla. In addition, monophyly is a guiding principle in comparing branching order with taxonomy. Here monophyly must be understood in a pragmatic way restricted to the given set of input data and the reference taxonomy. If all genomes from a taxon appear exclusively in a tree branch, the branch is said to be monophyletic.” R6 One cannot tell the rank of a node/leaf in a tree by simply looking at it. A reference taxonomy is alwys needed. We put the following in the “Material and Method” section to explain it: “Branching order in a tree by itself does not bring about taxonomic ranks, e,g, class or order.
| 2 | 1 |
Part 3.3 L221–222: Sure, but authors do not provide explanations about how do they know that a clade in a tree is a family, an order, a class, etc. There is a lack of criteria to reclassify the leaves.
| 1 | 2 |
life5010949_makarova
| 1 |
The phrase “3063 identical nucleotide positions” was copied from the caption of Figure 4 of the cited Nunoura et al. 2011 paper without much thinking. We simply deleted it.
| 2 | 1 |
L224: I don’t understand “3063 identical nucleotide positions”. Why identical?
| 1 | 2 |
life5010949_makarova
| 1 |
The whole paragraph has been rewritten as: “The nanosized archaean symbiont Nanoarchaeum equitans has a highly reduced genome (490,885 bp [44]). We note that the monophyly of Euryarchaeota was also violated by Nanoarchaeum in some 16S rRNA trees, see, e.g., Figure 4 in a 2009 paper [61] as well as (c) and (d) in our Figure 4. In fact, we have suggested skipping such tiny genomes when studying bacterial phylogeny, see, e.g., [29] and a note in the home page of the CVTree Web Server [21]. In the present case we may at most say that Nanoarchaeota probably makes a separate phylum, but its cutting into Euryarchaeota might be a side effect due to the tiny size of the highly reduced genome.”
| 2 | 1 |
L239–244: hard to read, please rephrase.
| 1 | 2 |
life5010949_makarova
| 1 |
As a method CVTree is independent of 16S rRNA analysis. First, it uses protein products in a genome instead of RNA segments in the genome. Second, it does not do sequence alignment. CVTree generates stable trees but cannot tell which branch corresponds to what taxon. Only after comparison with the existing classification and nomenclature one would be able to make connections with taxonomy. In this sense it does depend on 16S rRNA taxonomy. Anyway, CVTree does not challenge 16S rRNA analysis but makes it more convincing in most cases. The revealed discrepancies call for further study.
| 2 | 1 |
Conclusion I disagree that CVTree approach is independent of 16S, because authors are using the current accepted classification (which is mainly 16S-based) to validate the observed clades.
| 1 | 2 |
life5010949_makarova
| 1 |
In fact, genomic approaches are more effective at species level and below due to their high resolution power. At high ranks CVTree may be more effective in the sense that it does not require additional work. Suffice it to put genomes in CVTree web server and the branches come out, then compare them with a reference taxonomy.
| 2 | 1 |
Why at higher ranks, genomic approaches are more effective? That needs more explanation. And authors should also consider the large benefits of 16S data availability, specially at high ranks (genus and above) where the 16S has good resolution.
| 1 | 2 |
life5010949_makarova
| 1 |
Even “herenow” does not seem to be an correct English word; we changed it to “so far”.
| 2 | 1 |
L208: erenow → herenow
| 1 | 2 |
life5010949_makarova
| 1 |
We changed “is in contrast with” to “is distinct from” and added a phrase “focusing on taxonomy of higher ranks” at the end of a sentence. Now the sentences read: “This is distinct from phylogeny of species in a narrow range of taxa, e.g., that of vertebrates (a subphylum) or human versus close relatives (a few genera). Accordingly, the phylogeny should be compared with taxonomy at large, or, as Cavalier-Smith \cite{cavalier-smith2002} put it,with “megaclassificaton” of prokaryotes, focusing on taxonomy of higher ranks.” -
| 4 | 1 |
L35-38: I don’t get well the sentences which start from “This is in contrast ... ”. Please can you specify a bit more?
| 3 | 2 |
life5010949_makarova
| 1 |
done
| 4 | 1 |
L45: should provide → provides
| 3 | 2 |
life5010949_makarova
| 1 |
Moved to the conclusion section and the first word “Though” replaced by “In addition, since”
| 4 | 1 |
L47–49: move to conclusions?
| 3 | 2 |
life5010949_makarova
| 1 |
The whole paragraph has been deleted. This paragraph was added to the revised manuscript because one of the Reviewers asked “Does CVTree still require input genome data to be annotated to gene features, i.e., protein or CDS?” Well, this question reminds us that for many so-called “Permanent Draft” genomes it may be worthwhile returning to our early practice of using whole genome nucleotide sequences without distinguishing coding and non-coding segments. Although it did not lead to better results as compared with using translated protein products, but it is doable on un-annotated contigs. We will try this later.
| 4 | 1 |
L81–85: I think this paragraph is interrupting a bit the text flow. I suggest deletion.
| 3 | 2 |
life5010949_makarova
| 1 |
We have deleted all what appeared within the curly brackets and kept only “monophyletic Thaumarchaeota” as suggested. In fact, we could not tell how these words appeared there; there was none in our draft manuscript.
| 4 | 1 |
L244–245: I think the text within {} deviates the attention. I suggest delete that part, ending sentence with “monophyletic Thaumarchaeota” is also ok.
| 3 | 2 |
life5010949_makarova
| 1 |
Bending modulus is a material constant which does not depend on the size and shape of the object, but on the composition of the vesicle membrane and aqueous solution used (Lines 121–123 in the new version). We have inserted a definition of the membrane bending modulus (Lines 115–116) while the text regarding this issue extends between Lines 112–128.
| 2 | 1 |
It is an interesting topic, however, the presentation seems not to be clear. It would be better to have a short but clear description about what "bending elasticity modulus" is and its relevance to biological functions.
| 1 | 2 |
life5021101_makarova
| 1 |
As the referee suggested The Theory and Experimental section were organized into the Section “Materials and Methods”. Other sections were renumbered accordingly.
| 2 | 1 |
The theory section could be in the Method section with both principle and procedures to get kc values.
| 1 | 2 |
life5021101_makarova
| 1 |
As it was explained in the text our experimental method is developed for application to almost spherical vesicles. Vesicles with diameters between 20 and 40 micrometers were considered, whose deviations from spherical shape (fluctuation of the radius) were small in comparison with the mean sphere radius. We added the information concerning the shape and the size of the studied objects and added pictures of the equatorial cross section of a fluctuating vesicle (new Figure 3), as seen under the phase contrast microscope.
