A new undescribed clade of ancient phototrophic bacteria in metagenomic data

Protochlorophyllide and chlorophyllide reductases, two enzymes required for the synthesis of chlorophyll and bacteriochlorophyll precursors share significant sequence identity with an enzyme required for the synthesis of the Ni-tetrapyrrole, cofactor F430 of Methyl-coenzyme M reductase, a key enzyme in methanogenic and methanotrophic archaea. These enzymes are also closely related to nitrogenases, but the evolutionary relationships among the tetrapyrrole synthesis enzymes and nitrogenases have been difficult to resolve. Recent phylogenomic analysis have suggested that the last universal common ancestor was capable of nitrogen fixation and methanogenesis, raising puzzling questions about the early origin of photosynthesis and the early diversification of the tetrapyrrole biosynthesis enzymes.

I serendipitously stumbled upon two sequences annotated as ChlL subunits while searching for homologs to a subunit of the Ni-tetrapyrrole synthesis enzyme (CfbC) in a BLAST restricted to the domain Archaea.

The ChlL subunits appeared in the recently published genomes of of Candidatus Altiarchaeales archaeon WOR SM1 SCG and Candidatus Altiarchaeales archaeon WOR SM1 86-2, hereafter named Alti_SCG and Alti_86-2, respectively. An investigation of the genomic region surrounding the putative ChlL contained the two additional subunits of protochlorophyllide reductase, ChlB and ChlN, in the genome of Alti_86-2 but not in Alti_SCG1. However, after communication with the authors who sequenced the genomes of this strain it turns out that ChlB and N, in Alti_SCG1 are located in a different region of the genome. Though for some reason, the BLAST could not find them.

http://journal.frontiersin.org/article/10.3389/fmicb.2016.01221/full

The ChL proteins RefSeq Accession are ODS40814.1 and ODS39344.1

The sequence from strain Alti_SCG has 81% sequence identity to that if strain Alti_86-2 This suggests that they are quite a distance apart, but still within “the same phylum”. These strains have 83.1% sequence identity of the 16S rRNA, which suggests that these strains diverged already quite some time ago.

I have performed ML phylogenetic analysis of the sequences that I retrieved and performed a quick molecular clock analysis of ChlL. 

What you can see is that the Archeal sequences branch together with those of photosynthetic bacteria. They are indeed true subunits of protochlorophyllide reductase. This suggest that the proteins were acquired via horizontal gene transfer from a phototrophic bacterium.

Interestingly, the sequences do not branch within any of the known clades of phototrophic bacteria. From the phyllogeny of ChlN and B it would seem plausible that the sequences have some affinity to those in Heliobacteira, which may suggest that the source of the genes is a phototrophic Firmicutes distantly related to Heliobacteria... in fact, predating the divergence of Heliobacteria. This would be my most conservative guess. However, it may as well be a completely new phylum of bacteria.

One thing we can be sure of... the sequences do not match any of the known clades.

The molecular clock analysis, using a relaxed log-normal autocorrelated clock, with the CAT model, suggested that the event of horizontal gene transfer occurred about 1.3 +/- 0.4 billion years ago, as estimated by the divergence time of the ChlL from the two archeal strains.

From the position of the archaeal branches we can conclude that the "new clade" of phototrophic bacteria is an early evolving one... a really early one.

Fascinating stuff. This is a pretty rough and quick analysis though, I'll need some time to study this in more detail... might even need a grant. Any donors? ;) Haha, I wish, right?

Seeking answers in Asimov's Prelude to Foundation

Yesterday night as I was reading "Prelude to Foundation", the first prequel to Asimov's Foundation saga, I stumbled upon this marvelous quote:

“Why, he wondered, did so many people spend their lives not trying to find answers to questions—not even thinking of questions to begin with? Was there anything more exciting in life than seeking answers?”

― Isaac Asimov

I guess it is why I decided to be a scientist.

Prelude to Foundation, cover art by Tim White

Oxygen utilization in the Last Universal Common Ancestor

In the recent paper by Weiss et al 2016, in Nature Microbiology, eight oxygen-using enzymes were traced back to the last universal common ancestor. Listed below:

CtaD: Heme/copper-type cytochrome/quinol oxidase, subunit 1

SodC: Cu / Zn superoxide dismutase

BcpB: Peroxiredoxin

HmgA: Homogentisate 1,2-dioxygenase

KEGG Ortholog, K15746: hypothetical protein 

LigB: Aromatic ring-opening dioxygenase, LigB subunit

HpaB: Aromatic ring hydroxylase

Rr-2: Rubrerythrin

There were more proteins related to oxygen utilization in LUCA than those categorized in nitrogen metabolism (seven proteins) and energy metabolism (two proteins).

Three of these enzymes are used to deal with radical oxygen species (ROS), SodC, BcpB, and Rr-2.

