On the evolution of chlorophyll synthesis, methanogenesis, and nitrogen fixation. Was the ancestor of Bacteria photosynthetic?

Weiss et al. recently attempted to reconstruct the proteome of the Last Universal Common Ancestor (Weiss et al. 2016). One of the aspects that puzzled me the most about that work is that they suggested the LUCA was methanogenic and a nitrogen fixer, but there was absolutely no mention of photosynthesis.

There are three major groups of nitrogenase-like enzymes known. 1) The ones used for the synthesis of Ni-tetrapyrroles in methanogenic Archaea, 2) the ones used in Mg-tetrapyrroles in photosynthetic bacteria, and 3) proper nitrogenases. It is also likely that there are many uncharacterized enzymes belonging to this superfamily of proteins with unknown functions.

Under orthodox views on the evolution of photosynthesis it can be argued that those used in photosynthesis evolved from nitrogenases or those used in methanogenesis, but the phylogeny of these enzymes is inconsistent with that. It is inconsistent in such a way that the chlorophyll-synthesis enzymes do not seem to emerge from nitroegnases or those used in methanogenesis. What it is actually seen in their phylogeny is a deep divergence between the methanogenesis enzymes and those in photosynthesis: with nitrogenases being closer to the methanogenesis Ni-tetrapyrrole-synthesis enzymes. Therefore, there is a deep split between Bacteria/photosynthesis and Archaea/methanogenesis.

This is the thing, that if nitrogenase and the Ni-tetrapyrrole enzyme share a more recent common ancestor, and these were found in the LUCA, then the LUCA must have had also protochlorophyllide reductase. This may be hard to grasp, but it derives from the phylogenetic relationships of these enzymes. Phylogenetics 101. This is because the branch leading to protochlorophyllide reductase, in this case ChlL, should have diverged before the nitrogenase homolog (NifH) and the Ni-tetrapyrrole one (CbfC) had time to split.

The other way we can see this is that LUCA had the ancestral enzyme to these, and that their specialization occurred later. But there is no evidence that actually suggests the ancestor of these enzymes was more likely to be involved in methanogenesis or nitrogen fixation than photosynthesis.

What is more, I know that these enzymes have enough sequence identity to make it to the threshold of their analysis. And that is why I was puzzled, because I expected some of the chlorophyll synthesis enzymes to show up in their analysis, but apparently didn't...

As I was browsing through all 355 trees from the Weiss et al. work, I found this little gem! See image or the attached newick tree file.

That is indeed protochlorophyllide reductase subunit L splitting away from a NifH-like enzyme in methanogenic Archaea, which is probably a misannotated CbfC. Exactly as it should have been, and so it confirms that my puzzlement was not due to my lack of understanding of the evolution of these proteins.

The phylogeney on the bacteria part of the tree (red) matches perfectly that of ChlL and contains only phototrophs. It is indisputably ChlL and does not represent bacterial nitrogenases.

What does this mean? Well, if the authors work is informative in any way, it would mean that the split of the CbfC and ChlL is a Bacteria/Archaea split, which would make the ancestor to all bacteria photosynthetic. Nevertheless, that photosynthesis originated in the most recent common ancestor of all bacteria, or soon after that, is supported by the evolution of the photosynthetic reaction centres, as I concluded in my first review on the subject (Cardona 2015).

The reason this is not more widely understood or accepted is simply due to prevalent ideas on the evolution of photosynthesis, which are somewhat outdated and are now based more on speculation and personal opinions than on any real data... Something that I am working really hard to change.

If you want to have a look at the data by Weiss et al., have a look at this: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1007518#sec011

The tree in the image is numbered 2020 in the supplements.

References:

Cardona, T. (2015). "A fresh look at the evolution and diversification of photochemical reaction centers." Photosynthesis research 126(1): 111-134. DOI: 10.1007/s11120-014-0065-x.

Weiss, M. C., F. L. Sousa, N. Mrnjavac, S. Neukirchen, M. Roettger, S. Nelson-Sathi and W. F. Martin (2016). "The physiology and habitat of the last universal common ancestor." Nat Microbiol 1(9): 16116. DOI: 10.1038/nmicrobiol.2016.116.

Unified view for the evolution of oxygenic photosynthesis

I thought it would be a good idea to create a plot that outlines my perspective on the evolution of photosynthesis. It is based on my research and those by others. Please, keep on reading if you're interested. Make sure to open the attached figure.

So, when did oxygenic photosynthesis originated? Counterintuitively, the answer to this question does not depend so much on when Cyanobacteria originated. Oxygenic photosynthesis and Cyanobacteria are not strictly the same thing. It actually depends on when photosynthesis and the first reaction centres originated for the first time.

The y axis is time. On the right, we see a species tree of bacteria cantered around the diversity of Cyanobacteria. On the left side, we see a tree of reaction centre proteins.

