At the end of my PhD (2011), I started being interested in a potential novel order of methanogens that was only known through a few mcrA* sequences amplified from lakes1 and human gut2. At that time, six orders of methanogens were known (five of them for more than thirty years) and relatively well described in text books. I was thus extremely excited when it turned out that these mcrA sequences did indeed correspond to new methanogens3,4 (classified under a novel order named Methanomassiliicoccales). I spent several years studying these fascinating archaea being unique by strictly relying on reduction of methyl compounds with H2 for methanogenesis and in their potential role for human health5–7.
During that period, I often wondered: How many other lineages of methanogens could have been missed? What kind of substrates could they rely on? What can they teach us about the evolution of the methane metabolism, and the Archaea in general? With the increasing number of metagenomes available in public databases and the novel approaches to reconstruct genomes from metagenomes, moving forward on these issues had become possible without going through traditional culture approaches. In 2015, the study of Evans and collaborators8 was an electroshock for me. This study reported the presence of an MCR complex in two metagenome-assembled genomes (MAGs) affiliated to the Bathyarchaeota (MAGs BA1 & BA2), a different phylum than the Euryarchaeota to which all methanogens were previously known to belong.
Shortly after, I started a post-doc in Simonetta Gribaldo’s group at Institut Pasteur. My initial project aimed to study the evolution of Diaforarchaea, the superclass containing the Methanomassiliicoccales, but I decided to put the Methanomassiliicoccales aside for a moment to explore the “unknown diversity” of methanogens. For this, I screened thousands of metagenomes publicly available in the IMG/JGI database, to extract mcrA genes from a wide range of environments (Figure 1).
After removing sequences closely related to each other, I constructed a giant tree with all these McrA homologues. I think I will always remember the moment I examined this tree for the first time. It was December 2015 and I was in a train travelling back to my hometown, highlighting all the branches that corresponded to nothing previously known… it was crazy how many there were. Actually, at the order level, it appeared possible that until then, we only knew less than half of the total diversity of Archaea with an MCR complex, and this second half was full of surprises as we will see later. These novel lineages can be seen in turquoise in the tree presented in Figure 2, which is a simplified version of the original “giant McrA tree”.
At first, I thought we could publish a first report of this tree that was radically changing the vision of the diversity of archaea with a potential methane metabolism. However, after discussing with Simonetta, we decided to push the study further before publishing, in order to get a whole picture of the methane metabolism in these lineages and be able to dig deeper into their evolution. This took a while as it was not one but nine novel lineages with unusual enzymatic profiles for which methane metabolism was predicted, and this was followed by a large-scale comparative genomics and phylogenetic analyses. In the following three years, four of the lineages I was analyzing were described by other groups: Methanofastidiosa9, Verstraetearchaeota10, Methanonatronarchaeia11 and GoM-Arc112. This period has been quite frustrating and stressful, as we feared for the other yet unpublished lineages we had, but we don’t regret it. Especially now that we know that some of these MCR do not produce/activate methane (explained below). Another choice we had to do was about contacting the PIs who were responsible for the unpublished metagenomes we used. We decided it was important to do so, in order to be sure that we were not scooping them (in particular for PhD students or post-docs) on their own unpublished data. It was another stressful time because in a way it put the future of the study in their hands, as some (or all) of these scientists might have not wanted us to use their data. Fortunately, they all kindly and enthusiastically accepted to be part of the study. Beyond providing these invaluable data, they also contributed a critical reading of the paper draft. Most importantly, contacting them also opened several future collaborations. Only one of the metagenomes we used was already published, and afterward, I think we could have also associated Thomas Hess, its PI.