HmgA has a role in the catabolism of aromatic rings of tyrosine and phenylalanine. LigB and HpaB deal with the oxidation of aromatic rings and phenolic compounds.

The first one in the list, the heme/copper-type cytochrome/quinol oxidase, is required for aerobic respiration.

The hypothetical protein is actually, CrtZ; beta-carotene 3-hydroxylase (a.k.a. beta-carotene, NADH:oxygen 3-oxidoreductase), used in the conversion of betacarotene to zeaxanthin.

Similar results have been published before regarding the ROS enzymes and the dioxygen reductases. See Ouzounis et al 2006 for example, or Zamocky et al 2001, or Brochier-Armanet et al 2009. These are just a few examples...

Of course, the results of Weiss et al 2016 and those by others before can be interpreted in a number of ways and there has always been great debate. If we stick to the simplest explanation, Occam's Razor: I would say that the simplest explanation is that molecular oxygen was available to the last universal common ancestor.

Black Smoker, by NOAA [Public domain], via Wikimedia Commons

Rates of evolution of reaction center proteins and other proteins of photosynthesis

I have been doing molecular clock analysis of several proteins and enzymes relevant to photosynthesis. This has allowed me measure the rate of evolution. Several of this I have presented in my recent paper available in bioRxiv, which I have just submitted for peer-review. I present here a few more of other unpublished projects.

The rates are given as amino acid changes per site per billion years

Photosystem II core protein - D1: 0.088
Photosystem II core protein - D2: 0.12
Anoxygenic Type II RC core protein - L: 0.63
Anoxygenic Type II RC core protein - M: 0.60

Photosystem I core protein - PsaA: 0.13
Photosystem I core protein - PsaB: 0.15
Anoxygenic Type I RC protein of heliobacteria - PshA: 0.27
Anoxygenic Type I RC protein of the Chlorobi - PscA: 0.24

Mg-chelatase: ChlD/BchD: 0.60 (average from all phototrophs)
Putative Co-chelatase homolog to ChlD: 0.73 (average from archaea strains)

These were calculated using Phylobayes 3.3f under a CAT model using the birth-death model and soft bounds on the calibrations points, assuming an age for photosynthesis around 3.5 billion years ago.

If you want more details, please look at the paper above or send me an email.

Directed evolution of water oxidation catalysis for improved photosynthesis

Recently, a funding opportunity became available at Imperial open to all academic stuff at all levels. I participated with this little project about doing directed evolution on Photosystem II. The pre-proposal had to be really short so almost no considerations on the project can be really made. Only 1 proposal per department had to be put forward for the final round of selection. I ranked 3 out of 6...

The big issue I see with the project is that from an evolutionary perspective the rate of PSII water oxidation are limited not by the S cycle itself but by quinone exchange. The slow rates of quinone exchange at the same time are determined by the rates of quinone oxidation in the cytochrome b6f and other downstream processes.

It would be fun to prove that water oxidation can occur faster than it does. The directed evolution approach will probably have to also accelerate the rates of quinone exchange in PSII and also downstream in the thylakoid membrane and metabolic electron sink.

Summary of the project

Raising populations and greater incomes per capita will result in an unprecedented demand for food, fuel, and high-value products. This demand will not be met without an improvement of the efficiency of photosynthesis: the ultimate frontier in photosynthesis research. The engine that powers photosynthesis is called Photosystem II, a complex molecular machine that converts light into useful energy by decomposing water into protons, electrons, and oxygen. This chemical reaction is known as water oxidation and it is the source of all energy that sustains complex life and human societies. I hypothesise that Photosystem II has the potential to oxidise water several-fold faster than observed in known photosynthetic organisms. To test this hypothesis I will use directed evolution to select for variant Photosystem II with accelerated rates of water oxidation. The project aims to provide experimental support for the possibility of enhancing the catalytic efficiency of Photosystem II. The results of this innovative and high-risk project have the potential to be directly translated into strategies for the engineering of enhanced photosynthetic organisms.

Proposal 

It is likely that in the next decades the global demand for food, fibre, bioenergy, biopharmaceuticals, and other chemical precursors will not be met sustainably without significant improvements of the photosynthetic efficiency of crops and algae of biotechnological potential.^1,2

A radical and high-risk approach that could result in a significant enhancement of photosynthetic efficiency is the direct improvement of the rate of catalysis of Photosystem II, the light-driven water:plastoquinone oxidoreductase enzyme of oxygenic photosynthesis. I will employ directed evolution to screen and select for Photosystem II variants that display faster rates of water oxidation. The specific goal of the project is to demonstrate that faster rates of biological water oxidation are catalytically and thermodynamically possible.