Let’s focus first on the species tree. It has been recently suggested that the most recent common ancestor (MRCA) of Cyanobacteria capable of oxygenic photosynthesis postdate the Great Oxidation Event (GOE). Shih et al. (2017) suggested that the age of this ancestor is about 2.0 Ga. Similar results had been obtained before in other molecular clock studies, but remained unnoticed, see for example David and Alm (2011). These results have also been reproduced in newer analyses, see for example: (Marin et al. 2017, Betts et al. 2018). There is a real possibility that the MRCA of Cyanobacteria predated the GOE, see for example: (Sanchez-Baracaldo 2015, Sanchez-Baracaldo et al. 2017, Magnabosco et al. 2018). So, let’s not take sides and consider all possibilities.

Cyanobacteria, in the classic sense, are more closely related to the Melainabacteria and Sericytochromatia (Soo et al. 2017). But if we zoom out, the Cyanobacteria/Melainabcateria/Sericytochormatia (CMS) supergroup is thought to be contained within a much larger group that includes Chloroflexi, Actinobacteria, Firmicutes, and the Deinococcus-type. This larger group has been called Terrabacteria by some. I have seen many phylogenomic analysis that puts Cyanobacteria and Chloroflexi as each other’s closest relatives. The estimated time for the Cyanobacteria and Chloroflexi split has been calculated to have occurred about ~3.0 Ga ago by David and Alm (2011) and about ~2.7 Ga ago by Marin et al. (2017). Marin et al. (2017) also timed the MRCA of Terrabacteria at about 2.9 Ga.

Nevertheless, there are phylogenomic trees that put the branch leading to Cyanobacteria very basally in the tree of life of bacteria. See for example: (Hug et al. 2016, Yokono et al. 2018). Also, see the recent tree by Betts et al. (2018). This does not necessarily imply that the MRCA of Cyanobacteria is deeply branching with respect to other bacteria. However, I think overall, the “Terrabacteria” grouping has been reported more often than a basal CMS supergroup for example. Keep this in mind.

Now let’s have a look at the evolution of reaction centres.

Cyanobacteria are characterised by having Photosystem II and Photosystem I. PSII has a heterodimeric core made of the homologous subunits D1 and D2. This core is associated with the homologous core antenna proteins CP43 and CP47. PSI has a heterodimeric core made of the homologous subunits PsaA and PsaB.

The level of sequence identity (distance) between D1 and D2 in ALL cyanobacteria is just under 30%. Between CP43 and CP47 is just under 20%, and between PsaA and PsaB is just under 45%. In other words, the phylogenetic distance between D1 and D2, CP43 and CP47, and between PsaA and PsaB is very large.

The thing about Cyanobacteria is that they all inherited “standard” photosystems. That is to say, that the most recent common ancestor of Cyanobacteria already had photosystems with divergent heterodimeric cores. So, the duplication events leading to D1 and D2, CP43 and CP47, and PsaA and PsaB happened before the MRCA of Cyanobacteria (nodes marked red).

I have attempted to gain an understanding of the evolution of reaction centre proteins as a function of time. I have done that by comparing the levels of sequence identity and by applying molecular clocks under a wide range of evolutionary scenarios (Cardona 2016, Cardona 2018, Cardona et al. 2018).

What I have found is that the gene duplication event leading to D1 and D2, marked as D0 in the tree, is likely to have occurred more than 1 billion years before the MRCA of Cyanobacteria!

It sounds crazy, but it is not crazy at all. It is actually rather straight forward. We’re just not used to think this way about the evolution of photosynthesis. Don’t panic!

In this particular example, the span of time between the D0 duplication event and the MRCA of Cyanobacteria is called ΔT, see the figure.

The large ΔT is due to two facts of life that are pretty unambiguous. 1) The phylogenetic distance between D1 and D2 is VERY LARGE. 2) The rates of evolution of D1 and D2 are VERY SLOW. Therefore, it takes a very long time to span the distance between the D0 duplication event and the MRCA of Cyanobacteria. This is also true for the CP43/CP47 and the PsaA/PsaB duplications.

The rates of evolution of D1 and D2 are very slow, but these rates are not unusual in any way. The rates are just like those in any other highly conserved protein of bioenergetics involved in complex functions. Absolutely nothing peculiar about that. Have a look at Table 3 in Cardona et al. (2018), we have studied the rates of evolution of D1 and D2 in great detail and compared them to those of other proteins.

What is key however, is that the ancestral protein to D1 and D2, D0, likely made a photosystem that was capable of oxidizing water to oxygen or was well on its way towards the origin of water oxidation chemistry. Given the shared conserved traits between D1 and D2 we have a pretty solid idea of what D0 photosystem was capable of doing… and a photosystem made of D0 was not like other anoxygenic Type II reaction centres. That is for sure.

So the roots of oxygenic photosynthesis go deep. I find this conclusion inescapable.

I also found that the rate of evolution of L and M is about 5 times greater than D1 and D2. It appears as if D1 and D2 are actually the slowest evolving reaction centre proteins of all. This means that PSII is the most likely reaction centre to have retained ancestral traits. Counterintuitively as it seems, it is rather evident when you compare the structures of the photosystems… starting from the fact that like Type I reaction centres PSII has retained core antenna proteins and the core peripheral chlorophylls of D1 and D2. What is more, the position of the redox tyrosine residues is located at the ancestral entry point of electrons, as it is the case in homodimeric Type I reaction centres.

