Arcady R. Mushegian, Stowers Institute for Medical Research, Kansas City, MO, USA
This is the second of two thought-provoking manuscripts that discuss the hypothesis of the "Zn world" in which Life on Earth may have emerged. This manuscript is largely devoted to devising the ways of testing the traces of the Zn world in the present-day world of RNA and proteins. The computational experiments outlined in the manuscript are, in my opinion, the preliminary thoughts, which are unfortunately not set in a robust quantitative framework. More specifically:
Prediction 1. The elevated Zn2+ content of the primordial environments should be conserved inside modern cells
-- Please elaborate which content should be conserved – is it total concentration or free ion concentration? The latter is very low – is it because Zn2+ ion is nowadays toxic and needs to be sequestered? When in evolution did it become so?
Authors' response. In the revised version of the paper we have changed the order of predictions, this particular prediction has been moved to no. 5. We now explicitly indicate that it is the total concentration of Zn that should be conserved. Actually, zinc is relatively non-toxic, at least for humans who can tolerate it in fairly large amounts . We believe that the low levels offreeZn in the cells reflect certain Zn limitation of modern organisms and, perhaps, justify the currently popular inclusion of Zn-containing complexes into the vitamin sets.
Prediction 2. There should be ribozymes with Zn-dependent catalytic activities.
-- I think that an honest conclusion would be that direct evidence of this is weak, though synthetic ribozymes that can use Zn2+ ions are known.
Authors' response. Generally, we agree with the referee's comment. In more detail we consider this topic in our response to Koonin.
Prediction 1. RNA structures should be enriched in Zn2+ ions.
-- The way I understand the data, the presence of in the Zn2+ ions in known RNA structures in a form directly bound to RNA is not much different from Mn and is much less common than Mg. Computing the enrichment statistics here and everywhere else in the study may help, though I appreciate the difficult issues that have to do with the sampling.
Authors' response. In this work, to avoid a "self-serving" bias, we based on the data available either from the literature or from the publicly-accessible databases. We would like to emphasize that an accurate checking of each prediction is a task that would have required writing a separate article.
Prediction 3. Zn2+ ions should be associated with the evolutionarily oldest protein folds;
-- This again should be a quantitative argument, but I am not sure what it actually is: associated with the oldest folds more than with the younger ones? Associated more than other divalent cations? More than what should be expected by chance? More specifically: the particular set of ubiquitous folds that are taken as a (most likely, reasonable) proxy for the oldest folds are clearly rich in divalent cation-binding proteins. Many families of dinucleotide-binding Rossmanoids, nucleotidyltransferases and polymerases of different classes – all of them use Mg2+ in preference to everything else, and if there is no Mg2+ in the crystal growth media, occasional Zn2+ will substitute. In these classes, there is no such thing as "Zn or Mg".
Authors' response: We agree that a more systematic study of metal occurrence in the "oldest" and "youngest" folds would be highly desirable. Here we just counted metal ions that are present in the crystal structures. Thereby we did not check whether Mg2+was present in the crystal growth media or not (usually it was). The fact that Zn2+ions were seen in many structures in spite of the presence of Mg2+ions in crystallization media might indicate that in certain cases (e.g. in zinc fingers) Mg2+ions could not substitute for Zn2+ions.
Prediction 4. Enzymes with evolutionarily "old" functions should depend on Zn2+;
-- I suggest either obtaining Zerkle data and examining them more closely, or not discussing them at all.
Authors' response: We decided to retain the data of Zerkle and co-workers as unbiased evidence, but merged this prediction with the one on the function takeover from ribozymes by first enzymes. In the original version of the manuscript, while discussing the data of the data of Zerkle and co-workers, we have argued that they may have overestimated the fraction of iron-containing enzymes at the stage of the "very early life" (see also below for a related point made by Forterre). For example, we believe that iron-rich respiratory enzymes – assumed to be present from the very beginning by Zerkle and co-workers  – were not needed before the oxygenation of the atmosphere. In the revised manuscript we have dropped this discussion for the sake of brevity. These points deserve be considered in a separate publication.