Gene diversification will be accomplished using genome-wide random mutagenesis³ and iterative saturation mutagenesis⁴ of the core subunits of Photosystem II targeting the first and second coordination sphere of the Mn₄CaO₅ cluster, the exchangeable plastoquinone binding site, and the proton pathways. Cyanobacteria mutants will be screened for potential alterations in water oxidation photochemistry in a plate reader spectrometer using a range of oxygen sensitive dyes. Strains with potentially faster kinetics of water oxidation will be extensively characterised with the range of electrochemical, spectroscopic, and biochemical techniques available in my lab. Successful variants from both gene diversification strategies could be integrated using DNA shuffling.

                The accomplishment of improved catalytic efficiency of Photosystem II would be a tremendous breakthrough and should open a direct route for the technological realisation of enhanced photosynthesis in crops, eukaryotic algae, and cyanobacteria. Furthermore, it should expedite the development of artificial catalysts that mimic the water oxidation cycle, which still remains an outstanding technological challenge.^5,6

1. Ort, D. R. et al. Redesigning photosynthesis to sustainably meet global food and bioenergy demand. PNAS, 112, 8529-8536, (2015).

2. Tilman, D., Balzer, C., Hill, J. & Befort, B. L. Global food demand and the sustainable intensification of agriculture. PNAS, 108, 20260-20264, (2011).

3. Packer, M. S. & Liu, D. R. Methods for the directed evolution of proteins. Nat Rev Genet 16, 379-394, (2015).

4. Reetz, M. T. & Carballeira, J. D. Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat Protoc 2, 891-903, doi:10.1038/nprot.2007.72 (2007).

5. Zhang, C. X. et al. A synthetic Mn4Ca-cluster mimicking the oxygen-evolving center of photosynthesis. Science 348, 690-693, (2015).

6. Schulze, M., Kunz, V., Frischmann, P. D. & Wurthner, F. A supramolecular ruthenium macrocycle with high catalytic activity for water oxidation that mechanistically mimics Photosystem II. Nat Chem 8, 577-584, (2016).

The uncanny connections between methanogenesis and photosynthesis

I have become quite interested in the evolutionary relationship between methanogenesis in archaea and photosynthesis in bacteria.

The first evolutionary link comes from the homology between the two enzymes of chlorophyll and bacteriochlorophyll synthesis, Protochlorophyllide reductase (BchLNB or ChLNB) and Chlorophyllide reductase (BchXYZ), and the enzyme required for the synthesis of the Ni-tetrapyrrole cofactor, coenzyme F430, of methyl-coenzyme M reductase, which is essential for methanogenesis. 

Another enzyme of chlorophyll synthesis, BciB (8-vinyl reductase) may also be related to another enzyme of methanogenesis (FrhB). See this paper.

Now a new paper by Kono et al (2017) seems to find that this evolutionary link goes much deeper, as it seems that methanogenic archaea not only have a proper rubisco, but also have phosphoribulokinase (PRK), previously considered to be unique to photosynthetic organisms that use the Calvin-Benson-Bassham cycle for carbon fixation.

I know that some enzymes of methanogenesis seem to trace back to the last universal common ancestor (LUCA), but the same isn't true for photosynthesis. From this perspective it would seem that photosynthesis emerged perhaps by borrowing a few components from methanogenesis.

Recently, Martin et al (2017) said in a recent perspective: "Let us presume, just for a moment, that the first bacteria and archaea were acetogens and methanogens respectively. On an uninhabited planet, they have no competitors, and life multiplies quickly given ample growth substrates. The founders of their respective domains would have bubbled off into the ocean bottom waters to be spread around by currents and eventually to be introduced back into hydrothermal systems in the crust [...]"

I would add to this, that life could have only spread around the globe and take a hold during the early Archaean after the evolution of photosynthesis. In fact, I would go as far as to say that it was the innovation of photosynthesis what allowed the early microbes to escape the hydrothermal vents and the crust... but other may disagree! :)

Stromatolitic-chert member, Strelley Pool Formation, middle to lower Paleoarchean, 3.35 to 3.46 Ga. Photo by James St. Johns.

Just thinking about things can be a source of scientific progress

I just read "One step beyond a ribosome: The ancient anaerobic core", by Filipa L. Sousa, Shijulal Nelson-Sathi, William F. Martin, published in Biochimica et Biophysica Acta 1857 (2016) 1027–1038

It is a fascinating paper. I particularly enjoyed the following concluding line:

"With regard to the most primitive forms of microbial physiology, microbiologists reached the same conclusion 45 years ago, namely that methanogens and acetogens probably represent the most ancient lineages. We required 2000 genomes and powerful computers for our conclusions, while Decker et al. just thought about it. Evidently, just thinking about things can be a source of scientific progress."

I like to think about things, I like to think especially about photosynthesis ;)

Fossil stromatolite