I have tried to time the duplication leading to PsaA and PsaB as well (Cardona 2018), which is widely accepted to have occurred after the origin of oxygenic photosynthesis (Ben-Shem et al. 2004, Hohmann-Marriott and Blankenship 2008, Rutherford et al. 2012). It turns out that PsaA and PsaB are also evolving quite slowly, only slightly faster than D1 and D2, in such a way that the PsaA and PsaB duplication likely occurred long before the MRCA of Cyanobacteria too. It is expected that the duplication leading to CP43 and CP47 occurred simultaneously with the duplication of D1 and D2, as they make part of the same complex. The distance between CP43 and CP47 and their rates of evolution agrees with this.

The position of CP43 and CP47 in the tree of reaction centres is not well defined yet. That is why I have put the branch with dashes. That is the position that I think is better supported and has more explanatory power… but other scenarios are possible, all with interesting repercussions. I am currently working on a paper about this.

These three duplications that are unique to oxygenic photosynthesis are more likely to have occurred closer to the origin of reaction centre proteins than closer to the GOE, or after the GOE. Strong arguments supporting the premise that these duplications were driven by the optimisation of water oxidation and the evolution of photoprotective mechanisms to avoid the production of reactive oxygen species can be made. Such arguments can be applied to the initial divergence of anoxygenic and oxygenic specific reaction centre proteins (blue nodes, question marks), see for example (Orf et al. 2018). No matter how you look at it, water oxidation to oxygen likely started well before 3.0 Ga (blue wavy line), which is indeed supported by some geochemical evidence (Planavsky et al. 2014, Satkoski et al. 2015, Havig et al. 2017, Wang et al. 2018).

So how old are these duplications and initial divergences? As you can see in the plot, this depends on how old photosynthesis is. The older reaction centres are, the older the origin of water oxidation chemistry. If we assume that Cyanobacteria is much older than the GOE, then that would make the rates of evolution of reaction centre proteins even slower, which then would push the initial duplications specific to oxygenic photosynthesis (red nodes) even closer to the origin of reaction centres. This is a consequence of the two facts mentioned above, long distance and slow rates.

The origin of oxygenic photosynthesis started in an ancestor of Cyanobacteria… but this ancestor could have been the ancestor of a much greater diversity that could include other Terrabacteria, if that affiliation holds true. Betts et al. (2018) suggested that the MRCA of bacteria is only about 3.4 Ga old. David and Alm (2011) also suggested that the expansion of diversity in bacteria started about 3.4 Ga ago, peaking about 3.2 Ga ago.

I understand that the evidence for photosynthesis at 3.5 Ga (traditionally considered to be anoxygenic) is pretty strong. As far as I understand, the possibility that photosynthesis originated prior to 3.8 Ga cannot be ruled out yet (Rosing 1999, Rosing and Frei 2004, Czaja et al. 2013, Nisbet and Fowler 2014, Butterfield 2015).

Therefore, connect the dots.

If you have questions don’t hesitate to leave a comment.


References
Ben-Shem, A., F. Frolow and N. Nelson (2004). "Evolution of photosystem I - from symmetry through pseudosymmetry to asymmetry." FEBS Lett 564(3): 274-280. DOI: 10.1016/S0014-5793(04)00360-6.

Betts, H. C., M. N. Puttick, J. W. Clark, T. A. Williams, P. C. J. Donoghue and D. Pisani (2018). "Integrated genomic and fossil evidence illuminates life’s early evolution and eukaryote origin." Nature Ecology & Evolution. DOI: 10.1038/s41559-018-0644-x.

Butterfield, N. J. (2015). "Proterozoic photosynthesis - a critical review." Palaeontology 58(6): 953-972. DOI: 10.1111/pala.12211.

Cardona, T. (2016). "Reconstructing the origin of oxygenic photosynthesis: Do assembly and photoactivation recapitulate evolution?" Front Plant Sci 7: 257. DOI: 10.3389/fpls.2016.00257.

Cardona, T. (2018). "Early Archean origin of heterodimeric Photosystem I." Heliyon 4(3): e00548. DOI: 10.1016/j.heliyon.2018.e00548.

Cardona, T., P. Sanchez-Baracaldo, A. W. Rutherford and A. W. D. Larkum (2018). "Early Archean origin of Photosystem II." BioRxiv 109447. DOI: https://doi.org/10.1101/109447.

Czaja, A. D., C. M. Johnson, B. L. Beard, E. E. Roden, W. Q. Li and S. Moorbath (2013). "Biological Fe oxidation controlled deposition of banded iron formation in the ca. 3770 Ma Isua Supracrustal Belt (West Greenland)." Earth and Planetary Science Letters 363: 192-203. DOI: 10.1016/j.epsl.2012.12.025.

David, L. A. and E. J. Alm (2011). "Rapid evolutionary innovation during an Archaean genetic expansion." Nature 469(7328): 93-96. DOI: 10.1038/Nature09649.