Prediction 4a. The enzymes that emerged to take over the catalytic functions from ribozymes should be dependent on Zn2+
-- Why this would be the case? Transition from the RNA World to the Protein World may have occurred later than the Zn world, and other metals, like Mg2+, may have taken over the catalytic roles already. This seems to be better compatible with the evidence, does it not?
Authors' response: In the revised manuscript we argue, in response also to the comments of Koonin, that the Last Universal Common Ancestor still dwelled in Zn-rich habitats. Then the transition from the RNA World to the RNA/Protein World would also have proceeded in Zn-enriched environments. The amount of Mg2+in the sea water is high and apparently always has been high. However, since Mg2+is a poor Lewis acid, certain catalytic tasks require transition metals. Accordingly, we argue that many catalytic activities that are common for enzymes and ribozymes are catalysed by zinc-dependent enzymes.
Manuscript as a whole: There is no discussion of the effect of various concentrations of the Zn2+ ion on the stability of the phosphoester bond. There is related discussion of Fe2+ ions on pg 17, but not of Zn2+ ions.
Authors' response:The important topic of the Zn-catalyzed cleavage of phosphoester bonds (see also the comment by Forterre) is now included in the accompanying article  and is additionally discussed in the author's response to reviewers of that article.
pp. 26–31 are quite redundant with the first manuscript – consider shortening?
Authors' response: The redundant parts have been streamlined.
p. 34 – items (iv) and (v) sound very reasonable to me, but I do not see how they relate to Zn world. The same for items (vii) and (viii) on p. 35.
Item (iv): The fundamental difference between the membranes of Bacteria and Archaea indicates that modern membranes developed separately in the two domains. Then the Last Universal Common Ancestor (LUCA) must have had primitive (if any) membranes that could not be particularly ion-tight and should have enabled ionic equilibration with surrounding media [73, 138, 187, 206]. In the revised manuscript, we argue that the high Zn content of modern cells could be traced to the ionic equilibrium between the interior of the LUCA and its Zn-rich environment.
Item (v): We suggest that the separation of the major domains was driven by the drift of the ZnS-confined seafloor communities away from the continental phototrophic ones. If the LUCA had swimming gadgets, these communities could continuously mix and exchange genes, preventing the development of Bacteria- and Archaea-specific traits.
Items (vii) and (viii): Item (viii) became (ix) in the revised manuscript. The absence of eukaryote-specific, iron-processing machinery even in modern Eukarya strongly indicates that pro-eukarya could not rely upon iron-dependent redox metabolism. The only metabolic alternative would have been heterotrophy of some kind relying on the Zn-dependent hydrolases.
Reviewer's response in a second review
The distance that a swimming LUCA can cover: how does it compare with the velocity of continental drift?
Authors' response: The continental drift was unlikely to take place in Hadean. It is believed that at that time, cooling of the Earth, the main cause of contemporary continental drift, was mediated by numerous "hot spots" resembling modern Iceland . However, the importance of swimming for the gene exchange between the major domains could be evaluated by estimating the rate of "sinking" of the Zn-rich habitats. Using estimates of the atmospheric pressure of ca. 100 bar after condensation of the ocean at 4.4 Ga [52, 53]and 2–6 bar at 3.3 Ga and assuming a linear decrease of pressure with time, it is possible to calculate that the pressure may have decreased, on average, by 1 bar in ~10 million years. When the pressure falls below certain threshold value (ca. 10 bar), the highest temperature of hydrothermal fluids drops below ~200°C and they become depleted of Zn. Therefore, after the atmospheric CO2pressure dropped below 10 bar, the Zn-rich hot springs could function only at a certain depth, where the total pressure – of the atmosphere and the water column – remained above 10 bar. A 10-meter water column produces pressure of ca. 1 bar. Therefore the submersion of the Zn-rich hydrothermal systems should have proceeded with a rate of ca. 10 meters in 10 million years, or ca. 1 micron per year, slow enough to be overcome by any kind of swimming motility. Therefore, if LUCA and its immediate descendants could swim, the phototrophic, swimming organisms could occupy the surface water layer (the photic zone) just above the sea-floor communities. The sedimentation of these organisms would promote sharing of genes and would have prevented crystallization of the domain-specific traits. In contrast, non-swimming organisms would have stayed confined to their habitats, so that the sub-aerial phototrophic communities could not, at least initially, exchange genes with the see-floor, ZnS-confined communities.
p. 36 line 19: I do not see why these explanations are parsimonious or why this would necessarily be a good thing.
