Cultivating the Ancestors… (1|4)

by Christoph
 

...is a metaphysical affair across all human cultures. For experimental biologists, it is the more prosaic, physical task of cultivating – or 'culturing', but I won't digress into semantics – extant pro­karyotic relatives of the an­cestors of the eukaryotes in the lab. Or the 'most likely' ancestors of the eukaryotes, to be cautious when leaping back ~2 billion years in time. Now, meet Pro­methe­archaeum syntrophicum MK-D1 (4.46 Mbp) that you see on the Frontispiece, and its syntrophic companions, the sulfur-reducing proteobacterium Halode­sulfovibrio sp. (4.13 Mbp) and the methano­genic archae­on Me­tha­no­genium sp. (2.33 Mbp).
 

(click to enlarge)
© H. Imachi, M.K. Nobu, and JAMSTEC Scan­ning elec­tron microscopy image of P. syntrophicum strain MK-D1. Source

At the ASM General Meeting 2014 in Boston, Anja Spang introduced the Lokiarchaeota to the audience – including Elio and me – members of an archaeal phylum whose genomes "encode an expanded repertoire of eukaryotic signature proteins" (see here). Could the Lokiarchaeota "...bridge the gap between prokaryotes and eukaryotes" as stated in the title the of Spang et al. (2015) paper? In the STC post A Class Asks Ques­tions… from 2015, a corresponding author of this paper, Thijs Ettema, frankly admitted that the bridge would be somewhat shaky as long as they could not get the cells under the microscope and learn more about their metabolism through cultivation.
 

A Whiff of Taxonomy  What began as 'Lokiarcheota' was soon renamed 'Asgard' archaea (2017) or 'As­gardarcheota' (2017), and you can see how its various mem­bers, all given ancient Norse names and only known through DNA sequences, are distributed among subgroups of different taxonomic ranks on Wiki­pedia. With the first successful co‑cultivation and sequencing of the complete genome of Promethe­archaeum syntrophicum MK-D1, the topic of this post, it was finally possi­ble to ta­xo­nomically classify the entire 'Asgard' zoo as Clade 'Proteoarchaeota' in the Kingdom 'Promethearchaeati'. To help you navigate the Tree of Life (ToL), click here to see the approximate positions of P. synthrophicum in 'Loki.', of Halodesulfovibrio sp. in 'Thermodesulfobacteria', and of the future mito­chon­drion in 'Alphapro­teobacteria' (in the ToL, you see that the taxonomic names of prokaryotic groups have evolved faster than the species).
 

Cultivation of Promethearchaeum syntrophicum MK‑D1

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Figure 1. Schematic diagram and photographs of the continuous-flow bioreactor with polyurethane spon­ges as microbial habitats, called down-flow hanging sponge (DHS) reactor. (a) Syn­the­tic seawater tank, (b) distribu­tor, (c) gas collecting bag, (d) sponge carrier sampling port and (e) port for the pH/ORP sensor. Source. Hiro Imachi said on Bluesky: "I've been supplying 0.57 L of anaerobic medium to the reactor each day, which brings the total to 3733 L so far. Also, my reactor will be turning 18 on December 28th."

It took Hiroyuki Imachi, Masaru Nobu and their 22 collaborators at 海洋研究開発機構 (JAM­ST­EC), Yokosuka, Japan, more than 12 years from their detection of 16S rRNA genes of Lokiar­chae­ota in an active methane-seep sediment core (949C3) that was collected from the Omine Ridge, Nankai Trough off Kumano area, Japan at 2,533 m below the sea surface in 2006 to their publication of Prome­the­archaeum syntrophicum MK‑D1, titled Isolation of an archaeon at the prokaryote–eukary­ote interface, in 2020 (formally correct: 'Candidatus Promethe­archaeum syntrophicum' strain MK‑D1).
 

They engineered and operated a methane-fed continuous-flow bioreactor system (Figure 1) for more than 2,000 days to enrich microorgan­isms in the anaerobic (anoxic) marine methane-seep sediments from the drill core sample and found, by 16S rRNA gene analysis, various phy­lo­gene­tically diverse un­cul­tivated microorganisms, including Lokiarchaeota (see here, DSAG (MBG‑B) ). Samples of the bioreactor community were inoculated for further en­richment in serum bottles with simple substrates and basal medium, and, after ~1 year, they found faint cell turbidity in a culture containing casamino acids that was incubated (anoxically) at 20 °C. 16S rRNA gene analysis revealed a simple com­mu­nity that contained Me­tha­no­ge­ni­um sp., Halodesulfovibrio sp. and a small population of Lokiarchaeota, designated strain MK-D1.
 

Repeated subcultivation gradually enriched the archaeon MK-D1, which has a 30–60-day lag phase, an extremely slow growth rate with a doubling time of ~14–25 days, and low cell yield after 3 months to reach full growth: ~10⁵ 16S rRNA gene copies/mL, that is concentrations of bacteria you find in tap water. After six transfers and under optimized growth conditions, MK‑D1 reached 13% abun­dance in a tri-culture containing a Halodesulfovibrio sp. bacterium (85%) and a Me­thanogenium sp. ar­chaeon (2%). Analyses by FISH and SEM revealed a close physical associa­tion of the three microorganisms (see here), Through stable‑isotope probing the re­searchers discovered that MK-D1 can catabolize ten amino acids and peptides through syntrophic growth with Halodesulfovibrio sp. and Me­thanogenium sp. through interspecies H2 (and/or formate) transfer. Through additional subcultivation in me­dium supplemented with 20 amino acids and powdered milk, they could eliminate the Halodesulfovibrio sp. population, thus obtaining a pure co‑culture of strain MK-D1, now naming it Promethearchaeum syntrophicum, and Methanogenium sp. (see here, c).
 

