Contamination of genome projects with DNA from other organisms

I was blasting a protein named PsbO, also known as the 'manganese stabilizing protein' of Photosystem II. This is a protein found in cyanobacteria, algae, and plants, and it is important in photosynthesis. I was doing a phylogenetic tree and noted that one of the proteins originated from the recently sequenced non-photosynthetic bacterium Paenibacillus sp. IHB B 3415. A BLAST showed that the PsbO in this strain is identical to that in Camellia sinensis, the tea plant.

The chances of horizontal gene transfer from the chloroplast of the tea plant to Paenibacillus, I would say, is pretty close to 0%. So I imagine this is some form of contamination.

It is interesting that some of the investigators involved in the genome project are from Hill Area Tea Science Division, CSIR-Institute of Himalayan Bioresource Technology in Palampur, India.

I'm a little bit concerned. What is the chance of contamination to be present in genome projects? In the case of contamination from Eukaryote DNA into that of a bacterium, I guess it is not such a big deal because it can be easily spotted... but if you have contamination from another strain of bacteria, this might look like horizontal gene transfer and it may be not that simple to differentiate using just bioinformatics.

Update (April 20, 2015)
I contacted GenBank to report the issue, they investigated and this is what they told me:

"The submitter concurs with your assessment, so we have removed the contaminated contig JUEI01000195 from the public record." 

The PsbO protein of Photosystem II

The unusual D1 protein of Microcystis auriginosa TAIHU98

My coworkers and I recently published an article studying the phylogeny of all D1 subunits found in cyanobacteria. That study gave us some insight into the evolution of Photosystem II and water oxidation.

In that dataset I noticed a D1 that was quite aberrant present only in the genome of Microcystis aeruginosa TAIHU98. The genome of this strain was published in 2013.

D1 proteins are characterized by 5 transmembrane helices (A to E), between helices C and D, there is a parallel alpha helix, denominated CD (see below).

D1 protein from Thermosynechococcus vulcanus, PDB ID: 3WU2.

This anomalous D1 from M. aeruginosa TAIHU98 is predicted to have only 4 transmembrane helices. There is also what appears to be a 54 amino acids long 'sequence swap', in such a way that the original sequence has changed for a new sequence with no homology to any other protein known (as determined by BLASTing the 54 amino acid unique sequence).

Sequence alignment of a normal D1 and the unusual D1 in Microcystis aeruginosa TAIHU98 (click in the image to see larger). In blue I have highlighted the usual transmembrane helices, in red I highlighted the unique sequence in Microcystis. In purple I highlighted Y161, H191 and the ligands to the manganese cluster.

This unique sequence cut the second transmembrane helix (B) in half and the third transmembrane helix (C) disappeared completely. Instead, it is predicted using the TMHMM tool and the ΔG prediction server, that a brand new transmembrane helix exists in this protein made from a bit of the new sequence insertion and from the CD parallel helix. This becomes then the second helix.

Transmembrane helix prediction using TMHMM 2.0 for the weird D1 in M. aeruginosa TAIHU98

Transmembrane helix prediction using TMHMM 2.0 for a normal D1 (PsbA1 from T. elongatus BP-1)

If this D1 is inserted into the membrane then the last two transmembrane helices should be inverted in comparison with normal D1, due to the absence of helix B.

The 54 amino acid sequence swap also eliminated the redox tyrosine Y161 and the high-affinity manganese binding site, D170. All other ligands to the manganese cluster have remained unchanged.

The 'sequence swap' is actually caused by three nucleotide insertions into the psbA gene, the first one causes a frame shift, and the third insertion takes it back to normal, see the image below.

Three insertions into the psbA gene of this Mycrocystis strain (Query) caused the 'sequence swap' in the D1 protein, in comparison with the D1 from T. elongatus. The insertions are highlighted in purple.

Taking this into consideration it is unlikely that this sequence is incorporated into Photosystem II, but who knows really.

A curious thing is that in this 54 amino acid sequence there are 7 cysteines. Is this a sign that this sequence has a new function as a Fe-S protein?

In my published phylogeny of D1, this sequence clustered with other D1 from Microcystis and does not have an ancient origin, suggesting that these radical alterations occurred in this particular strain of Microcystis only.

This implies that the gain of new protein functions and the drastic redesign of proteins could evolved really fast.

Conserved lipids in photochemical reaction centers

Back in 2001, Wakeham et al. (2001) wondered whether a lipid molecule found in the Type II reaction center from Rhodobacter sphaeroides (Fig. 1) was conserved in all anoxygenic Type II reaction centers.

Fig. 1. Cardiolipin binding in the Type II RC from Rhodobacter sphaeroides, 1qov. The M subunit is shown in orange and the L subunit in gray. Cardiolipin is displayed as spheres.

