Lokiarchaeon with exceptionally high activity

A serendipitous discovery provides insights regarding the physiology of Lokiarchaeon, an enigmatic archaeon that is a close relative of eukaryotes
Lokiarchaeon with exceptionally high activity

In July 2018, I traveled to Namibia for an oceanographic research expedition on board the german reserach vessel METEOR to the Benguela upwelling system (BUS).  The BUS is one of the most productive regions of the world's oceans, and as a result there is a high sedimentation rate and very low concentrations of dissolved oxygen in the bottom waters overlying the sediments.   We didn't have much sampling time, and had to work through the night in order to obtain the samples for all of the scientific participants.  However, everyone got their samples which was an impressive feat and largely due to the organization of Tim Ferdelman (MPI Bremen) who was the chief scientist of the cruise.  As an american, I was blown away at how well organized and smoothly run the sampling was on the ship, the phrase "german efficiency" apparently also applies to oceanography.  I think they should start offering cross-cultural training courses in oceanography !

While the sampling was focused on the water column, we were able to obtain a quality 30 cm long sediment core with a nicely preserved sediment seawater interface from below the hypoxic waters.  This was actually pretty tricky, since the seafloor surface was very soft it was difficult to get the multi-core rig to land on the surface without sinking into the mud.  Luckily we had Tim Ferdelman on board who is a sediment coring expert.  Finally, after several attempts, we had success at the last station along the continental shelf transect before transiting to the Canary Islands.  Perfect for microbiology, the METEOR is outfitted with three cold rooms that are connected with airtight doors, all at different temperatures (10, 4, and -20 degrees C).  Thus, it was possible to section the core in the 4 degree room and freeze the sections immediately in the -20 C room, which is important to get the best possible gene expression results (temperature changes has a huge effect on the activity of seafloor microbial communities).
While we were sectioning the core, we found many polychaete worms and shrimps that were burrowing vertically through the anoxic mud.  A truly startling observation, given the extremely high concentrations of toxic hydrogen sulfide!  Here is a cross section of the core, the green color comes from the iron minerals Glauconite that forms under low oxygen conditions in marine sediments

Cross section of the Namibian sediment core

After flash-freezing the sections, I used the 10 C room to set up some anaerobic incubations with 13C-labeled bicarbonate and 13C-labeled diatomaceous extracellular polysaccharides (dEPS) that I had brought with me.  This has the advantage that, the samples are not exposed to higher temperatures during experimental setup and should retain more of their in situ activities.  After returning from the cruise I was awarded a small grant from the German national science foundation to process the samples, and my research group was able to apply qPCR, 16S rRNA gene sequencing, metagenomics, metatranscriptomics, and quantitative stable isotope probing (qSIP) to the core.  And, we were able to integrate these results with the geochemical profiles of nitrate and sulfide measured by our collaboration partners in Oldenburg and Bremen.

Now for the moment of serendipity that led to our publication.  The main goal of our expedition to BUS was to investigate microbial sources of trace greenhouse gases nitrous oxide and methane from low oxygen marine waters, particularly one group of microbes called 'ANME-2d' that is believed to couple nitrate reduction with methane oxidation.  As I was screening through the gene expression results I noticed a relatively small number of expressed genes that had highest similarity to ANME-2d.  To double check their true origin, I BLASTed the predicted peptides against NCBI-nr and surprisingly all had reasonably high similarities (>60% amino acids shared) to the candidate archaeal taxon "Lokiarchaeota" (or Lokiarchaeon).  Then I thought to myself, "wait a minute, does my database have the Lokiarchaeon genomes"?  After a double check I realized that no Lokiarchaeon genomes were in my database, and none of the other genomes from the Asgard archaea candidate superphylum were in there either.  So, I added all of the Asgard genomes including all of the available Lokiarchaeon genomes and ran the analysis again.   When I got the results, it was one of those 'ah ha' moments where you sit back in your chair.  In the metatranscriptomes, there were now thousands of hits with highest similarity to Lokiarchaeon genes - which were clearly enriched in the deeper sulfidic samples.  This was completely unexpected because the abundance of Lokiarchaeon in the 16S rRNA gene data was less than 0.1%, and in the metagenomes less than 2% of the community - but in the metatranscriptomes they were more than 30% of all expressed genes!  After normalizing the gene expression, Lokiarchaeon and Bathyarchaeota had orders of magnitude higher transcriptional activity compared to all other groups of bacteria.  Thus, these anoxic Namibian sediments that are rich in organic matter and sulfide apparently promote an exceptionally high activity of these Archaea.

We then used qSIP to quantify how much carbon Lokiarchaeon fixed as CO2 and how much it utilized from the organic matter (dEPS).   And, sure enough, as was observed in the gene expression results, Lokiarchaeon utilized way more dEPS and CO2 compare to most bacteria.  We could then conclude with high certainty, that the metabolic activity implied in the metatranscriptomes was translated into increased physiological activity and growth.  Putting all the data together, it was clear that Lokiarchaeon combines both fermentation of organic matter, and H2-dependent CO2 reduction, a physiology referred to as homoacetogenesis.  The H2 released during fermentation is used for CO2 reduction, which ultimately improves bioenergetic efficiency during anaerobic metabolism.  This might explain why Lokiarchaeon (and the Bathyarchaeota, which are also homoacetogens) can maintain higher levels of activity under anoxic conditions compared most of the other Bacteria that were in the core.  Our results also fit nicely with the recent study by Imachi et al (prepint on Bio Archives), that showed Lokiarchaeon likes to ferment amino acids.  Of all the genes expressed by Lokiarchaeon for substrate transport, genes encoding transmembrane transporters for amino acids were by far the most expressed.   Thus, it seems that Lokiarchaeon behaves similar in the culture experiments by Imachi et al, as it does in the natural environment.    

My main take away in this blog post is to update your database regularly with new genomes as they become available.  If I had not double checked and re-added all of the Asgard genomes to my database, I would have missed this entire discovery!

 I was aware of the studies recently published that suggested Lokiarchaeon and other Asgard archaea are thought to be some of the closest living relatives of the host cell that acquired the mitochondrion at the origin of eukaryotes.   So, I figured the exceptionally high activity of Lokiarchaeon in these sediments might be relevant not only to microbial ecologists, but also to evolutionary biologists as well.  Since, the ecology of the host cell that acquired the mitochondrion at the origin of eukaryotes must be relevant for the mechanisms behind how this all happened.  In settings such at the BUS where there is periodic ventilation of anoxic bottom waters, dependence of the Asgard archaeal host cell on an H2 producing syntrophic partner at the seafloor surface may have been promoted due to a reduction in available H2.   Below is a graphical summary of this idea (Asgard cell artwork modified from Eme and Ettema 2018 "The eukaryotic ancestor shapes up"), the idea of the emergence of the first eukaryote from archaeal cell fusions (enclosing the mitochondrial endosymbiont) comes from the Martin et al 2017 review on phagotrophy (Martin et al 2017 MMBR).

Of course, the graphic summary below is hugely speculative.  Nevertheless, if Lokiarchaeon is truly a close living relative of the last eukaryotic common ancestor (LECA), then at a minimum our results still imply that anoxic and sulfide rich marine sediments would have been a habitat promoting the activity of LECA and possibly eukaryogenesis.  This would fit with the conditions at the time, since ocean anoxia was widespread during the Proterozoic eon when eukaryotes evolved ca. 2 billion years ago. 

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