Havig, J. R., T. L. Hamilton, A. Bachan and L. R. Kump (2017). "Sulfur and carbon isotopic evidence for metabolic pathway evolution and a four-stepped Earth system progression across the Archean and Paleoproterozoic." Earth-Sci Rev 174: 1-21. DOI: https://doi.org/10.1016/j.earscirev.2017.06.014.

Hohmann-Marriott, M. F. and R. E. Blankenship (2008). Anoxygenic Type-I photosystems and evolution of photosynthetic reaction centers. Photosynthetic Protein Complexes. P. Fromme, Wiley-VCH Verlag GmbH & Co. KGaA: 295-324.

Hug, L. A., B. J. Baker, K. Anantharaman, C. T. Brown, A. J. Probst, C. J. Castelle, C. N. Butterfield, A. W. Hernsdorf, Y. Amano, K. Ise, Y. Suzuki, N. Dudek, D. A. Relman, K. M. Finstad, R. Amundson, B. C. Thomas and J. F. Banfield (2016). "A new view of the tree of life." Nat Microbiol 1: 16048. DOI: 10.1038/nmicrobiol.2016.48.

Magnabosco, C., K. R. Moore, J. M. Wolfe and G. P. Fournier (2018). "Dating phototropic microbial lineages with reticulate gene histories." Geobiology. DOI: 10.1111/gbi.12273.

Marin, J., F. U. Battistuzzi, A. C. Brown and S. B. Hedges (2017). "The timetree of prokaryotes: New insights into their evolution and speciation." Mol Biol Evol 34: 437-446. DOI: 10.1093/molbev/msw245.

Nisbet, E. G. and C. F. R. Fowler (2014). The early history of life. Treatise on Geochemistry. K. D. M. and W. H. Schlesinger. Amsterdam, Elsevier Science. 10: 1-42.

Orf, G. S., C. Gisriel and K. E. Redding (2018). "Evolution of photosynthetic reaction centers: insights from the structure of the heliobacterial reaction center." Photosynthesis research. DOI: 10.1007/s11120-018-0503-2.

Planavsky, N. J., D. Asael, A. Hofmann, C. T. Reinhard, S. V. Lalonde, A. Knudsen, X. Wang, F. Ossa Ossa, E. Pecoits, A. J. B. Smith, N. J. Beukes, A. Bekker, T. M. Johnson, K. O. Konhauser, T. W. Lyons and O. J. Rouxel (2014). "Evidence for oxygenic photosynthesis half a billion years before the Great Oxidation Event." Nat Geosci 7(4): 283-286. DOI: 10.1038/ngeo2122.

Rosing, M. T. (1999). "C-13-depleted carbon microparticles in > 3700-Ma sea-floor sedimentary rocks from west Greenland." Science 283(5402): 674-676. DOI: 10.1126/science.283.5402.674.

Rosing, M. T. and R. Frei (2004). "U-rich Archaean sea-floor sediments from Greenland - indications of > 3700 Ma oxygenic photosynthesis." Earth and Planetary Science Letters 217(3-4): 237-244. DOI: 10.1016/S0012-821x(03)00609-5.

Rutherford, A. W., A. Osyczka and F. Rappaport (2012). "Back-reactions, short-circuits, leaks and other energy wasteful reactions in biological electron transfer: redox tuning to survive life in O2." FEBS Lett 586(5): 603-616. DOI: 10.1016/j.febslet.2011.12.039.

Sanchez-Baracaldo, P. (2015). "Origin of marine planktonic cyanobacteria." Sci Rep 5: 17418. DOI: 10.1038/srep17418.

Sanchez-Baracaldo, P., J. A. Raven, D. Pisani and A. H. Knoll (2017). "Early photosynthetic eukaryotes inhabited low-salinity habitats." Proc Natl Acad Sci USA. DOI: 10.1073/pnas.1620089114.

Satkoski, A. M., N. J. Beukes, W. Li, B. L. Beard and C. M. Johnson (2015). "A redox-stratified ocean 3.2 billion years ago." Earth and Planetary Science Letters 430: 43-53.

Shih, P. M., J. Hemp, L. M. Ward, N. J. Matzke and W. W. Fischer (2017). "Crown group Oxyphotobacteria postdate the rise of oxygen." Geobiology 15(1): 19-29. DOI: 10.1111/gbi.12200.

Soo, R. M., J. Hemp, D. H. Parks, W. W. Fischer and P. Hugenholtz (2017). "On the origins of oxygenic photosynthesis and aerobic respiration in Cyanobacteria." Science 355(6332): 1436-1440. DOI: 10.1126/science.aal3794.

Wang, X. L., N. J. Planavsky, A. Hofmann, E. E. Saupe, B. P. De Corte, P. Philippot, S. V. LaLonde, N. E. Jemison, H. J. Zou, F. O. Ossa, K. Rybacki, N. Alfimova, M. J. Larson, H. Tsikos, P. W. Fralick, T. M. Johnson, A. C. Knudsen, C. T. Reinhard and K. O. Konhauser (2018). "A Mesoarchean shift in uranium isotope systematics." Geochim Cosmochim Ac 238: 438-452. DOI: 10.1016/j.gca.2018.07.024.