| 2 | 1 |
However, not all results seem to be presented. For example, it is not clear what kind of images were recorded, which were used to calculate the kc values, what the sizes/shapes of the fluctuating vesicles looked like. How these observations would be different or similar to other types of vesicles.
| 1 | 2 |
life5021101_makarova
| 1 |
We have rewritten the introductory paragraph (Lines 46–60).
| 2 | 1 |
The first sentence in the introduction section should be corrected as cell should not be considered to be “the building block of life”. It’s better to re-write the “basic motivation” for the study.
| 1 | 2 |
life5021101_makarova
| 1 |
Thank you for the comment, there was a mistake. We have tidied and updated the references.
| 2 | 1 |
There are two reference lists, one for 1–31 and another from 1–34. There are repeated ones. It’s not clear why the case was.
| 1 | 2 |
life5021101_makarova
| 1 |
We thank the reviewer for further comments and hope that we can clarify the issues as given below. We have made changes in the manuscript (marked red) and added one new reference (ref. [35]).
| 4 | 1 |
However, the comment of the reviewer is notable and we state in the conclusion of the manuscript (Lines 229–232) that at room temperature the thermophilicity is not necessarily related to the bending elasticity.
| 3 | 2 |
life5021101_makarova
| 1 |
In principal in order to obtain elastic constants you need to apply force and measure the deformation that this force causes. If we want to obtain the bending elasticity of lipid vesicle we need a force with a very small power. In the case of thermally induced shape fluctuations as a force we use the Brownian (thermal) motion of water molecules, bombarding the membrane. This force is stochastic (we do not know its instant value), but the mean value of it is proportional to the temperature. As a result of this stochastic bombardment the lipid vesicle deforms (changes its shape or fluctuates). We acquire a big amount of pictures of fluctuating vesicle in order to get the mean value of the deformation that our force causes. To extract mean we need to have stationary conditions over the time, so the mean is taken, that is why it is really important to have constant temperature throughout the experiment.
| 4 | 1 |
If only measured at one temperature, how would it be possible to draw a conclusion with “thermally induced shape fluctuations”?
| 3 | 2 |
life5021101_makarova
| 1 |
Using our experimental system we can measure bending elasticity also at higher temperatures (up to approximately 40 degrees C).
| 4 | 1 |
Is it possible to do the measurement at elevated temperatures?
| 3 | 2 |
life5021101_makarova
| 1 |
The bending elasticity modulus depends on the temperature below and near the phase transition temperature, but far above the phase transition temperature (this is the case in our experiment) the bending elasticity modulus is practically constant. See: Temperature and Chain Length Effects on Bending Elasticity of Phosphatidylcholine Bilayers. Fernandez-Puente, I. Bivas, M. D. Mitov and P. Meleard, Europhys. Lett., 28, 181 (1994). We have added this text to the Discussion (Lines 234–237) and also added the reference to the reference list (ref.
| 4 | 1 |
Would kc be temperature dependent? If yes, what would be the valid range?
| 3 | 2 |
life5021101_makarova
| 1 |
It is not a problem for us to make pictures of a fluctuating vesicle at different temperatures, but these photos would not be related to the manuscript.
| 4 | 1 |
The photo added merely shows the shape, but is it possible to show a few time related changes of the shape at different temperatures?
| 3 | 2 |
life5021101_makarova
| 1 |
We thank the referee for pointing to negligent mistakes in numbering. We have corrected wrong numbering of subsections.
| 4 | 1 |
The numbering from Lines 306–307 may not be correct as there are two 2.2.
| 3 | 2 |
life5021101_makarova
| 1 |
As the referee suggested, we have added data for the bending elasticity modulus of another lipid type—POPC lipid membrane. Also, we have added a new figure (Figure 3).
| 2 | 1 |
In addition, the number of tables and figures are too low for article. If could, the author should add other experimental data.
| 1 | 2 |
life5021101_makarova
| 1 |
A number of improvements of the experimental procedure for the thermally induced shape fluctuation method were used in this study: the stroboscopic illumination was improved on several steps to achieve a better experimental conditions; a thermostatic stage was used to set and control a constant temperature, the analysing procedure was improved by adding strict objective criteria for qualification of the vesicle as a whole as well as for acceptance or rejection of a given contour of the sequence of recorded images and the white noise contribution to the amplitudes of thermal shape fluctuations was R3 evaluated and taken into account. We have given short description of the improvements made and for each of them cited the corresponding articles for detailed explanation. (Lines 181–184; 187–193).
| 2 | 1 |
In Page 1, Lines 27–28, the author described “the experimental set-up was improved …”. However, the authors have already reported the measurements of bending elasticity using stroboscopic illumination in author’s previous papers (such as Genova et al., J. Optoelectro. Adv. Mat., 2005). Describe how the experimental set-up was improved in this paper more precisely.
| 1 | 2 |
life5021101_makarova
| 1 |
SOPC was chosen as both lipid types have transition temperature far below the temperature at experiments. In the revised version, we measured the bending modulus for another lipid type (POPC), measured and analysed by the same method for the same experimental conditions.
| 2 | 1 |
As a comparison of archaeal lipids, SOPC was used in this study. SOPC contains an unsaturated bond in its alkyl chains and its transition temperature seems to be quite low compared with archaeal lipids. What is the reason why the authors selected the SOPC for the representative of eukaryotic lipids?
| 1 | 2 |
life5021101_makarova
| 1 |
We have corrected the wrong number.
| 2 | 1 |
In page 4, between line 108 and 109, this equation should be numbered as (6).
| 1 | 2 |
life5021101_makarova
| 1 |
The aim of the present study was to obtain the bending elasticity modulus of the archaeal membrane at fixed temperature (27 degrees C) far above the phase transition temperature. We have not measured the temperature dependence of the bending constant.
| 2 | 1 |
Describe the temperature condition for measurement of fluctuation of GVs. Are there any effects of temperature on the fluctuation of GVs?
| 1 | 2 |
life5021101_makarova
| 1 |
In this work we present the bending elasticity modulus of archaeal lipid membrane in pure water environment. We added the details concerning the water purification (Lines 173–175). In other works the influence of different admixtures in the aqueous solution around the membrane was studied and the values depending on the type and the concentration of it for given lipid were reported. As the referee suggested we added such information with the corresponding references in the introduction section (Lines 57–60).
