Since the discovery of archaeoplankton in 1992, the euryarchaeotal Marine Group II (MGII) remains uncultured and less understood than other planktonic archaea. We characterized the seasonal dynamics of MGII populations in the southern North Sea on a genomic and microscopic level over the course of four years. We recovered 34 metagenome-assembled genomes (MAGs) of MGIIa and MGIIb that corroborated proteorhodopsin-based photoheterotrophic lifestyles. However, MGIIa and MGIIb MAG genome sizes differed considerably (~1.9 vs. ~1.4 Mbp), as did their transporter, peptidase, flagella and sulfate assimilation gene repertoires. MGIIb populations were characteristic of winter samples, whereas MGIIa accounted for up to 23% of the community at the beginning of summer. Both clades consisted of annually recurring, sequence-discrete populations with low intra-population sequence diversity. Oligotyping of filtered cell-size fractions and microscopy consistently suggested that MGII cells were predominantly free-living. Cells were coccoid and ~0.7 µm in diameter, likely resulting in grazing avoidance. Based on multiple lines of evidence, we propose distinct niche adaptations of MGIIa and MGIIb Euryarchaeota populations that are characteristic of summer and winter conditions in the coastal North Sea.

Protein inhibition is a natural regulatory process to control cellular metabolic fluxes. PII-family signal-transducing effectors are in this matter key regulators of the nitrogen metabolism. Their interaction with their various targets is governed by the cellular nitrogen level and the energy charge. Structural studies on GlnK, a PII-family inhibitor of the ammonium transporters (Amt), showed that the T-loops responsible for channel obstruction are displaced upon the binding of 2-oxoglutarate, magnesium and ATP in a conserved cleft. However, GlnK from Methanocaldococcus jannaschii was shown to bind 2-oxoglutarate on the tip of its T-loop, causing a moderate disruption to GlnK–Amt interaction, raising the question if methanogenic archaea use a singular adaptive strategy. Here we show that membrane fractions of Methanothermococcus thermolithotrophicus released GlnKs only in the presence of Mg-ATP and 2-oxoglutarate. This observation led us to structurally characterize the two GlnK isoforms apo or in complex with ligands. Together, our results show that the 2-oxoglutarate binding interface is conserved in GlnKs from Methanococcales, including Methanocaldococcus jannaschii, emphasizing the importance of a free carboxy-terminal group to facilitate ligand binding and to provoke the shift of the T-loop positions.

Methanogenic archaea operate an ancient, if not primordial, metabolic pathway that releases methane as an end-product. This last step is orchestrated by the methyl-coenzyme M reductase (MCR), which uses a nickel-containing F430-cofactor as the catalyst. MCR astounds the scientific world by its unique reaction chemistry, its numerous post-translational modifications, and its importance in biotechnology not only for production but also for capturing the greenhouse gas methane. In this report, we investigated MCR natively isolated from Methermicoccus shengliensis. This methanogen was isolated from a high-temperature oil reservoir and has recently been shown to convert lignin and coal derivatives into methane through a process called methoxydotrophic methanogenesis. A methoxydotrophic culture was obtained by growing M. shengliensis with 3,4,5-trimethoxybenzoate as the main carbon and energy source. Under these conditions, MCR represents more than 12% of the total protein content. The native MCR structure refined at a resolution of 1.6-Å precisely depicts the organization of a dimer of heterotrimers. Despite subtle surface remodeling and complete conservation of its active site with other homologues, MCR from the thermophile M. shengliensis contains the most limited number of post-translational modifications reported so far, questioning their physiological relevance in other relatives.

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Ketol-acid reductoisomerase (KARI) orchestrates the biosynthesis of branched-chain amino acids, an elementary reaction in prototrophic organisms as well as a valuable process in biotechnology. Bacterial KARIs belonging to class I organise as dimers or dodecamers and were intensively studied to understand their remarkable specificity towards NADH or NADPH, but also to develop antibiotics. Here, we present the first structural study on a KARI natively isolated from a methanogenic archaea. The dodecameric structure of 0.44-MDa was obtained in two different conformations, an open and close state refined to a resolution of 2.2-Å and 2.1-Å, respectively. These structures illustrate the conformational movement required for substrate and coenzyme binding. While the close state presents the complete NADP bound in front of a partially occupied Mg2+-site, the Mg2+-free open state contains a tartrate at the nicotinamide location and a bound NADP with the adenine-nicotinamide protruding out of the active site. Structural comparisons show a very high conservation of the active site environment and detailed analyses point towards few specific residues required for the dodecamerisation. These residues are not conserved in other dodecameric KARIs that stabilise their trimeric interface differently, suggesting that dodecamerisation, the cellular role of which is still unknown, might have occurred several times in the evolution of KARIs.

Glutamine synthetases catalyze the ATP-dependent ammonium assimilation, the initial step of nitrogen acquisition that must be tightly regulated to fit cellular needs. While their catalytic mechanisms and regulation are well-characterized in bacteria and eukaryotes, only limited knowledge exists about the archaeal representatives. Here, we natively purified the glutamine synthetases fromMethanothermococcus thermolithotrophicusandMethermicoccus shengliensis, two thermophilic methanogens belonging to different orders. Biochemical investigations combined with X-ray crystallography unveiled the first structures of archaeal glutamine synthetases and highlighted differences in their regulation. The enzyme fromM. thermolithotrophicusis inactive in its resting state and employs 2-oxoglutarate as an on-switch. The 2-oxoglutarate acts as a sensor of cellular nitrogen deficiency, and its reported cellular concentration remarkably overlays with that required for the enzyme activation. Its binding to an allosteric pocket leads to the reconfiguration of the active site and promotes a catalytically competent state. The homolog fromM. shengliensisdoes not harbor the 2-oxoglutarate binding motif and, consequently, is 2-oxoglutarate insensitive. Instead, it is directly feedback-inhibited by glutamine, as shown for bacterial homologs. The glutamine inhibition depends on a key arginine residue from the Asp50ʹ-loop. The arginine is substituted by a glycine inM. thermolithotrophicus, abolishing the inhibitory effect. While the effectors are surprisingly different, the molecular switch controlling the glutamine synthetase activity is fundamentally the same and depends on the correct positioning of the Asp50ʹ-loop and a catalytic arginine. Residue conservation suggests that both regulation mechanisms are widespread and not mutually exclusive across archaea.