Authors' response:Parsimony is a good thing. If one finds an explanation that simultaneously covers several unresolved items, the probability that this hypothesis captures some actual features of a natural phenomenon is higher than when one has to come up with a separate explanation for each unsolved item.
Reviewer's response in a second review: You explained why you think parsimony is good but not why you think these explanations are parsimonious.
Authors' response: The Zn world concept may be considered a parsimonious explanation of the three items listed in section on testing the explanatory power of the Zn world concept. because it explains them all. Alternatively, one could suggest separate explanation(s) for each item. For example, the prevalence of Zn in modern enzymes (Zn paradox) might be explained by the emergence of life within Zn-rich hydrothermal settings of sea floor. Then, however, the unique photochemical traits of nucleic acids would still remain unexplained. These unique traits, separately, were suggested to reflect the emergence of life in some illuminated settings [78, 119, 120]. If we now try to find a parsimonious explanation for the Zn paradox and the unique photochemistry of nucleic acids, the seemingly unique solution is the emergence of first life forms in some illuminated, Zn-rich settings. However, Zn could be present on primeval Earth only as ZnS, which, when illuminated, can reduce CO2. Hence a parsimonious explanation just for two items "bore" a solution to the problem of abiogenic CO2reduction as well.
Eugene V. Koonin, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA
This paper strives to test the hypothesis that is put forward in the accompanying paper by Mulkidjanian, namely that life originated in a photosynthetic "Zn world" where Zn sulfide catalyzed a variety of synthetic reaction fuelled by the energy of UV radiation . The idea of the pivotal role of Zn ions (and ZnS in particular) in the earliest stages of the evolution of life is highly attractive and generally plausible. However, in this manuscript, Mulkidjanian and Galperin put the plank very high by formulating several predictions that they claim to serve as Popperian tests of the "Zn world" hypothesis. In principle, the intention to test the hypothesis in a formal Popperian setting is indeed commendable. In practice, it is well known that it is hard to strictly adhere to Popperian criteria, and this paper is no exception. Unfortunately, as I see it, all the formulated predictions are weak, not unique to the "Zn World" hypothesis, and the nature of the evidence cited as being compatible with the predictions is such that one is prompted to ask "so what?" on most occasions. I briefly address the predictions and their purported tests one by one (the numbering of the predictions is mine – I think the authors should try to be more consistent to facilitate reading).
In relation to the popular view on the importance of iron for the primeval metabolism, our article gives a following answer to the "so what?" question: "the early life, most likely, developed in Zn-rich settings, its emergence in Fe-rich settings is unlikely. Free Zn2+ions, however, because of their poor solubility in primordial seawater, could have become available only if some process led to their continuous release – e.g. primordial abiogenic photosynthesis, as we suggest".
The rationale beyond our decision to formulate a set of testable predictions was simple (see also ref. ): if several predictions prove to be correct, then the tested hypothesis could be fairly countered only by an alternative hypothesis that would explain all those predicted observations. Hence the number of different predictions has a value of its own.
The following paragraphs respond to the specific comments of the referee that are related to particular predictions; the more general comment is addressed at the end of our response.
1. High content of Zn in modern cells – predicted on the basis of the "chemical conservation" principle. The principle is extremely general and might not be of huge heuristic value but suppose we accept it. The problem with this prediction is that it is not specific at all. Yes, of course, there are many Zn-dependent enzymes in all cells, and Zn is concentrated compared to any environment. Is it relatively more abundant than other divalent cations? There is no answer in the paper. But, even if it was, does this link Zn to primordial stages of evolution? Or, if it was not, would that falsify the Zn world hypothesis? I doubt that either of these propositions holds. The prediction just is not specific enough.