| 2 | 1 |
In this paper, bending elasticity modulus was measured in pure water. Are there any effects of ionic strength or composition of solution on the fluctuation of GVs?
| 1 | 2 |
life5021101_makarova
| 1 |
As I understand the referee had no further comments.
| 4 | 1 |
The manuscript was thoroughly revised according to reviewer’s suggestions and comments. Now this revised manuscript can be acceptable for publication.
| 3 | 2 |
life5021101_makarova
| 1 |
Bending modulus is a material constant which does not depend on the size and shape of the object, but on the composition of the vesicle membrane and aqueous solution used (Lines 121–123 in the new version). We have inserted a definition of the membrane bending modulus (Lines 115–116) while the text regarding this issue extends between Lines 112–128.
| 2 | 1 |
It would be better to have a short but clear description about what "bending elasticity modulus" is and its relevance to biological functions.
| 1 | 2 |
life5021101_perova
| 1 |
As the referee suggested The Theory and Experimental section were organized into the Section “Materials and Methods”. Other sections were renumbered accordingly.
| 2 | 1 |
The theory section could be in the Method section with both principle and procedures to get kc values.
| 1 | 2 |
life5021101_perova
| 1 |
As it was explained in the text our experimental method is developed for application to almost spherical vesicles. Vesicles with diameters between 20 and 40 micrometers were considered, whose deviations from spherical shape (fluctuation of the radius) were small in comparison with the mean sphere radius. We added the information concerning the shape and the size of the studied objects and added pictures of the equatorial cross section of a fluctuating vesicle (new Figure 3), as seen under the phase contrast microscope.
| 2 | 1 |
However, not all results seem to be presented. For example, it is not clear what kind of images were recorded, which were used to calculate the kc values, what the sizes/shapes of the fluctuating vesicles looked like. How these observations would be different or similar to other types of vesicles.
| 1 | 2 |
life5021101_perova
| 1 |
It would also be helpful to clarify/expand the description about the drug delivery system using the archaeal vesicle. The first sentence in the introduction section should be corrected as cell should not be considered to be “the building block of life”. It’s better to re-write the “basic motivation” for the study.
| 2 | 1 |
We have rewritten the introductory paragraph (Lines 46–60).
| 1 | 2 |
life5021101_perova
| 1 |
Thank you for the comment, there was a mistake. We have tidied and updated the references.
| 2 | 1 |
There are two reference lists, one for 1–31 and another from 1–34. There are repeated ones. It’s not clear why the case was.
| 1 | 2 |
life5021101_perova
| 1 |
We thank the reviewer for further comments and hope that we can clarify the issues as given below. We have made changes in the manuscript (marked red) and added one new reference (ref. [35]).
| 4 | 1 |
It is also not clear about the “thermally induced shape fluctuations”, such as the temperatures and incubation length and etc., especially this is a hyperthermophilic archaeal membrane vesicle, which would be expected to be more thermostable than those eukaryotic ones. The observed fluctuations of the membrane do not destabilize it. However, the comment of the reviewer is notable and we state in the conclusion of the manuscript (Lines 229–232) that at room temperature the thermophilicity is not necessarily related to the bending elasticity.
| 3 | 2 |
life5021101_perova
| 1 |
In principal in order to obtain elastic constants you need to apply force and measure the deformation that this force causes. If we want to obtain the bending elasticity of lipid vesicle we need a force with a very small power. In the case of thermally induced shape fluctuations as a force we use the Brownian (thermal) motion of water molecules, bombarding the membrane. This force is stochastic (we do not know its instant value), but the mean value of it is proportional to the temperature. As a result of this stochastic bombardment the lipid vesicle deforms (changes its shape or fluctuates). We acquire a big amount of pictures of fluctuating vesicle in order to get the mean value of the deformation that our force causes. To extract mean we need to have stationary conditions over the time, so the mean is taken, that is why it is really important to have constant temperature throughout the experiment.
| 4 | 1 |
If only measured at one temperature, how would it be possible to draw a conclusion with “thermally induced shape fluctuations”?
| 3 | 2 |
life5021101_perova
| 1 |
Using our experimental system we can measure bending elasticity also at higher temperatures (up to approximately 40 degrees C).
| 4 | 1 |
Is it possible to do the measurement at elevated temperatures?
| 3 | 2 |
life5021101_perova
| 1 |
The bending elasticity modulus depends on the temperature below and near the phase transition temperature, but far above the phase transition temperature (this is the case in our experiment) the bending elasticity modulus is practically constant. See: Temperature and Chain Length Effects on Bending Elasticity of Phosphatidylcholine Bilayers. Fernandez-Puente, I. Bivas, M. D. Mitov and P. Meleard, Europhys. Lett., 28, 181 (1994). We have added this text to the Discussion (Lines 234–237) and also added the reference to the reference list (ref.
| 4 | 1 |
Would kc be temperature dependent? If yes, what would be the valid range?
| 3 | 2 |
life5021101_perova
| 1 |
It is not a problem for us to make pictures of a fluctuating vesicle at different temperatures, but these photos would not be related to the manuscript.
| 4 | 1 |
The photo added merely shows the shape, but is it possible to show a few time related changes of the shape at different temperatures?
| 3 | 2 |
life5021101_perova
| 1 |
We thank the referee for pointing to negligent mistakes in numbering. We have corrected wrong numbering of subsections.
| 4 | 1 |
The numbering from Lines 306–307 may not be correct as there are two 2.2.
| 3 | 2 |
life5021101_perova
| 1 |
As the referee suggested, we have added data for the bending elasticity modulus of another lipid type—POPC lipid membrane. Also, we have added a new figure (Figure 3).
| 2 | 1 |
This manuscript described the measurement of bending elasticity of giant vesicles (GVs) prepared by archaea-derived lipids. Stroboscopic illumination measurements of GVs and theoretical models about shape fluctuation of GVs revealed that the archaeal lipid-based GVs showed the almost same value of bending elasticity modulus with synthetic lipid-based GVs. According to the authors, this is the first report for the measurement of bending elasticity modulus for archaeal lipid-based GVs. The measurement itself is novel, but same methods and theories have been reported in the authors’ previous papers. In addition, the number of tables and figures are too low for article. If could, the author should add other experimental data.