Yokono, M., S. Satoh and A. Tanaka (2018). "Comparative analyses of whole-genome protein sequences from multiple organisms." Sci Rep 8. DOI:10.1038/s41598-018-25090-8.

Searching for new Type I reaction centre proteins in metagenomes

Testing my new-found metagenome-searching skills I decided to look for Type I reaction centre core subunits from Heliobacteria. This is because there are less than a handful of PshA sequences from this fascinating group of organisms, and only one complete and published sequenced genome.

Judging from the massive phylogenetic distance between the PshA core subunit of the reaction centre from Heliobacteria and the next closest relative (the PscA from Chlorobi/Acidobacteria), one must assume that a significant biodiversity should have existed spanning this distance, even if one or the other obtained phototrophy via horizontal gene transfer.

I limited my search to about 2000 metagenomes. I narrowed down my selection to those using in the metagenome title: “microbial dark matter”. I am not sure however if all of these belong to a singular project or if these have come from different/independent labs or projects.

I have always wondered however, if in these humongous datasets there are any novel phototrophs still unknown to science.

I used the PshA sequence from Heliobacterium modesticaldum as query.

The BLAST did not retrieve new sequence from Heliobacteria nor Acidobacteria, but did retrieve quite a few sequences from phototrophic Chlorobi and Cyanobacteria, see the attached figures. No sequences outside the known phyla of phototrophs were found, which is kind of sad. I had great expectations.

PscA from phototrophic Chlorobi

255 complete or almost complete sequences were obtained, which I then used to build a Maximum Likelihood tree. I did not have a look at fragmented sequences.

There was one almost complete sequence of a PsaA subunit from a new strain close to Gloeobacter.

It had 82% sequence identity to the PsaA of G. violaceus and G. kilaueensis. In comparison, the PsaA of these last two share 88% sequence identity. As another point of comparison, the level of sequence identity for PsaA between a red algae, C. merolae, and A. thaliana is 82%.

PsaA, the early branches. ML tree. In bold the metagenome sequnces

At this level of sequence divergence, it should be a new genus/species. I name this strain Protogloeobacter cardonensis. Kidding.

The metagenome where this particular sequence was found is the following:

Hot spring sediment bacterial and archeal communities from British Columbia, Canada, to study Microbial Dark Matter (Phase II) - Larsen N4 metaG (Released on 2016-05-27)

There were also quite a few sequences from the early-branching hot spring Synechococcus type. In addition, a PsaA/PsaB pair for another Gloeomargarita strain and a PsaA/PsaB pair of isoforms of the far-red light acclimation response from a form of Fischerella.

If you want the sequences or would like to see the full tree, let me know.

The atypical D1 sequence of Gloeobacter kilaueensis: looking for another one in metagenomes

The evolution of D1 proteins is complicated. It is characterized by many gene duplication events occurring at every taxonomic level. Some of these duplications could potentially predate the most recent common ancestor of all described cyanobacteria.

See our previous work on this:

Article Origin and Evolution of Water Oxidation before the Last Comm...

Some of the earliest duplications, we suggested, gave rise to the atypical D1 forms, of which we have described three forms. What I have called Group 0, Group 1, and Group 2 D1.

Group 0 is made of a single sequence, found exclusively in the genome of Gloeobacter kilaueensis. G. kilaueensis has additionally 5 standard D1 forms. There may be a D1 fragment encoded in the genome of the early branching Synechococcus sp. PCC 7336, have a look at this:

http://tanaiscience.blogspot.com/2015/10/evolution-of-oxygenic-photosynthesis.html

Group 1 is the super-rogue D1 also known as chlorophyll f synthase (or PsbA4).

Group 2 is the rogue D1: function unknown/unconfirmed.

A recent preprint by Grettenberger et al., described a new type of early branching cyanobacteria, which was named Aurora. The genome of this cyanobacterium was assembled from a metagenome of a microbial mat found in lake Vanda in Antarctica. It is more than 90% complete. This strain seems to be distantly related to Gloeobacter. As far as I understand, it is not clear however if this strain is an early-branching cyanobacterium sister to Gloeobacter, or whether it predates Gloeobacter, being therefore a sister branch to all described cyanobacteria.

This is the preprint:

Preprint Insights into the evolution of oxygenic photosynthesis from ...

Aurura vandensis has a PSII with a subunit composition similar to that of Gloeobacter. Only one D1 was reported in the preprint, and this is a standard form of D1, a Group 4.

Excited by this, I wondered if I could find another Group 0 sequence in the available metagenomes. Another G0, similar to that from G. kilaueensis.

So, I did a BLAST to all JGI environmental metagenomes: these were a total of 12361. I left out metagenomes categorized as “engineered” or “host-associated”.

To do a BLAST in so many metagenomes directly on the JGI site, it is necessary to split the data into sets of maximum 500 metagenomes. That gives 25 sets of metagenomes that needed to be BLASTed.

My query sequence was the very atypical G0 sequence from G. kilaueensis.

In the first set I obtained more than 30000 hits, which must include D1, D2, L, and M subunits; both complete and partial sequences. The cut-off E-value was 1e-5.