1) In the revised manuscript we state explicitly that zinc is indeed relatively more abundant in cells than other divalent transition metals – if compared to the chemical composition of sea water – see Table 1in the accompanying article.
2) We added a new section devoted to the metallome of the Last Universal Common Ancestor. where we link the content of Zn in modern cells to primordial stages of evolution.
3) If this particular prediction would not prove to be correct, the hypothesis would not be falsified yet, but strongly weakened; the final outcome would then depend on the tests of other predictions. If they all would fail, the hypothesis would be falsified indeed. Fortunately for us, this is not the case.
2. There should be ribozymes with Zn-dependent activity. Provided the RNA world hypothesis is accepted, there could be a more serious prediction here. However, I think the authors are too permissive in their formulation. A strong prediction would be that ribozymes are, mostly, Zn-dependent or that certain classes of ribozymes that are most relevant to the origin of cells, such as polymerases and ligases, should be Zn-dependent. The way it stands, the prediction is too vague, whereas the data are very uncertain as the authors admit, even as they check this prediction in their favor.
Authors' response: We admit that the test of this prediction has led to the least conclusive results. This has several reasons. (a) The metal specificity of ribozymes is low, such that Mg2+ions can occupy almost any divalent metal binding site; (b) the incubation media that are used by RNA scientists usually contain no other divalent cations besides the large amounts of Mg2+(G. Yusupova, personal communication); and (c) there are only few types of natural ribozymes discovered so far, although, as we discuss in the manuscript, some their representatives show Zn-specific activity.
Among the native ribozymes, only the type I and type II introns can operate as ligases. Their activity requires divalent metals, but seems to be Mg-specific . Generally, the activity of the known natural ribozymes is limited to the cleavage and formation of phosphoester bonds. The enzymes with this kind of activity are also Mg-dependent.
There are no natural ribozymes with polymerase activity. The artificially selected ones were shown to be Mg-dependent – since they were selected in a medium that contained Mg2+as the only divalent cation . We are not aware about attempts to select ribozyme with a polymerase activity by using mixed Zn/Mg media. Such an approach proved to be successful when a ribozyme for aldol reaction – which is chemically more demanding than polymerization of nucleotides – was selected. In a selection medium that contained 5 mM MgCl2and 0.3 mM ZnSO4a Zn-dependent ribozyme was readily obtained . The Zn2+ions were added to the selection medium since protein aldolases are Zn-dependent. As long as protein polymerases are Zn-dependent as well, we dare to predict that a selection of a Zn-dependent ribozyme polymerase is just a matter of a worthwhile trial. Generally, the hypothetical ribozymes of the RNA World should have been able to catalyze various chemical transformations; the example of enzymes shows that Zn2+ions may have been involved as catalysts in many of them.
RNA structures should be enriched in Zn compared to other transition metals. Again, this might make sense in the context of the RNA World. However, there are few RNA structures containing any transitional metals including Zn. The authors present a variety of post hoc considerations to explain the paucity of these ions – all this might be true but as a result, the argument does not seem to be convincing. An interesting observation in this section is the absence of Fe in any RNA structures, and the authors' interpretation that iron is likely to catalyze RNA cleavage makes sense. However, the data that are cited in support of this idea, on ribozyme activation by iron (ref. 179), logically suggest the opposite of their argument, namely, that iron is tolerated by RNA molecules, at least molecules like ribozymes that seem to be most relevant for the RNA World.