Similar lipid binding sites in Photosystem II (Fig. 2 and 3) and in Photosystem I (Fig. 4 and 5) can be seen in the crystal structures. However, unlike the reaction center from R. sphaeroides, which appears to bind only one cardiolipin, Photosystem II and Photosystem I bind lipids symmetrically on both sides of the reaction center. In the crystal structure from plant Photosystem I only one 1,2-distearoyl-monogalactosyl-diglyceride (3lw5) was found in a similar position to the one in Synechococcys elongatus. I suspect the symmetrical counterpart was not seen because of the low resolution.

Fig. 2. 1,2-dipalmitoyl-phosphatidyl-glycerol and sulfoquinovosyldiacylglycerol binding in Photosystem II from Thermosynechococcus vulcanus, 3wu2. The D1 subunit is shown in orange and D2 in gray. The lipids are displayed as spheres.

Fig. 3. Symmetrical binding of lipids in Photosystem II.

Fig. 4. Binding of 1,2-distearoyl-monogalactosyl-diglyceride in Photosystem I from Synechococcus elongatus, 1jb0. The PsaB subunit is shown in orange and the PsaA in gray. The lipid is shown as spheres.  The antenna domain has been omitted for clarity.

Fig. 5. Like in Photosystem II, the lipids bind symmetrically in Photosystem I.

It appears then that the binding of lipids in that position is a conserved feature of all reaction centers and might have existed in the primordial reaction center at the dawn of photosynthesis. It is possible that in each type of reaction center the role of these lipids have changed. I found a paper that suggested that cardiolipin affects charge recombination in R. sphaeroides (Giustini et al. 2005).  But in Photosystem II the role of this lipids might be more related to assembly and repair, besides specific structural roles (Mizusawa and Wada 2012). The role of these lipids found in Photosystem I is less clear but it has been suggested that they might influence the phylloquinones in some manner (Fromme et al. 2001).

References
Fromme, P., Jordan, P. & Krauss, N. Structure of Photosystem I. BBA-Bioenergetics 1507, 5-31 (2001).

Giustini, M. et al. Influence of cardiolipin on the functionality of the QA site of the photosynthetic bacterial reaction center. J Phys Chem B 109, 21187-21196 (2005).

 Mizusawa, N. & Wada, H. The role of lipids in photosystem II. BBA-Bioenergetics 1817, 194-208 (2012).

Wakeham, M. C., Sessions, R. B., Jones, M. R. & Fyfe, P. K. Is there a conserved interaction between cardiolipin and the type II bacterial reaction center? Biophys J 80, 1395-1405 (2001).

Photosynthetic constraints on fuel from microbes

The question is, are biofuels really a solution to the global energy crisis? Are they really sustainable? The main issue is the low photosynthetic efficiency of solar energy conversion to fuel or biomass. The efficiency is so low that the amount of energy obtained in the biofuel is apparently lower than the energy that was invested to produce it.

Perhaps it is possible to enhance photosynthesis to the point that biofuels become truly a solution... in our recent opinion paper we have discussed some recent approaches at improving photosynthesis.

Photosynthetic constraints on fuel from microbes

Grown algae - By IGV Biotech (Own work) [CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons

The origin of oxygenic photosynthesis in Cyanobacteria (some thoughts)

The last common ancestor to all extant cyanobacteria had already evolved the capability to oxidize water as the earliest known diverging species of cyanobacteria of the genus Gloeobacter have a fully evolved Photosystem II. For example, Gloeobacter violaceous has five D1, D2, the CP43 and CP47, the Cyt b559, Cyt c550, PsbO, L, M, J, K, H, T, X, and P [1-3].

It can be deduced with confidence that oxygenic photosynthesis evolved before the last common ancestor of extant cyanobacteria. It suggests a period of evolution that saw the transformation of a simple Type II reaction center incapable of water oxidation into the sophisticated Photosystem II.

The question is: for how long before the last common ancestor of extant cyanobacteria was water oxidation possible? Could water oxidation by a primitive Photosystem II have evolved fast?

The Great Oxygenation Event occurred around 2.3-2.4 Ga ago. I think this time correspond to the major radiation of cyanobacteria… when the major clade of cyanobacteria evolved, but after the evolution of the Gloeobacter genus and early evolving Synechococcus (e.g. Yellowstone strains).

The closest relatives of cyanobacteria that are incapable of oxygenic photosynthesis are the melainabacteria [4, 5]. It appears though, oxygenic photosynthesis appeared some time in between the divergence of the melainabacteria and the last common ancestor of the cyanobacteria. Before this, there is the divergence of the chloroflexi from the lineage that led to cyanobacteria [6]. Because the chloroflexi are phototrophs I dare to suggest that the chloroflexi and the cyanobacteria shared a photosynthetic ancestor that was incapable of water oxidation [7].

Based on molecular clock analysis it’s been estimated that the major phyla of bacteria radiated around 3.2 Ga [8, 9].

Some of the earliest evidence for oxygen on earth date to ~3.0 Ga [10, 11].

So, I speculate that there is about 200 million years for water oxidation to have evolved, starting from the major radiation of bacteria and culminating with the first significant amounts of oxygen detected in the geochemical record.