None of the 25 sets produced a sequence similar to the G0 sequence. Nothing close to it. The closest identity was 54%, usually to other standard forms of D1. No sequence alignment included the C-terminus, which is kind of special in the G0 sequence. Some of the metagenome sets gave a top hit to super-rogue D1 sequences, but the level of sequence identity between G0 and the other atypical forms is also just over 50%. This makes sense if the phylogenetic tree that we published in the paper above is correct, as it would imply that the G0 sequence is as close to the other atypical sequences, as it is to the standard forms of D1.

This is because we suggested based on the phylogeny of D1, that Group 1 to Group 4 would make a monophyletic group to the exclusion of the G0 sequence. But, phylogenetic trees are susceptible to artifacts, so having more G0 sequences could potentially improve the D1 phylogeny.

Each search for each of the metagenome sets produced more than 30k hits: that means that I could have obtained more than 750k hits in these 12361 metagenomes! But not a second G0 sequence?

I have to say that I did not examine every sequence in detail (of course)… waaay too many. So there may have been a partial sequence close to G0 that did not score high due to its very short length. If there was another G. kialueensis somewhere else I would have expected at least some identical sequences, but nothing at all!

I thought that Gloeobacter was not that uncommon after all:

Article The Primitive Thylakoid-Less Cyanobacterium Gloeobacter Is a...

Would anyone be interested in repeating this search? :)

This is the link to the G0 sequence: https://www.ncbi.nlm.nih.gov/protein/AGY58976.1

Now, with the recent eruption of Kilauea this unique strain of Gloeobacter may have just gone extinct.

Little paper on the evolution of Photosystem I and oxygenic photosynthesis is out now

A short update. The little manuscript detailing the evolution of Photosystem I and the implications for the evolution of oxygenic photosynthesis is now available in the open access journal Heliyon, have a look:

https://www.heliyon.com/article/e00548/

A press release accompanied the paper. Have a look too if interested: https://bit.ly/2FZKXkA

The main conclusion of the paper is that the duplication event that allowed Photosystem I to become a heterodimer occurred long before the most recent common ancestor of cyanobacteria. It is has been hypothesized that the reason why Photosystem I is a heterodimer in oxygenic photosynthesis has to do with oxygen and avoiding reactive oxygen species. So, if the duplication occurred after the evolution of water oxidation to oxygen it would imply that the earliest stages in the evolution of oxygenic photosynthesis appear much early than currently accepted.

The funny thing is that according to my plans, I was hoping for the Photosystem II version of the manuscript to be published first, link below. I have had to experience however everything that is horrible and beautiful about peer-review, and everything in between, in the process. The manuscript is still there fighting its way to freedom:

https://www.biorxiv.org/content/early/2017/09/15/109447

If you like the paper show your support by sharing the preprint or leaving a comment!

Cyanobacteria doing photosynthesis

Photosystem I and the evolution of oxygenic photosynthesis

I want to understand when and how oxygenic photosynthesis originated. Sometime ago I posted an evolutionary analysis of the core proteins of Photosystem II, which is still undergoing peer-review.

Check it out here if you have not seen it already:

https://www.biorxiv.org/content/early/2017/09/15/109447

Basically, all reaction centres are made of a dimer of homologous core proteins. All Type II reaction centres have a heterodimeric core, meaning that each monomer is different. Photosystem II, the water oxidising enzyme, has a core made of D1 and D2. D1 and D2 share slightly under 30% sequence identity and it is in D1 that the manganese cluster that oxidises water to oxygen is located.

It is pretty evident when the sequence and structure of D1 and D2 are compared, that the homodimeric Photosystem II (before the divergence of D1 and D2) was able to do some highly-oxidising photochemistry on both sides. Indeed, it looks like there was some kind of manganese cluster also in the D2 side at some point in time. It has been suggested before that this homodimeric Photosystem II was able to oxidise water. So if I can time when the gene duplication event that led to D1 and D2 happened, then I can have a pretty good idea of when water oxidation appeared for the first time. That is the subject of the above mentioned paper.

Type I reaction centres come in two versions: with homodimeric cores, meaning that the core is made of two copies of the same subunit; or with heterodimeric cores. The homdimeric Type I reaction centres are found only in anoxygenic phototrophs, and the heterodimeric Type I reaction centres are found only in oxygenic phototrophs: Cyanobacteria and photosynthetic eukaryotes. This heterodimeric Type I reaction centre is also known as Photosystem I.

It is hypothesised that the reason why Photosystem I is heterodimeric in oxygenic photosynthesis has something to do with oxygen… and well, there is no other alternative hypothesis that I know of.

The core of Photosystem I is made of two subunits, PsaA and PsaB. They share about 45% sequence identity. So, if oxygen is responsible for the heterodimerisation of Photosystem I; that means that the gene duplication event that led to PsaA and PsaB had to occur AFTER the evolution of water oxidation to oxygen.

That is the subject of the new manuscript:

https://www.biorxiv.org/content/early/2017/11/27/225888

I found out, like it was the case for Photosystem II, that the divergence of PsaA and PsaB is a lot older than anyone could have imagined… from my calculations, I would say that such gene duplication event occurred minimum 3.4 billion years ago, but it is likely much older than that.