| 1 | 2 |
life5021101_perova
| 1 |
A number of improvements of the experimental procedure for the thermally induced shape fluctuation method were used in this study: the stroboscopic illumination was improved on several steps to achieve a better experimental conditions; a thermostatic stage was used to set and control a constant temperature, the analysing procedure was improved by adding strict objective criteria for qualification of the vesicle as a whole as well as for acceptance or rejection of a given contour of the sequence of recorded images and the white noise contribution to the amplitudes of thermal shape fluctuations was R3 evaluated and taken into account. We have given short description of the improvements made and for each of them cited the corresponding articles for detailed explanation. (Lines 181–184; 187–193).
| 2 | 1 |
I also have questions and comments for acceptance. The list of comments is as follows. In Page 1, Lines 27–28, the author described “the experimental set-up was improved …”. However, the authors have already reported the measurements of bending elasticity using stroboscopic illumination in author’s previous papers (such as Genova et al., J. Optoelectro. Adv. Mat., 2005). Describe how the experimental set-up was improved in this paper more precisely.
| 1 | 2 |
life5021101_perova
| 1 |
SOPC was chosen as both lipid types have transition temperature far below the temperature at experiments. In the revised version, we measured the bending modulus for another lipid type (POPC), measured and analysed by the same method for the same experimental conditions.
| 2 | 1 |
As a comparison of archaeal lipids, SOPC was used in this study. SOPC contains an unsaturated bond in its alkyl chains and its transition temperature seems to be quite low compared with archaeal lipids. What is the reason why the authors selected the SOPC for the representative of eukaryotic lipids?
| 1 | 2 |
life5021101_perova
| 1 |
We have corrected the wrong number.
| 2 | 1 |
In page 4, between line 108 and 109, this equation should be numbered as (6).
| 1 | 2 |
life5021101_perova
| 1 |
The aim of the present study was to obtain the bending elasticity modulus of the archaeal membrane at fixed temperature (27 degrees C) far above the phase transition temperature. We have not measured the temperature dependence of the bending constant.
| 2 | 1 |
Describe the temperature condition for measurement of fluctuation of GVs. Are there any effects of temperature on the fluctuation of GVs?
| 1 | 2 |
life5021101_perova
| 1 |
In this work we present the bending elasticity modulus of archaeal lipid membrane in pure water environment. We added the details concerning the water purification (Lines 173–175). In other works the influence of different admixtures in the aqueous solution around the membrane was studied and the values depending on the type and the concentration of it for given lipid were reported. As the referee suggested we added such information with the corresponding references in the introduction section (Lines 57–60).
| 2 | 1 |
In this paper, bending elasticity modulus was measured in pure water. Are there any effects of ionic strength or composition of solution on the fluctuation of GVs?
| 1 | 2 |
life5021101_perova
| 1 |
As I understand the referee had no further comments.
| 4 | 1 |
The manuscript was thoroughly revised according to reviewer’s suggestions and comments. Now this revised manuscript can be acceptable for publication.
| 3 | 2 |
life5021101_perova
| 1 |
We have rephrased the four paragraphs where the referee found that the described examples are not always easy to follow.
| 2 | 1 |
However, being experts in their field the authors might not be aware that for readers less familiar with the metabolism/physiology of archaea, the examples are not always easy to follow, such as examples given on p. 7 starting line 196 (The main difference here is between a ferredoxin-dependent and/or a NAD-dependent decarboxylation of pyruvate into acetyl-CoA?/A few lines later when I read that “halophilic archaea do not contain the coenzyme methanopterin”, I had to go back and reread the preceding sentences to get an idea of the argument. ), p. 10 starting Line 299, and p. 12 starting Line 354.
| 1 | 2 |
life5021427_makarova
| 1 |
We have added a short “outlook-type” paragraph towards the end of the conclusions. In this paragraph we mention three approaches that will facilitate gene discovery, among them large-scale screening techniques as exemplified by the insertion mutant library pointed out by the referee.
| 2 | 1 |
There is now available a great resource for the gene discovery in Haloferax volcanii (Kiljunen et al 2014: BMC Biology). A comprehensive insertion mutant library has been generated for this model archaeon in order to facilitate gene discovery. This should be mentitioned and discussed in the text; i.e., how this resource could be used to assign functions for genes.
| 1 | 2 |
life5021427_makarova
| 1 |
We have rephrased the four paragraphs where the referee found that the described examples are not always easy to follow.
| 2 | 1 |
Giving specific examples is certainly a good idea. However, being experts in their field the authors might not be aware that for readers less familiar with the metabolism/physiology of archaea, the examples are not always easy to follow, such as examples given on p. 7 starting line 196 (The main difference here is between a ferredoxin-dependent and/or a NAD-dependent decarboxylation of pyruvate into acetyl-CoA?/A few lines later when I read that “halophilic archaea do not contain the coenzyme methanopterin”, I had to go back and reread the preceding sentences to get an idea of the argument. ), p. 10 starting Line 299, and p. 12 starting Line 354.
| 1 | 2 |
life5021427_perova
| 1 |
We have added a short “outlook-type” paragraph towards the end of the conclusions. In this paragraph we mention three approaches that will facilitate gene discovery, among them large-scale screening techniques as exemplified by the insertion mutant library pointed out by the referee.
| 2 | 1 |
There is now available a great resource for the gene discovery in Haloferax volcanii (Kiljunen et al 2014: BMC Biology). A comprehensive insertion mutant library has been generated for this model archaeon in order to facilitate gene discovery. This should be mentitioned and discussed in the text; i.e., how this resource could be used to assign functions for genes.