Evidence of oxygenic photosynthesis before 3.2 Ga… are difficult to reconcile with the overall evolution of bacteria. IF for some unexpected reason strong evidence for oxygenic photosynthesis around 3.8 Ga is found. This should imply panspermia… maybe a rock containing a huge diversity of prokaryotes with all sorts of archaea and bacteria (including fully evolved cyanobacteria) hit earth during the late heavy bombardment. But I don’t think that makes much sense given the available evidence :-)

Sequence of events in the evolution of water oxidation

1. Saw, J.H.W., et al., Cultivation and Complete Genome Sequencing of Gloeobacter kilaueensis sp nov., from a Lava Cave in Kilauea Caldera, Hawai'i. Plos One, 2013. 8(10).

2. Koyama, K., et al., Oxygen evolution in the thylakoid-lacking cyanobacterium Gloeobacter violaceus PCC 7421. Biochim Biophys Acta, 2008. 1777(4): p. 369-78.

3. Kaneko, T., et al., Complete genome structure of the unicellular cyanobacterium Gloeobacter violaceus PCC 7421. Plant and Cell Physiology, 2004. 45: p. S129-S129.

4. Soo, R.M., et al., An Expanded Genomic Representation of the Phylum Cyanobacteria. Genome Biology and Evolution, 2014. 6(5): p. 1031-1045.

5. Di Rienzi, S.C., et al., The human gut and groundwater harbor non-photosynthetic bacteria belonging to a new candidate phylum sibling to Cyanobacteria. Elife, 2013. 2.

6. Segata, N., et al., PhyloPhlAn is a new method for improved phylogenetic and taxonomic placement of microbes. Nature Communications, 2013. 4.

7. Cardona, T., A fresh look at the evolution and diversification of photochemical reaction centers. Photosynth Res, 2014.Advanced access, 18 of Dec.

8. David, L.A. and E.J. Alm, Rapid evolutionary innovation during an Archaean genetic expansion. Nature, 2011. 469(7328): p. 93-96.

9. Battistuzzi, F.U. and S.B. Hedges, A major clade of prokaryotes with ancient adaptations to life on land. Molecular Biology and Evolution, 2009. 26(2): p. 335-343.

10. Crowe, S.A., et al., Atmospheric oxygenation three billion years ago. Nature, 2013. 501(7468): p. 535-8. 11. Planavsky, N.J., et al., Evidence for oxygenic photosynthesis half a billion years before the Great Oxidation Event. Nature Geosci, 2014. 7(4): p. 283-286.

Reflexions on the origin of photochemical reaction centers

My review paper dealing with the evolution of photosynthesis has been published now in Photosynthesis Research. Please, take a look:

A fresh look at the evolution and diversification of photochemical reaction centers

Phylogenetic tree of prokaryotes

Stages in the evolution of photosynthesis

There are four stages from the origin of life until the appearance of oxygenic photosynthesis.

Stage 1

From no photosynthesis to the origin of chlorophyll and the first reaction center protein.

There had to be a period in time before the origin of chlorophyll synthesis and photosynthesis. A series of proteins evolved to turn a porphyrin precursor into chlorophyll and bacteriochlorophyll. Enzymes with homology to nitrogenases were recruited for this purpose. Proteins capable of binding chlorophyll or its precursors had to evolve before photosynthesis. This include a membrane protein that would evolve into a reaction center capable of light-driven charge separation.    

Stage 2

The first homodimeric reaction center diverges into two classes presumably both homodimeric: a Type I and Type II reaction centers.

Stage 3

The primordial Type II reaction center protein diverges into two distinct classes. One class of protein is the ancestral to the PufL and PufM proteins in photosynthetic Proteobacteria and Chloroflexi. The other class is the ancestral to D1 and D2 proteins in a lineage that would be the progenitors of Cyanobacteria. 

Photosystem I (PDB ID: 1jb0)

Stage 4

Starting with a homodimeric Type II reaction center, in the lineage that would be a progenitor of the Cyanobacteria, a gene duplication occurs that drives the divergence of D1 from D2. At some moment that could have started even before D1 and D2 diverged, the capacity to oxidize water appeared. The homodimeric Type II reaction center then becomes heterodimeric and gains incredible complexity to evolve into the Photosystem II characteristic of Cyanobacteria.

The last common cyanobacterial ancestor had already a fully developed oxygenic photosynthetic machinery implying and unknown biota of cyanobacteria, this is because the earliest branching cyanobacteria from the genus Gloeobacter, already possesses a fully developed Photosystem II and Photosystem I. This implies an unknown biota preceding the last common cyanobacterial ancestor.

Photosystem II (PDB ID: 3arc)

The key question is, how long it took from the origin of photosynthesis to the appearance of water oxidation. If we assume that the first water splitters originated around 2.7 to 3.0 billion years ago, and the origin of photosynthesis between 3.8 to 3.3 billion years ago, the completion of these four stages could have taken anything from 300 million to 1.1 billion years.

Knowing the speed at which bacteria evolves and the lengths of time we are talking about, hundreds of millions of years, each one of these stages should have given rise to a significant diversity. Most of which appears to have gone extinct.