Because PsaA and PsaB are more similar to each other than D1 and D2, it would seem as if the gene duplication event that led to PsaA and PsaB occurred long after the gene duplication that led to D1 and D2… but that may not necessarily be the case: that is only an illusion. Each duplication could have followed each other almost immediately: from this perpesctive, it may even be possible that heterodimeric Photosystem I predates heterodimeric Photosystem II, since the latter was oxidising water in a homodimeric form!

One thing that needs to be taken into account is that PsaA and PsaB are each about 730 to 750 amino acids long, while D1 and D2 are only about 360 amino acids long.

So in the case of D1 and D2: 70% sequence divergence is equivalent to about 250 amino acid differences along the entire sequence.

In the case of PsaA and PsaB, 55% sequence divergence is equivalent to about 440 amino acid differences along the entire sequence.

So consider the following example: if we assume that D1, D2, PsaA, and PsaB evolve at exactly the same rate: measured as amino acid substitutions PER POSITION per unit of time... a change of amino acids at 100 positions would take each protein the same amount of time, but in the case of D1 and D2, that would represent a change in sequence identity of 27%; while in PsaA and PsaB it would represent a change of only 13%!

In real life, however, PsaA and PsaB are evolving at a different pace than D1 and D2... but the difference is not huge.

So the fact that the level of sequence identity of PsaA and PsaB is significantly higher than D1 in D2 does not mean that the gene duplication event had to occur much later in Photosystem I than in Photosystem II. It is only an illusion caused by the fact that PsaA and PsaB are much longer than D1 and D2 and by the fact that sequence similarity in percentage is not necessarily the best measurement of sequence divergence.

Therefore, when the rates of evolution are taken into account and if we add to this the significant level of sequence divergence between PsaA and PsaB, it turns out that this gene duplication event happened a lot deeper in time than anyone could have guessed. Nevertheless, it is consistent with my hypothesis that water oxidation originated rapidly after (or at) the origin of photosynthesis, in the very early Archean.

Origin of water oxidation at the divergence of Type I and Type II reaction centres

Introduction

My friends, the way we think about the evolution of photosynthesis is about to change irreversibly.

I want to share with you some awesome stuff regarding the recent structure of the homodimeric Type I reaction centre by Gisriel et al. (2017) and what I think it all means for the origin of oxygenic photosynthesis. Huge thanks to all the authors. I know it must have been an unbelievable effort.

I have had a chance now to play with the structure a bit. What great pleasure! The structure is amazing and I have seen something that intrigues me enormously. Please, keep reading.

This is the link to the paper describing the structure:

http://science.sciencemag.org/content/357/6355/1021.full

This is a link to the pdb files:

https://www.rcsb.org/pdb/explore/explore.do?structureId=5V8K

We need first a bit of background though:

In a recent letter to the editor of the Journal of Molecular Evolution I argued that the peculiar structural characteristics of Photosystem II are better explained if water oxidation originated at the divergence of Type I and Type II reaction centres (Cardona, 2017).

Article Photosystem II is a Chimera of Reaction Centers

What are these peculiar characteristics?

I find quite peculiar that Photosystem II is made of a core, which originated form a Type II reaction centre (D1 and D2) and an antenna, which originated from a Type I reaction centre (CP43 and CP47).

Even more peculiar still is the fact that the CP43 subunit offers a direct ligand to the Mn4CaO5 cluster.

Another peculiar trait about Photosystem II is that D1 and D2 coordinate each a peripheral chlorophyll, ChlZ-D1 and ChlZ-D2. These peripheral chlorophylls and their binding sites are also conserved in Type I reaction centres, but are not found in anoxygenic Type II reaction centres. This means that the most ancestral reaction centre, before the divergence of Type I and Type II, had these peripheral pigments.

The implication of these peculiarities is that an interaction of ancestral Type I and Type II reaction centres is required for the origin of the Mn4CaO5 cluster. A second implication is that this interaction is continuous since the origin of both types of reaction centres, and therefore since very early after the emergence of photosynthesis and the first reaction centres.

So, given the fact that Photosystem II and Photosystem I 'working in series' is the hallmark of oxygenic photosynthesis. And add to this the fact that Photosystem II is a chimera of Type I and Type II reaction centres… it does not take a huge leap forward to think that the initial divergence of both types of reaction centres is actually linked to the origin of water oxidation chemistry.

Think about this for a moment.

This would actually mean that the earliest stages in the evolution of photosynthesis are related to the origin of water oxidation chemistry. In other words, this would mean that oxygenic photosynthesis traces back to the very early stages in the evolution of photochemical reaction centres (Cardona, 2017).

It sounds crazy, right?

Photosystem II and the homodimeric Type I reaction centre

So, what about the homodimeric Type I reaction centre? What about it?