| 1 | 2 |
life5021427_perova
| 1 |
The authors would like to thank for this helpful comment. We changed the subsection as described below. With stating that argon-nitrogen mixtures mainly emit high amounts of radiation, the authors want to emphasize, that the sterilization efficiency is based on radiation rather than interaction of radicals with the sample. In low-pressure argon-nitrogen plasmas, the only significant possible radical is atomic nitrogen. As atomic nitrogen is not known for high etching efficiency towards organic materials, the main reason for inactivation of D. radiodurans is based on radiation effects. Furthermore, Argon-Nitrogen plasmas emit high amounts of radiation compared to Argon-Oxygen plasmas, as shown for example in [5]. The data in [5] is not comparable in absolute values, as the system was changed afterwards. Absolutely calibrated spectra were measured and have been submitted in another publication. The authors think that estimating the inactivation efficiency towards D. radiodurans based on the UV dose cannot be performed on a reliable bases due to the following reasons: First, the inactivation efficiency towards spores is not only depending on the overall dose of the emitted radiation, but especially on the dose per wavelength. This has been demonstrated by Munakata et al. for B. subtilis spores (Munakata et al., Photochemistry and Photobiology, Blackwell Publishing Ltd, 1991, 54, 761-768). To the authors’ knowledge, there is no publication measuring the wavelength depending inactivation efficiency of B. radiodurans in the VUV. Second, the samples are mixed with Mars Analog Soil and most of the samples are covered with it. This leads to a very inhomogeneous exposure of the samples which can only be estimated arbitrarily. To address the reviewer’s comments, subsection 2.3 has been modified as follows: An argon-nitrogen mixture (100:5 sccm) was used with continuous power of 500 W, as it emits significantly higher amounts of radiation in the bactericidal wavelength region from 100 nm to 400 nm compared to an argon-oxygen mixture (100:5 sccm) [5]. The absolute values presented in [5] are not applicable to the results shown here, due to modification of the plasma system concerning the power coupling. Nevertheless, the significantly higher emission of the argon-nitrogen mixture is shown. Absolute values of the radiation dose in the range from 100 nm to 400 nm of the system configuration used in this study were measured and submitted [6]. A possible radical that can interact with the samples is atomic nitrogen. As atomic nitrogen is not known for etching of organic materials, we assume that inactivation in the argon-nitrogen mixture is based on radiation effects. [6] Raguse, M.; Fiebrandt, M.; Denis, B.; Stapelmann, K.; Eichenberger, P.; Driks, A.; Eaton, P.; Awakowicz, P.; Moeller, R. Understanding of the importance of the spore coat structure and pigmentation in the Bacillus subtilis spore resistance to low pressure plasma sterilization. Submitted to Journal of Physics D: Applied Physics.
| 2 | 1 |
The plasma conditions are not described sufficiently. It is only stated that the argon-nitrogen mixture ‘mainly emits high amounts of radiation in the wavelength region from 100 nm to 400 nm’ without specifying what high fluences at the sample site are. The term amount is not applicable here. With the knowledge of the spectral irradiance in the UV range from 100 to 400 nm the authors could have made a calculation to estimate the inactivation of D. radiodurans by the UV component of the plasma.
| 1 | 2 |
life6020022_makarova
| 1 |
The authors thank the referee for this comment that demonstrates that the authors need to clarify. However, to the authors it is not clear which potentially inactivating components of the plasma are not specified, as radicals and radiation are mentioned and the temperature is kept below 80°C to prevent inactivation effects due to heating. As the samples are placed in an ICP discharge, ions towards the samples are only accelerated by several tenth of eV. Thus, sputtering of the sample due to ion bombardment can be neglected. Low-pressure plasmas dissociate molecular gases up to several percent. Thus, the authors wanted to emphasize, that the atomic oxygen density and therewith the atomic oxygen flux to the samples is in the order of a few percent of the overall gas flux. Quantitative and even qualitative analysis of atomic oxygen densities by optical emission spectroscopy is not an easy task as several parameters like gas temperature, electron density and electron temperature are necessary. This is even more challenging in pulsed mode, as the plasma does not achieve a steady state due to switching it on and off to prevent intensive heating of the plasma. Determination of the gas densities and plasma parameters is currently addressed in extensive analysis of plasmas with different gas compositions but not finished and published yet. Nevertheless, the authors’ state, that a relevant amount of atomic oxygen species is produced in the low pressure argon-oxygen plasma as the minimum energy for dissociation of molecular oxygen is around 5 eV. The ionization energy for argon is 16 eV and 13 eV for molecular oxygen. Thus, sufficient electrons with enough energy to dissociate molecular oxygen will be present in the plasma for dissociation up to a few percent. In the authors’ view, estimating the mean free path of the atomic oxygen in the plasma is not necessary, as recombination of radicals in low pressure plasmas in the regime of a few Pascal takes place at the surfaces. Thus, any radical formed will be lost at the walls and not on its way to a surface. Estimating the mean free path of the radical in the Mars Analog Soil can only be performed on an arbitrary basis as the sticking and recombination coefficients of atomic oxygen are not known for the Mars Analog Soil. Thus, any calculation of the mean free path of the radical is based on estimated data and could only be verified by experiments. To address the reviewer’s comments, subsection 2.3 has been modified as follows: Only JSC Mars-1A analog soil was used in the second trial. In this case, an argon-oxygen mixture (100:5 sccm) was used as it produces reactive oxygen species due to dissociation of molecular oxygen in the plasma. Since radiation-based sterilization is faster than sterilization due to oxidation, the treatment time was increased. As this leads to heating of the sample above 80 °C in the continuous plasma, we used a pulsed mode, switching the plasma on and off at a frequency of 1 kHz and a duty cycle of 10 %. To account for this fact, the treatment time was increased to 45 min, yielding a plasma exposure time of 4.5 min. Furthermore, the power was increased to 1500 W, yielding a mean power of 150 W, as it is only applied 10 % of the time. The plasma conditions are not matched to each other as the main objective was to observe to what degree the cells of D. radiodurans were affected by radiation or reactive species and not which component is more efficient. Thus, sputtering of the sample due to ion bombardment can be neglected as possible inactivation mechanism.
| 2 | 1 |
The other potentially inactivating components of the plasma are not specified in the manuscript. For the argon-oxygen gas mixture it is stated that ‘high amounts of reactive oxygen species are formed in the plasma’ again without any qualitative and quantitative description. Estimations of the average free length of path for the inactivating plasma components might have been helpful for experiment planning.
| 1 | 2 |
life6020022_makarova
| 1 |
The grain size distributions of the three Mars regolith simulants have been added to the text in form of two additional tables (table 2: P-MRS and S-MRS; and table 3: JSC Mars-1A), as well as a new citation for the grain size distribution of JSC Mars-1A [8].
| 2 | 1 |
Three different Mars regolith simulants were used. The mineralogical composition is given in table, but not the grain size distribution. Knowing the grain sizes and assuming a homogeneous distributrion of cells and grains or a measurement of the real distribution an assessment can be made about the shading effect of the grains with respect to UV radiation.
| 1 | 2 |
life6020022_makarova
| 1 |
To assess the shading effect of the regolith is difficult. As described we used a very thin layer of regolith particles, but did not measure them according to their shading effect but according to the weight, which was the same for all types of regolith. This experiment was meant as a test of the sterilization method adjusted to our purposes, as described in the introduction section. Therefore, we did not plan it according to the maximal theoretically achievable sterilization effect, but to design a life detection mission to Mars. [8] Wan, L., Wendner, R., & Cusatis, G. A Novel Material for In Situ Construction on Mars: Experiments and Numerical Simulations. 2016, arXiv preprint arXiv:1512.05461
| 2 | 1 |
The authors should have estimated the maximal theoretically achievable effect and to plan the experiments accordingly.