Authors' response: This is a very important point, so we have revisited the experimental protocols in ref.  (no. 179 in the original manuscript). In these experiments, it was checked whether diverse metals could activate a particular ribozyme, which was pre-selected for the ability to be activated by Mn2+, Co2+, Ni2+, Zn2+and Cd2+. Thirty eight diverse metal chlorides were added at 100 μM to the samples that contained precursor RNA and a ribozyme, the reaction was terminated after 5 min incubation at 23°C by the addition of a buffer supplemented with EDTA to the final concentration of 40 mM. The reaction products were then electrophoretically separated and the extent of the precursor RNA cleavage was checked. Of the 38 metals tested, only Fe2+ions were able to induce cleavage of the precursor RNA. However, this procedure seems to be inappropriate for Fe2+ions. In such a set-up there is no way to discriminate whether the precursor RNA had been cleaved by the Fe2+-activated ribozyme during the 5 min incubation or by the Fe2+-EDTA complexesafterthe anticipated reaction termination. In fact, Fe2+-EDTA complexes are widely used as cleavage agents for RNA . Because of this uncertainty, we have decided to drop the discussion of the work of Zivartis et al. .
3. Zn ions should be present in oldest protein folds. I will refrain from the criticism of the work of Bourne's group – let us assume that the inference of the oldest folds there is reasonable. Zn is found in a rather small minority of representative structures, albeit from many folds. This is hardly surprising given that, oldest or not, these are indeed very common folds. Perhaps, a comparison with "less ancient" folds would help (it is unclear why such a comparison is not included) but then, again, I am rather skeptical as there can be many reasons why some protein structures contain a particular metal whereas others do not. There is just not bridge from here to the "Zn World".
Authors' response: We agree that checking of the metal content of the "less ancient" folds is a worthwhile task. This task, however, is beyond our current capabilities since the total number of fold superfamilies is about 1300. We, however, would be happy if somebody carried out this work. As already noted, we have added a new section on the metallome of the LUCA where we tried to build a bridge from the traits of modern proteins to the primeval Zn world.
4. Occurrence of Zn in enzymes with the oldest functions. I find this line of argument more interesting than the preceding 4 lines. It is therefore somewhat unfortunate that the authors do not perform any analysis of their own but rather limit themselves to the citation of  where the exact list of "primordial enzymes" is not given. This lack of concreteness seriously weakens this potentially relevant argument.
Authors' response: In this work we deliberately relied on the data sets obtained by others, to avoid a potential bias. Whatever mistakes could have been made by the authors of those data sets, neither of them had any intention to provide supporting evidence for the Zn world concept.
5. Presence of Zn ions in enzymes that could have taken over ribozyme activities. The argument here is long and convoluted, and I am afraid I cannot conclude that there is any strong data presented.
Authors' response: We shortened this section and merged it with the section on the Zn-dependence of evolutionarily old functions.
In the beginning of the next section, it is claimed that the Zn World hypothesis "has successfully passed all six falsification tests". I am afraid that I cannot condone this statement. To me, there is only one serious falsification test here, #4. Indeed, if there were no Zn-dependent enzymes among the ones assumed to be primordial, that could be construed to falsify the Zn World hypothesis. Unfortunately, as noticed above, the analysis of this prediction in the paper is not the strongest. The rest of the predictions, to me, either do not follow from the Zn World hypothesis or are too weak and vague – self-serving, within the framework of the Popperian paradigm – to provide any argument in support of the hypothesis (this is not quite Popperian language but, at the end of the day, when a hypothesis passes several falsification tests, this result constitutes support, and I think this is how the authors see the situation).
Authors' response: In response to reviewers' comments, we have added a new section on the metallome of the LUCA, which discusses the link between the relatively high Zn levels in modern cells, enzymes, and RNA structures and the primordial stages of evolution. We argue that the Zn enrichment of modern cells/RNA structures/enzymes is evolutionarily relevant and supports the Zn world hypothesis. In addition, we now analyze the metal content of the proteins that supposedly were present in the LUCA. To minimize the authors' bias, we have used the data set that was obtained by the Reviewer himself . We show that proteins that supposedly were present in the LUCA predominantly contain Mg2+and two transition metals, Zn and Mn.