AMAZINGLY, the structure of the homodimeric Type I reaction centre has a Ca-binding site with a number of intriguing parallels to the Mn4CaO5 cluster of Photosystem II:

1. It is positioned exactly where the redox Tyr-His pair is found in D1 and D2 (See Figures 1 and 2).

2. It is connected to the C-terminus by L605 and V608. V608 is the last amino acid in the sequence. In D1 of Photosystem II, the Mn4CaO5 cluster is coordinated by D342 and A344. A344 is the last amino acid of the processed D1 and it ligates not only Mn, but also the Ca!!!!!!!!!

3. It has a connection to the antenna domain, via N263, which is within the 5th and 6th helices. N263 connects to the Ca via two water molecules. In PSII, the CP43 antenna residue E354 offers a ligand to two Mn atoms and it is in the loop connecting the 5th and 6th helices of the antenna! A totally homologous site is also found in D2 and CP47, but phenylalanine residues are found instead of ligands.

4. At the overlapping position where TyrZ/TyrD is, there is a coordinating aspartate.

Figure 1: Panel A shows the Ca-binding site from the reaction centre of H. modesticaldum. In grey I show the connections from the core domain, and in orange the connections from the antenna domain. Panel B shows a schematized version of the Ca-binding site and in italics I have highlighted the parallels with the Mn4CaO5 cluster. In Panel C I show PSII for comparison; and in Panel D I overlap D1 (orange) and PshA (grey). The yellow atom is the Ca of the H. modesticaldum reaction centre.

Figure 2: It shows a comparison of PSII and the homodimeric reaction centre. No doubt that the Ca-binding site and the Mn4CaO5 cluster occupy homologous positions.

Implications for the evolution of water oxidation and the origin of photosynthesis

The main implication is that the most ancestral reaction centre before the divergence of Type I and Type II reaction centres had, at the very least, a Ca-binding site like the one in the structure of H. modesticaldum.

This is strong evidence that the divergence of Type I and Type II reaction centres was due to the development of the structural and energetic requirements to support water oxidation chemistry and the emergence of the oxygen-evolving complex.

This explains why the Mn4CaO5 cluster has a Ca atom! It was there to begin with.

This explains why the Mn4CaO5 cluster has a ligand from the C-terminus. It was there to begin with!

This explains why the Mn4CaO5 cluster has a ligand from the antenna. Guess what? It was there to begin with!

This explains why the site is also mirrored in D2 and CP47, because it all started symmetrically on both sides, as suggested by Rutherford and Faller (2003).

In other words, this implies that the connection from the C-terminus and the antenna domain to the cluster site has been continuous since the emergence of the first reaction centres. Just as I mentioned in my letter!

This also implies that the emergence of water oxidation can be traced to the earliest events in the evolution of photosynthesis. It implies that water oxidation likely predates the diversification of most groups of phototrophs, including Cyanobacteria!

This implies that Cyanobacteria are the only bacteria to have retained water oxidation chemistry, but a greater diversity of oxygenic phototrophs must have predated them.

This is also in perfect agreement with the conclusions of my molecular clock analysis that suggests water oxidation started long before the most recent common ancestor of Cyanobacteria:

Article Molecular evidence for the early evolution of photosynthetic...

(I have uploaded a new version of this article to the BiorXiv (http://www.biorxiv.org/content/early/2017/09/15/109447)

This implies that the anoxygenic Type II reaction centres likely evolved from a water-oxidizing Type II reaction centre before the gene duplication event that led to D1 and D2.

I had mentioned earlier that the ancestral Type II reaction centre (before D1, D2 , L, and M) already had some of the components that were needed to evolve the water-oxidizing complex (Cardona, 2015, 2016; Cardona et al., 2015). This validate those observations too.

In the near future, I hope to write something more substantial about this. Put all these ideas together in a nice review: go a bit deeper. In the meantime, it would be nice to discuss what you all think of this madness!

Don't hesitate to leave comments, especially if you strongly disagree and think this is all bonkers.

References

Cardona, T. (2015). A fresh look at the evolution and diversification of photochemical reaction centers. Photosynth Res, 126(1), 111-134. doi:10.1007/s11120-014-0065-x

Cardona, T. (2016). Reconstructing the origin of oxygenic photosynthesis: Do assembly and photoactivation recapitulate evolution? Frontiers in Plant Science, 7, 257. doi:10.3389/fpls.2016.00257

Cardona, T. (2017). Photosystem II is a chimera of reaction centers. Journal of Molecular Evolution, 84(2-3), 149-151. doi:10.1007/s00239-017-9784-x

Cardona, T., Murray, J. W., & Rutherford, A. W. (2015). Origin and evolution of water oxidation before the last common ancestor of the cyanobacteria. Mol Biol Evol, 32(5), 1310-1328. doi:10.1093/molbev/msv024

Gisriel, C., Sarrou, I., Ferlez, B., Golbeck, J. H., Redding, K. E., & Fromme, R. (2017). Structure of a symmetric photosynthetic reaction center-photosystem. Science, 357(6355), 1021-1025. doi:10.1126/science.aan5611

Rutherford, A. W., & Faller, P. (2003). Photosystem ii: Evolutionary perspectives. Philos Trans R Soc Lond B Biol Sci, 358(1429), 245-253. doi:10.1098/rstb.2002.1186