| 1 | 2 |
life6020022_makarova
| 1 |
The title has been changed.
| 2 | 1 |
First of all it is necessary to change the title and at least remove “The testing”.
| 1 | 2 |
life6020022_makarova
| 1 |
In the introduction we already described the problem of the destructiveness of heat sterilization like autoclaving on the organic contents of a soil, which was our motivation to test an alternative method such as low temperature plasma sterilization. D. radiodurans was used as test organism because of its resistance towards UV radiation, which is a part of the sterilizing effect of plasma sterilization. The appropriate passages in the introduction section have been edited.
| 2 | 1 |
The introduction part requires more arguments to prove choice of the problem and the research object (D. radiodurans).
| 1 | 2 |
life6020022_makarova
| 1 |
Since we did not intend to test the influences of the Martian Analog soils on D. radiodurans, but the sterilizing effect of the low temperature plasma treatment on cells intermixed with soil, we did not make a control to determine the effects of the Mars analog soils. The starting values of cell numbers were estimated on samples prepared the same way as the samples for the plasma sterilization (intermixed with soils and desiccated as described in the method section). They were incubated on TYG agar plates like the plasma samples after the treatment. This explanation was missing in the text and has now been added to the appropriate passages in the method section (Chapter 2.2.) for a better understanding.
| 2 | 1 |
In addition, I think, a control consisting of D. radiodurans cells before and after sterilization is missing to determine the role of the Martian Analog soils.
| 1 | 2 |
life6020022_makarova
| 1 |
The names are written in italics now, the mistake must have happened during the editing of the text.
| 2 | 1 |
Lines 31-32: Bacillus subtilis and B.pumilus should be written in italic.
| 1 | 2 |
life6020022_makarova
| 1 |
Done, thank you.
| 2 | 1 |
Line 38: Deinococcus radiodurans should be written in italic. While writing D. radiodurans R1 has always necessary to note that this is type strain R1T.
| 1 | 2 |
life6020022_makarova
| 1 |
Yes, we just used one medium (TYG), so we changed it to ‘medium’.
| 2 | 1 |
Line 41: Medium instead of media. If I understood correctly, you used only one medium.
| 1 | 2 |
life6020022_makarova
| 1 |
The writing has been changed accordingly.
| 2 | 1 |
Line 50: R1T (=ATCC 13939T=DSM 20539T) instead of R1 ATCC13939/DSM20539 Reply:
| 1 | 2 |
life6020022_makarova
| 1 |
Since the bottles containing the Martian Analog soils were autoclaved before use, they should be sterile. We also made a separate test to check for sterility after autoclaving the Martian Analog soils, which has been negative (meaning no contamination could be detected via plating of the autoclaved JSC Mars-1A regolith on TYG medium).
| 2 | 1 |
Line 57: Are you sure that the Martian Analog soils were sterile before using?
| 1 | 2 |
life6020022_makarova
| 1 |
An explanation has been added to the text passage.
| 2 | 1 |
Line 78: Please explain in the text what is “a vacuum treatment control”.
| 1 | 2 |
life6020022_makarova
| 1 |
The differences might be caused due to the finer grain size of the other regolith used - particularly P-MRS, which possibly increased the shielding effect. This has been added to the discussion section.
| 2 | 1 |
Discussion How do you explain the difference between the results when using JSC Mars-1A analog and the other soils?
| 1 | 2 |
life6020022_makarova
| 1 |
The authors would like to thank for this helpful comment. We changed the subsection as described below. With stating that argon-nitrogen mixtures mainly emit high amounts of radiation, the authors want to emphasize, that the sterilization efficiency is based on radiation rather than interaction of radicals with the sample. In low-pressure argon-nitrogen plasmas, the only significant possible radical is atomic nitrogen. As atomic nitrogen is not known for high etching efficiency towards organic materials, the main reason for inactivation of D. radiodurans is based on radiation effects. Furthermore, Argon-Nitrogen plasmas emit high amounts of radiation compared to Argon-Oxygen plasmas, as shown for example in [5]. The data in [5] is not comparable in absolute values, as the system was changed afterwards. Absolutely calibrated spectra were measured and have been submitted in another publication. The authors think that estimating the inactivation efficiency towards D. radiodurans based on the UV dose cannot be performed on a reliable bases due to the following reasons: First, the inactivation efficiency towards spores is not only depending on the overall dose of the emitted radiation, but especially on the dose per wavelength. This has been demonstrated by Munakata et al. for B. subtilis spores (Munakata et al., Photochemistry and Photobiology, Blackwell Publishing Ltd, 1991, 54, 761-768). To the authors’ knowledge, there is no publication measuring the wavelength depending inactivation efficiency of B. radiodurans in the VUV. Second, the samples are mixed with Mars Analog Soil and most of the samples are covered with it. This leads to a very inhomogeneous exposure of the samples which can only be estimated arbitrarily. To address the reviewer’s comments, subsection 2.3 has been modified as follows: An argon-nitrogen mixture (100:5 sccm) was used with continuous power of 500 W, as it emits significantly higher amounts of radiation in the bactericidal wavelength region from 100 nm to 400 nm compared to an argon-oxygen mixture (100:5 sccm) [5]. The absolute values presented in [5] are not applicable to the results shown here, due to modification of the plasma system concerning the power coupling. Nevertheless, the significantly higher emission of the argon-nitrogen mixture is shown. Absolute values of the radiation dose in the range from 100 nm to 400 nm of the system configuration used in this study were measured and submitted [6]. A possible radical that can interact with the samples is atomic nitrogen. As atomic nitrogen is not known for etching of organic materials, we assume that inactivation in the argon-nitrogen mixture is based on radiation effects. [6] Raguse, M.; Fiebrandt, M.; Denis, B.; Stapelmann, K.; Eichenberger, P.; Driks, A.; Eaton, P.; Awakowicz, P.; Moeller, R. Understanding of the importance of the spore coat structure and pigmentation in the Bacillus subtilis spore resistance to low pressure plasma sterilization. Submitted to Journal of Physics D: Applied Physics.