Our predictions are not "self serving" in the Popperian sense – to our knowledge he has never used such a term in relation to the falsification tests. The tests can be either tautological or not – we cared to make predictions that are not related to the premises on which the hypothesis is built.
Our predictions, however, are obviously "self-serving" in the common sense of word – we try to gather support for own hypothesis. Of course, we would be happy to see this hypothesis scrutinized by others.
I must note that I am generally sympathetic with the Zn World hypothesis and agree with the authors that substituting ZnS for FeS in the model of early evolution within networks of inorganic compartments is a promising idea. Moreover, I think the paper does include an argument that is not only compatible with this hypothesis but can be considered supportive. This is the so-called "Zn paradox" discussed in section 2.4.3. Indeed, properties of transition metals do not seem to explain why Zn is a cofactor for a disproportional number of diverse enzymes, and this is a provocative observation. Indeed, if a biological pattern does not have a functional or mechanistic explanation, one starts suspecting that it could be explained by the legacy of early stages of evolution – in this case, the Zn World. Perhaps, this argument could be addressed in somewhat greater detail.
Authors' response: The respective section has been expanded. We additionally note that while prevalence of Zn in certain types of enzymes could be attributed to the catalytic properties of Zn2+ions, their ubiquitous involvement as structural elements [128, 210, 243] had no known explanation until now. The Zn world concept explains the paradoxical prevalence of Zn ions both as catalysts and as structural elements by the shaping – and folding – of first proteins in Zn-rich habitats.
I am rather frustrated with the discussion of the purported implications of the Zn World hypothesis for the divergence of the domains of life and subsequent evolution. I am not inclined to discuss these in much detail but I think the majority are far-fetched and some just do not seem to be serious like conclusions based on the higher content of Zn-dependent enzymes in eukaryotes compared to archaea and bacteria or the conclusions on symbiosis etc. [(ix) and (vii) in section 2.4.5, respectively] etc. In my opinion, it is highly desirable to simply drop this section.
Authors' response: We have decided to retain some of this discussion; the respective part of the article has been, however, shortened and rewritten for the sake of clarity.
To summarize, I think the accompanying paper by Mulkidjanian together with this paper present a new, interesting, chemically plausible and overall promising hypothesis on the settings of primordial evolution. However, I think the authors do a disservice to their own good idea by heavily overloading the hypothesis with (at best) tangentially relevant arguments that they counter-productively attempt to present in a formal Popperian setting (I suppose, at least, in part, this approach is borrowed from Wächtershäuser but here it is taken to much greater and entirely unnecessary lengths). The paper is also full of implications that do not seem to really follow from the hypothesis but are discussed at great lengths. I would note that this paper (and I think the accompanying one as well) are heavily "overspecified", to the extent that there is a danger to completely drawn an interesting idea in a muddy waters of weak argumentation and excessive discussion. In my opinion, both papers could be reduced to a single, perhaps, 10 pages long article on the hypothesis that would discuss the relevant photochemistry and photophysics along with the interesting "Zn paradox". In that form, it would be a valuable contribution to the origin of life literature.
Authors' response: Regular scientific papers are usually addressed to a handful of experts in a narrow research field. The potential audience of a paper on the origin of a life might be much broader and could include readers with different backgrounds, from mathematics to geology. Accordingly, their understanding of which arguments are convincing and which are only tangentially relevant might differ as well. In the view of this broad and unevenly trained potential audience, we have chosen a presentation style that, while being more diffuse as compared to routine scientific papers, might reach more readers.
A specific and relatively minor comment on horizontal gene transfer:
"The rigorous vertical inheritance of the genes responsible for information processing, in particular of the ribosome machinery , might indicate that these genes, at least at the LUCA stage, already formed the non-shared genetic cores of the first organisms"
The vertical inheritance of ribosomal protein genes let alone other components of information processing systems is by no account "rigorous", horizontal transfer of these genes is common enough [201, 202] even if less common than horizontal transfer of genes for metabolic enzymes, although even that trend has been questioned .
Authors' response: These references are now cited.