Note  Masaru Nobu briefly summarized the isolation of P. syntrophicum MK-D1 in his recorded contribution to the JGI2021 Meeting from 02:50–04:15 min.
 

Morphology and metabolism of P. syntrophicum MK‑D1

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Figure 2. Microscopy characterization and lipid com­position of MK-D1 (Click here for panels a and e). a–c, SEM images of MK-D1. Single cell (a), aggregated cells covered with EPS-like materials (b) and a dividing cell with polar chains of blebs (c). d, Cryo-electron tomo­gra­phy image of MK-D1. The top-right inset image shows a magnification of the boxed area to show the cell enve­lope structure. e, Cryo-EM image of large membrane vesicles attached to and surrounding MK-D1 cells. f, Ul­trathin section of an MK-D1 cell and a membrane ve­sicle. The bottom-right inset image shows a magnified view of the membrane vesic­le. g, h, SEM images of MK-D1 cells producing long branching (g) and straight (h) membrane protrusions. i, Ultrathin section of a MK-D1 cell with protrusions. Scale bars, 1 μm (b, c, g, h), 500 nm (a, d, e, i) and 200 nm (f). a–c, g, h, SEM images are representative of n = 122 recorded images that were obtained from 4 inde­pendent observations from 4 cul­ture samples. d, e, Cryo-EM images are representative of n = 14 recorded images that were taken from two inde­pendent observations from two culture samples. f, i, The ultrathin section images are representative of n = 131 recorded images that were obtained from six in­dependent observations from six culture samples. White arrows in the images indicate large membrane ve­­sicles. Source

Under the microscope, MK‑D1 cells are small cocci with an average diameter of 550 nm, and mostly found embedded in extracellular polymer substances (EPS) as aggregates (Figure 2, b). In samples from co‑cultures, MK‑D1 cells are easily distinguished from Methano­genium sp. cells, which are highly irregular coccoid cells of ≥2 μm (see here, d+e). Divid­ing MK‑D1 cells have less EPS and a ring-like structure around the cells (Figure 2, c). Cryo-EM and TEM analyses revealed that the cells contain no visible organelle-like inclusions, the hallmarks of eukaryotic cells. MK‑D1 cells are very "blebby" and produce membrane vesi­cles of 50–280 nm in diame­ter (Figure 2, a–f) and chains of blebs (Figure 2, c). Given the even distance between the inner and outer layers of the cell envelope (Figure 2, d), the MK-D1 cell envelope may be composed of a membrane and a surrounding S-layer, given the presence of four genes en­coding putative S-layer proteins in its genome, and stalk-like precursor structures on the surface of the mem­brane vesicles (Figure 2, e).
 

MK-D1 cells not in the process of division form several, occasionally branching mem­brane-based cytosol-connected protrusions of various lengths with diameters of 80–100 nm (Figure 2,  g+h; Frontispiece). These "tiny arms" are distinct from those of other archaea, which are known to use protrusions to form elaborate networks and intracellular connections ("nano­tubes").
 

With 4.46 Mbp, the genome of Promethearchaeum syntrophicum MK-D1 is in the size range of other free-living archaea. It encodes most of the genes necessary to synthesize ether-type li­pids – except a recognizable gene for GGGP synthase – and lacks genes for ester-type lipid syn­thesis. MK-D1 has thus a typical archaeal membrane with C20-phytane and C40-biphytanes as confirmed by analysis of the lipid composition in its co-culture with Methano­geni­um sp.
 

Imachi, Nobu et al. (2020) found that P. syntrophicum MK-D1 can degrade ten amino acids anaerobically and use them for growth, as confirmed by monitoring the depletion of amino acids in growing co-cultures, and produces both H2 and formate (HCO2^–) from amino acids for interspecies electron transfer. The latter was confirmed by quantifying the uptake of a mixture of ¹³C and ¹⁵N-labelled amino acids through NanoSIMS. The genome only encodes one NiFe hydrogenase, MvhADG–HdrABC, and molybdopterin-depen­dent formate dehydrogenase, FdhA, which suggests that MK-D1 can produce and syntrophically transfer H2 and formate using these enzymes. A first among cultured archaea.
 

MK-D1 can degrade 2-oxoacids hydrolytically (through 2-oxoacid-formate lyases) or oxidatively (through 2-oxoacid:ferredoxin oxidoreductases) to yield acyl-CoA intermediates that can be further degraded for ATP generation. In the hydrolytic path, the carboxylate group of the amino acid is released as formate that can be directly handed off to partnering methanogenic archaea or sulfate-reducing bacteria (SRB). In the oxidative path, 2-oxoacid oxidation is coupled with release of amino acid carboxylate as CO2 and reduction of ferredoxin, which can be re-oxidized through H⁺ and/or CO2 reduction to H2 and formate, respectively. MK-D1 can probably switch between syntrophic interaction through 2-oxoacid hydrolysis and oxidation depending on the partner(s).
 

In the forthcoming second part of Cultivating our Ancestors..., I will outline how getting to know Promethearchaeum syntro­phicum MK-D1 has led to completely new ideas about eukaryogenesis. In parts 3+4 then, I will show that P. syntrophicum is not a maverick among the 'Asgard' archaea, but a member of a family that occurs all over the world with similar physiological and cellular properties, including dependence on syntrophy, a penchant for blebbing, and those characteristic protrusions, the "tiny arms".
 

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