| 2 | 1 |
The plasma conditions are not described sufficiently. It is only stated that the argon-nitrogen mixture ‘mainly emits high amounts of radiation in the wavelength region from 100 nm to 400 nm’ without specifying what high fluences at the sample site are. The term amount is not applicable here. With the knowledge of the spectral irradiance in the UV range from 100 to 400 nm the authors could have made a calculation to estimate the inactivation of D. radiodurans by the UV component of the plasma.
| 1 | 2 |
life6020022_perova
| 1 |
The authors thank the referee for this comment that demonstrates that the authors need to clarify. However, to the authors it is not clear which potentially inactivating components of the plasma are not specified, as radicals and radiation are mentioned and the temperature is kept below 80°C to prevent inactivation effects due to heating. As the samples are placed in an ICP discharge, ions towards the samples are only accelerated by several tenth of eV. Thus, sputtering of the sample due to ion bombardment can be neglected. Low-pressure plasmas dissociate molecular gases up to several percent. Thus, the authors wanted to emphasize, that the atomic oxygen density and therewith the atomic oxygen flux to the samples is in the order of a few percent of the overall gas flux. Quantitative and even qualitative analysis of atomic oxygen densities by optical emission spectroscopy is not an easy task as several parameters like gas temperature, electron density and electron temperature are necessary. This is even more challenging in pulsed mode, as the plasma does not achieve a steady state due to switching it on and off to prevent intensive heating of the plasma. Determination of the gas densities and plasma parameters is currently addressed in extensive analysis of plasmas with different gas compositions but not finished and published yet. Nevertheless, the authors’ state, that a relevant amount of atomic oxygen species is produced in the low pressure argon-oxygen plasma as the minimum energy for dissociation of molecular oxygen is around 5 eV. The ionization energy for argon is 16 eV and 13 eV for molecular oxygen. Thus, sufficient electrons with enough energy to dissociate molecular oxygen will be present in the plasma for dissociation up to a few percent. In the authors’ view, estimating the mean free path of the atomic oxygen in the plasma is not necessary, as recombination of radicals in low pressure plasmas in the regime of a few Pascal takes place at the surfaces. Thus, any radical formed will be lost at the walls and not on its way to a surface. Estimating the mean free path of the radical in the Mars Analog Soil can only be performed on an arbitrary basis as the sticking and recombination coefficients of atomic oxygen are not known for the Mars Analog Soil. Thus, any calculation of the mean free path of the radical is based on estimated data and could only be verified by experiments. To address the reviewer’s comments, subsection 2.3 has been modified as follows: Only JSC Mars-1A analog soil was used in the second trial. In this case, an argon-oxygen mixture (100:5 sccm) was used as it produces reactive oxygen species due to dissociation of molecular oxygen in the plasma. Since radiation-based sterilization is faster than sterilization due to oxidation, the treatment time was increased. As this leads to heating of the sample above 80 °C in the continuous plasma, we used a pulsed mode, switching the plasma on and off at a frequency of 1 kHz and a duty cycle of 10 %. To account for this fact, the treatment time was increased to 45 min, yielding a plasma exposure time of 4.5 min. Furthermore, the power was increased to 1500 W, yielding a mean power of 150 W, as it is only applied 10 % of the time. The plasma conditions are not matched to each other as the main objective was to observe to what degree the cells of D. radiodurans were affected by radiation or reactive species and not which component is more efficient. Thus, sputtering of the sample due to ion bombardment can be neglected as possible inactivation mechanism.
| 2 | 1 |
The other potentially inactivating components of the plasma are not specified in the manuscript. For the argon-oxygen gas mixture it is stated that ‘high amounts of reactive oxygen species are formed in the plasma’ again without any qualitative and quantitative description. Estimations of the average free length of path for the inactivating plasma components might have been helpful for experiment planning.
| 1 | 2 |
life6020022_perova
| 1 |
The grain size distributions of the three Mars regolith simulants have been added to the text in form of two additional tables (table 2: P-MRS and S-MRS; and table 3: JSC Mars-1A), as well as a new citation for the grain size distribution of JSC Mars-1A [8]. To assess the shading effect of the regolith is difficult. As described we used a very thin layer of regolith particles, but did not measure them according to their shading effect but according to the weight, which was the same for all types of regolith. This experiment was meant as a test of the sterilization method adjusted to our purposes, as described in the introduction section. Therefore, we did not plan it according to the maximal theoretically achievable sterilization effect, but to design a life detection mission to Mars. [8] Wan, L., Wendner, R., & Cusatis, G. A Novel Material for In Situ Construction on Mars: Experiments and Numerical Simulations. 2016, arXiv preprint arXiv:1512.05461
| 2 | 1 |
Three different Mars regolith simulants were used. The mineralogical composition is given in table, but not the grain size distribution. Knowing the grain sizes and assuming a homogeneous distributrion of cells and grains or a measurement of the real distribution an assessment can be made about the shading effect of the grains with respect to UV radiation.
| 1 | 2 |
life6020022_perova
| 1 |
The title has been changed.
| 2 | 1 |
First of all it is necessary to change the title and at least remove “The testing”.
| 1 | 2 |
life6020022_perova
| 1 |
In the introduction we already described the problem of the destructiveness of heat sterilization like autoclaving on the organic contents of a soil, which was our motivation to test an alternative method such as low temperature plasma sterilization. D. radiodurans was used as test organism because of its resistance towards UV radiation, which is a part of the sterilizing effect of plasma sterilization. The appropriate passages in the introduction section have been edited.
| 2 | 1 |
First of all it is necessary to change the title and at least remove “The testing”.
| 1 | 2 |
life6020022_perova
| 1 |
Since we did not intend to test the influences of the Martian Analog soils on D. radiodurans, but the sterilizing effect of the low temperature plasma treatment on cells intermixed with soil, we did not make a control to determine the effects of the Mars analog soils. The starting values of cell numbers were estimated on samples prepared the same way as the samples for the plasma sterilization (intermixed with soils and desiccated as described in the method section). They were incubated on TYG agar plates like the plasma samples after the treatment. This explanation was missing in the text and has now been added to the appropriate passages in the method section (Chapter 2.2.) for a better understanding.
| 2 | 1 |
The introduction part requires more arguments to prove choice of the problem and the research object (D. radiodurans).
| 1 | 2 |
life6020022_perova
| 1 |
Done, thank you.
| 2 | 1 |
Line 38: Deinococcus radiodurans should be written in italic. While writing D. radiodurans R1 has always necessary to note that this is type strain R1T.