Patrick Forterre, Institut Pasteur, Département de Microbiologie, Unité de Biologie Moléculaire du Gène chez les Extrêmophiles, Paris, and Université Paris-Sud, Institut de Génétique et Microbiologie, Orsay, France
The origin of life remains a major unsolved problem in science, and no consensus exists in the community of people who try to tackle this problem, despite the great number of theories that have already been proposed. In an accompanying paper, Mulkidjanian proposed a new theory in which the first organisms were photosynthetizers, that used zinc sulfide (ZnS) formed in sub-aerial setting to capture solar energy for abiotic reduction of CO2. In this paper, Mulkidjanian and Galperin look in modern cells for support of this theory. By extensively reviewing the literature, they made six observations that could be viewed as supporting the idea that Zinc has indeed played a major role in early life evolution. In particular, they conclude from their survey of the experimental literature that ribozymes can work using Zinc as cofactor instead of magnesium, and that zinc is enriched in proteins with "old folds" or "old functions". This part of the paper is very interesting and can push the biochemists to look more carefully at the real metal dependency of the protein they are studying. These observations convince me that somehow Zinc should have played an important role in early biological evolution. An interesting observation is that the fraction of Zn-containing enzymes is higher in Eukarya than in Bacteria or Archaea. If the hypothesis of the author is correct, this supports heretical ideas according to which the proto-eukaryotic lineage might be more ancient than streamlined lineages of Archaea and Bacteria . The authors call the ancestor of Archaea and Eukarya, a proto-archaeon or a pro-eukaryotic archaeon. This is based on the idea that evolution always goes from simple to complex and that streamlining in the archaeal domain has been much less important than complexification in the eukaryotic domain, something not so clear at the molecular level. Considering that Archaea are probably monophyletic , I think that the authors should avoid these confusing terms (proto-archaeon or a pro-eukaryotic archaeon) and only refer to the pro-eukaryotic ancestor.
Authors' response: Throughout the manuscript, the pro-eukaryotic archaeon has been renamed pro-eukaryote.
More importantly considering the objective of this paper, I have a fundamental problem with the idea of an early Zinc world. Indeed, from my own lab experience, it is clear that Zinc has the ability to strongly induce cleavage of DNA, especially at high temperature . Zinc has even a stronger deleterious effect on RNA. The cleavage of polyribonucleotide by Zinc has been extensively studies by Butzow and Eichhorn in the last century. These authors have shown in particular that RNA is especially prone to Zinc-induced degradation and even argued that the higher resistance of DNA to metal-induce degradation explains why DNA replaced RNA as cellular genetic material in the course of evolution . The high sensitivity of RNA to Zinc should have created a great problem for early RNA-based systems (before the invention of proteins that could protect RNA against Zinc-induced hydrolysis). In any case, if the Zinc world really occurred it should have done so at low temperature. The Zinc world hypothesis therefore strongly supports the idea of a cold origin of life. It raises the possibility that life could have only invades high temperature environment after the emergence of DNA and modern complex proteins. In any case, this paper is important because it should stimulate investigators to resume the study the effect of Zinc on RNA stability in the presence of minerals, lipids and/or peptides, and to analyze the activity of various ribozymes in the presence of this metal at different temperatures.
Authors' response: The possibility of the Zn-catalyzed cleavage, which was also addressed in the comment by Mushegian, is indeed a very important topic. We now consider this point in detail in the accompanying paper  and in the response to the Mushegian's comment to that paper. We argue that the ability of proteins to bind to the 2'-OH group of ribose could protect RNA molecules from Zn-catalyzed cleavage and may have driven the emergence of the first proteins. We are grateful to the Reviewer for pointing out the very useful reference to the work by Butzow and Eichhorn.