Once the genomes corresponding to these mcrA were reconstructed, placed in a reference tree of Archaea and their metabolism predicted, two things were really striking. First, these new lineages were branching all over the tree of Archaea, strongly supporting the hypothesis that methane metabolism is ancient and could have even been present in the last common ancestor of this domain of life. Second, none of these novel archaea had the genetic potential for the so far most common type of methanogenesis (reduction of CO2 with H2). Instead, most of them were predicted to rely on the reduction of various methyl-compounds with H2, the same type of methanogenesis pathway than Methanomassiliicoccales. Therefore, this type of methanogenesis, previously considered uncommon, now appeared to have even a wider phylogenetic distribution than methanogenesis based on the reduction of CO2 with H2. Interestingly, energy conservation mechanisms associated to the reduction of methyl-compounds with H2 are substantially different across the novel lineages, some being not yet completely resolved, and other likely involving novel uncharacterized enzymatic complexes.
While I was analyzing these genomes, I had a second shock: the publication by Laso-Pérez and collaborators13 revealing that divergent MCR complexes (MCR-like complexes) are responsible for the activation of short-chain alkanes (butane, propane). This means that the MCR complex, an enzymatic complex that looks like nothing else in nature and that was thought to be exclusively involved in methane formation (methanogenesis) or activation (methanotrophy), could have in fact evolved to use a wider range of substrates. So, the long branches in the MCR phylogeny (Figure 2) likely correspond to MCR-like complexes activating short-chain alkanes. To test this hypothesis, I looked at the residues previously described to have an important role in the catalytic site of the canonical MCR and indeed found that several were different in most MCR-like sequences. Notably, the replacement of large aromatic amino-acids by smaller ones might be associated to the accommodation of larger substrates than methane (i.e. butane or propane) in the MCR-like complex catalytic site. Three of the MAGs of our study contain MCR-like sequences. Strikingly, two of them also have the genes coding for a canonical MCR, meaning that they possibly have both a short-chain alkane and a methane metabolism, something never reported before. These novel genomes coding for a canonical MCR and an MCR-like complex are part a novel class of archaea for which we proposed the name of Methanoliparia because they are often present in oil-rich environments. These two mostly complete genomes were lacking the same genes needed to transfer the electron from methane or short-chain alkane oxidation to a syntrophic partner or an inorganic electron acceptor. This led us to propose that these archaea, by their own, could couple short-chain alkane oxidation to methanogenesis. This process is only known to occur through syntrophic partnership between archaea and bacteria and would therefore represent a novel type of methanogenesis, which will be exciting to prove experimentally.
In order to have a wider perspective on the evolution of methane and short-chain alkane metabolisms, we decided to integrate in the analysis all currently known MAGs of archaeal methanotrophs (as well as new ones provided by Jillian Banfield and collaborators). In particular, I was curious to see the distribution in these MAGs of 38 markers previously thought being specifically related to methanogenesis. It was surprising to find out that methanogens, methanotrophs and short-chain alkane oxidizers indeed share many of these markers, indicating that these metabolisms are evolutionarily tightly linked. Moreover, methanotrophs and short chain alkane oxidizers have generally very similar way to transfer electrons and conserve energy, and in two instances, lineages of methanotrophs and short-chain alkane oxidizers are closely related in the reference archaeal phylogeny. This led us to propose that several evolutionary transitions occurred between methanotrophs and short-chain alkane oxidizers, and in both directions. From the bulk of our results, we draw a potential scenario for the origin and evolution of methane metabolisms, with an early emergence of methanogenesis, followed by multiple losses and modifications of this metabolism during archaeal diversification. This illustrates nicely how metabolic diversity can arise through tinkering of a common set of enzymes.
A last thing I would like to point out is that similarly to other studies reconstructing MAGs from metagenomic data, we made a lot of hypotheses on metabolisms potentially at work in those novel lineages. It is clear that environmental sequence analysis is just a step, not an end in the understanding of the metabolisms and environmental roles of these novel microorganisms, and these data now need to be integrated with culture and enzyme structure resolution. But now at least, we know that these lineages exist, we know where to find them and we have many working hypotheses to grow them and characterize them.
*a methanogenesis/methanotrophy marker gene, coding for one of the three subunits of the methyl-coenzyme M reductase complex (MCR) that produces/activates methane.
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