| 1 | 2 |
life6020022_perova
| 1 |
Yes, we just used one medium (TYG), so we changed it to ‘medium’.
| 2 | 1 |
Line 41: Medium instead of media. If I understood correctly, you used only one medium.
| 1 | 2 |
life6020022_perova
| 1 |
The writing has been changed accordingly.
| 2 | 1 |
Line 50: R1T (=ATCC 13939T=DSM 20539T) instead of R1 ATCC13939/DSM20539 Reply:
| 1 | 2 |
life6020022_perova
| 1 |
Since the bottles containing the Martian Analog soils were autoclaved before use, they should be sterile. We also made a separate test to check for sterility after autoclaving the Martian Analog soils, which has been negative (meaning no contamination could be detected via plating of the autoclaved JSC Mars-1A regolith on TYG medium).
| 2 | 1 |
Line 57: Are you sure that the Martian Analog soils were sterile before using?
| 1 | 2 |
life6020022_perova
| 1 |
An explanation has been added to the text passage.
| 2 | 1 |
Line 78: Please explain in the text what is “a vacuum treatment control”.
| 1 | 2 |
life6020022_perova
| 1 |
The differences might be caused due to the finer grain size of the other regolith used - particularly P-MRS, which possibly increased the shielding effect. This has been added to the discussion section.
| 2 | 1 |
How do you explain the difference between the results when using JSC Mars-1A analog and the other soils?
| 1 | 2 |
life6020022_perova
| 1 |
I added a description at line 38 – 39.
| 2 | 1 |
line 38 - The linking of 'other molecules' to 'phenotype' here is a little odd. There may be a link from one gene to one protein, but one protein is not really a phenotype. There are many quantitative traits affected by many genes.
| 1 | 2 |
life6030029_makarova
| 1 |
I added a description at line 73 – 74.
| 2 | 1 |
line 73 - the point is made that current RNA World theories do not seem compatible with a hydrothermal origin of life. But it is not clear where this leads us. Maybe life did not begin with RNA. Maybe life did not begin with hydrothermal vents. Maybe there is some slightly different environment that we would still call hydrothermal in which RNA is sufficiently stable. Maybe there is some slightly different nucleic acid-like polymer that is stable in hydrothermal conditions. Too many unknowns here to be a useful point.
| 1 | 2 |
life6030029_makarova
| 1 |
This comment is in relation to the first comment by reviewer 2. Thus, I added description in lines 99 – 101 and 111 – 112. And I shorten this part to appreciate the comment by reviewer 2.
| 2 | 1 |
lines 93-106 - I mostly agree with this paragraph but it is not said very clearly. Certainly we need to distinguish between long peptides formed spontaneously and those that are translated. But several questions still remain - were there long peptides before long RNA strands? Was there a way of reproducibly making the same amino acid sequence without translating it from RNA? If not, then is there any way that random non-encoded peptides could be useful to RNAs?
| 1 | 2 |
life6030029_makarova
| 1 |
Actually, the comparison was made for cell-based life-like systems. So, I removed 2 references regarding civilization from this part, and add words at line 243 and 276 – 277. And, I simplify descriptions as shown in line 272.
| 2 | 1 |
The idea in caption to Fig 4 that building blocks do not interact directly with the environment seems interesting and plausible, but not very well defined. Are you saying a cell interacts with the environment but a single gene does not? Or a multicellular organism interacts but one cell does not? The citations 52 and 53 are in fields that are not related to biology and origin of life. So I think this idea needs to be explained and justified in a biological context.
| 1 | 2 |
life6030029_makarova
| 1 |
This part is important. While it was difficult to simplify, but I tried to simplify section 2.3 as possible and added some descriptions.
| 2 | 1 |
Fig 5 - drawing parallels between systems of different levels of complexity (from prokaryotes up to civilizations) is interesting, but it is a bit distracting at this point. The abstract promised to talk about RNA World and the origin of life. At this point the article seems to have strayed a long way from this intention. From the origin of life viewpoint, even the simplest of these (the prokaryote) is still very complex. The issue for the origin of life is how to get to a prokaryote. I don't really think that looking at social insects and human societies will help much in understanding the origin of life. The ideas of CCSI and CMIO seem interesting, but they are discussed with the high-level examples (pages 10-11), not with molecular and cellular examples. Probably a lot of this could be simplified. It is only when we get to paragraph 3.1 that we get to the point of the article.
| 1 | 2 |
life6030029_makarova
| 1 |
It is not essential that the whole circular network to be incorporated in a life-like system. A part of the network is accelerate by a newly evolved ribozyme is important. I tried to describe this, but it seems not sufficient. So I added carefully descriptions at lines 455 – 457, 466 – 471, 483 – 48
| 2 | 1 |
Fig 7 - Viewing a metabolism as a cycle seems to be only half the story. Nutrients go in and waste comes out. This is a throughput, not a cycle. Also the diagram does not indicate whether the ribozymes are made by the cycle. I think there is some room for improvement in this diagram.
| 1 | 2 |
life6030029_makarova
| 1 |
I added a description at lines 538 – 540.
| 2 | 1 |
Fig 8 is very reminiscent of Fig 1 of Wu and Higgs (2009) J Mol Evol 69:541-554 and Fig 1 of Wu and Higgs (2011) Astrobiology 11:895-906.
| 1 | 2 |
life6030029_makarova
| 1 |
As I mentioned above, I added carefully descriptions at lines 455 – 457, 466 – 471, 483 – 488.
| 2 | 1 |
Once again in Fig 8 I think that the cycle is over-emphasized and the throughput is not included. For example there must be ways of making nucleotides from scratch. It cannot be true that the only source of nucleotides is by degrading oligomers.
| 1 | 2 |
life6030029_makarova
| 1 |
Formation of membrane is very important as reviewer 1 mentioned. I added a description at lines 542 – 544, 483 – 488, 544 – 548. line 554 - The case that two genes were essential initially is not made.
| 2 | 1 |
Section 3.4 - 'There is a debate whether CMIO could have formed by a CCSI consisting entirely or mainly of RNA'. This section seems to mix up two important questions. (i) Are RNA catalysts sufficient? Do we need other kinds of biomolecules like protein catalysts? (ii) Do we need the RNA system to be enclosed in a cell membrane or other kind of compartment? If so, can RNA control the growth and division of the membrane? These issues need to be considered separately.
| 1 | 2 |
life6030029_makarova
| 1 |
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