Although I found this paper really interesting, I think that the authors were misguided in connecting their Zinc world hypothesis to the Martin and co-workers hydrothermal vent scenario for the origin of life, in which, as described by Mulkidjanian and Galperin: "Bacteria and Archaea are descendents of two distinct populations that thrive around hydrothermal vents". The plural of vents in that description is misleading. Indeed, in that scenario, life should have originated and evolved up to modern cells in a single chimney, since life forms were trapped in mineral cages until the advent of Bacteria and Archaea. If the conditions were favourable to the development of a Zinc-world in cold hydrothermal settings, many independent primitive Zinc worlds should have emerged more or less simultaneously at many places at the earth surface. In the one chimney scenario, this means that all hydrothermal vents at the earth surface should have produce living organisms, but that Archaea and Bacteria originated from the same chimney (out of millions) and wiped out all other cells that were produced by other chimneys!! This seems ridiculous. Even if all ancestral chimneys were connected into a single giant chimney all around the globe, living organisms could not have moved and competed from one part of this giant chimney to another since they were trapped in mineralized cages (ZnS compartments), so LUCA and its descendents should have originated from the same region of this giant chimney, against wiping out cells that were produced by other parts of this chimney. In general, I don't think that one can trace the origin of the three domains to the evolution of the vent systems and I don't buy the geochemical scenario proposed by the authors for the origin of the three cellular domains. To explain the formation of the three domains, one should understand why three types of molecular biology (for instance three versions of the ribosomes) originated from LUCA, and this cannot be related simply to considerations based on various metabolisms.
Authors' response: The expression "around hydrothermal vents" has been replaced by "within a deep sea hydrothermal vent".
In contrast to Martin and co-workers, we think about the first biotopes as rings of precipitated ZnS/MnS particles around chimneys of continental hot springs. Based on the typical structure of ancient volcanogenic massive sulfide deposits , we can envision that these rings intersected and formed a continuous net of photosynthesizing and inhabited settings, most likely occupying the bottom lands of primeval hydrothermal fields. The life in these biotopes was confined to the illuminated ZnS surface, so the first life forms could be moved within these biotopes by water. Moreover, the illuminated ZnS compartments should have been fragile and break continuously because of photocorrosion.
Further, we speculate that upon the separation of the main lineages not the organisms themselves, but the geologic settings that they inhabited started to move away from each other. With the decrease in the CO2atmospheric pressure, inhabited ZnS-rich settings at hydrothermal vents would move deeper and deeper into the ocean, away from the continental phototrophic communities. As we believe, exactly the initial lack of swimming motility of the first organisms enabled their separate evolution and crystallization of the domain-specific features in the phototrophic communities (future Bacteria), on the one hand, and chemotrophic/heterotrophic communities on the sea floor (future Archaea/Eukarya), on the other hand.
Generally speaking I think that the authors, as many other scientists working in the origin of life fields, tend to underestimate the various evolutionary steps that were required to go from the first proto-cells to LUCA and later on to modern cells (Archaea, Bacteria and Eucarya). This is clear when the authors speak of proteins that have been "attributed to the very early life", including in that category DNA repair and replication proteins. For me these proteins do not testify for the very early life because they could have only appeared after the evolution of very sophisticated ribosomes, at a very late period of early life evolution. If the first life forms indeed originated in ZnS compartments of an hydrothermal vent, I would suggest that the cells that emerged from these vents were primitive cells that started to compete with each others as free living cells in ancestral water ponds and/or in shallow waters of the ancestral oceans. They might have been pre-RNA cells or cells that found a way to protect their RNA against Zinc-induced degradation. This does not dismiss the possibility that the first proteins that replaced ribozymes after the invention of ribosomes mainly inherited zinc ions from the catalytic centers of these ribozymes, or that cells of that time (second age of the RNA world) were still thriving in a rich zinc world.
Authors' response. We agree that attribution of DNA repair and replication proteins to the very early life, as done by Zerkle et al. , is controversial. In the revised manuscript we still present their data but do not discuss them (see also our response to the respective comment of Mushegian). We fully agree with the Reviewer that acquisition of Zn2+ions may have preceded the emergence of DNA repair proteins. In the revised version we added a new section on the metallome of the LUCA, where we argue that the first organisms could have thrived in the Zn-rich environments up to the stage of the LUCA.