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Although methanogens were recently discovered to occur in aerated soils, alpine regions have not been extensively studied for their presence so far. Here, the abundance of archaea and the methanogenic guilds Methanosarcinales, Methanococcales, Methanobacteriales, Methanomicrobiales and Methanocella spp. was studied at 16 coniferous forest and 14 grassland sites located at the montane and subalpine belts of the Northern Limestone Alps (calcareous) and the Austrian Central Alps (siliceous) using quantitative real-time PCR. Abundance of archaea, methanogens and the methanogenic potentials were significantly higher in grasslands than in forests. Furthermore, methanogenic potentials of calcareous soils were higher due to pH. Methanococcales, Methanomicrobiales and Methanocella spp. were detected in all collected samples, which indicates that they are autochthonous, while Methanobacteriales were absent from 4 out of 16 forest soils. Methanosarcinales were absent from 10 out of 16 forest soils and 2 out of 14 grassland soils. Nevertheless, together with Methanococcales they represented the majority of the 16S rRNA gene copies quantified from the grassland soils. Contrarily, forest soils were clearly dominated by Methanococcales. Our results indicate a higher diversity of methanogens in well-aerated soils than previously believed and that pH mainly influences their abundances and activities. Various methanogenic guilds were found to be present in aerated forest and grassland soils located at the montane and subalpine belts of the European Alps. Graphical Abstract Figure. Various methanogenic guilds were found to be present in aerated forest and grassland soils located at the montane and subalpine belts of the European Alps.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and Cas proteins represent an adaptive microbial immunity system against viruses and plasmids. Cas3 proteins have been proposed to play a key role in the CRISPR mechanism through the direct cleavage of invasive DNA. Here, we show that the Cas3 HD domain protein MJ0384 from Methanocaldococcus jannaschii cleaves endonucleolytically and exonucleolytically (3′–5′) single‐stranded DNAs and RNAs, as well as 3′‐flaps, splayed arms, and R‐loops. The degradation of branched DNA substrates by MJ0384 is stimulated by the Cas3 helicase MJ0383 and ATP. The crystal structure of MJ0384 revealed the active site with two bound metal cations and together with site‐directed mutagenesis suggested a catalytic mechanism. Our studies suggest that the Cas3 HD nucleases working together with the Cas3 helicases can completely degrade invasive DNAs through the combination of endo‐ and exonuclease activities. Cas3 is an essential protein required for CRISPR‐based bacteriophage immunity in bacteria. This study describes the structure, biochemical activity, and catalytic mechanism of Cas3.

Several methanogenic archaea lack cysteinyl-transfer RNA (tRNA) synthetase (CysRS), the essential enzyme that provides Cys-tRNA[superscript Cys] for translation in most organisms. Partial purification of the corresponding activity from Methanocaldococcus jannaschii indicated that tRNA[superscript Cys] becomes acylated with O-phosphoserine (Sep) but not with cysteine. Further analyses identified a class II-type O-phosphoseryl-tRNA synthetase (SepRS) and Sep-tRNA:Cys-tRNA synthase (SepCysS). SepRS specifically forms Sep-tRNA[superscript Cys], which is then converted to Cys-tRNA[superscript Cys] by SepCysS. Comparative genomic analyses suggest that this pathway, encoded in all organisms lacking CysRS, can also act as the sole route for cysteine biosynthesis. This was proven for Methanococcus maripaludis, where deletion of the SepRS-encoding gene resulted in cysteine auxotrophy. As the conversions of Sep-tRNA to Cys-tRNA or to selenocysteinyl-tRNA are chemically analogous, the catalytic activity of SepCysS provides a means by which both cysteine and selenocysteine may have originally been added to the genetic code.

Mobilome of hyperthermophilic archaea dwelling in deep-sea hydrothermal vents is poorly characterized. To gain insight into genetic diversity and dynamics of mobile genetic elements in these environments we have sequenced five new plasmids from different Thermococcus strains that have been isolated from geographically remote hydrothermal vents. The plasmids were ascribed to two subfamilies, pTN2-like and pEXT9a-like. Gene content and phylogenetic analyses illuminated a robust connection between pTN2-like plasmids and Pyrococcus abyssi virus 1 (PAV1), with roughly half of the viral genome being composed of genes that have homologues in plasmids. Unexpectedly, pEXT9a-like plasmids were found to be closely related to the previously sequenced plasmid pMETVU01 from Methanocaldococcus vulcanius M7. Our data suggests that the latter observation is most compatible with an unprecedented horizontal transfer of a pEXT9a-like plasmid from Thermococcales to Methanococcales. Gene content analysis revealed that thermococcal plasmids encode Hfq-like proteins and toxin-antitoxin (TA) systems of two different families, VapBC and RelBE. Notably, although abundant in archaeal genomes, to our knowledge, TA and hfq-like genes have not been previously found in archaeal plasmids or viruses. Finally, the plasmids described here might prove to be useful in developing new genetic tools for hyperthermophiles.

Genes encoding DNA replication proteins have been frequently exchanged between cells and mobile elements, such as viruses or plasmids. This raises potential problems to reconstruct their history. Here, we combine phylogenetic and genomic context analyses to study the evolution of the replicative minichromosome maintenance (MCM) helicases in Archaea. Several archaeal genomes encode more than one copy of the mcm gene. Genome context analysis reveals that most of these additional copies are encoded within mobile elements. Exhaustive analysis of these elements reveals diverse groups of integrated archaeal plasmids or viruses, including several head-and-tail proviruses. Some MCMs encoded by mobile elements are structurally distinct from their cellular counterparts, with one case of novel domain organization. Both genome context and phylogenetic analysis indicate that MCM encoded by mobile elements were recruited from cellular genomes. An accelerated evolution and a dramatic expansion of methanococcal MCMs suggest a host-to-virus-to-host transfer loop, possibly triggered by the loss of the archaeal initiator protein Cdc6 in Methanococcales. Surprisingly, despite extensive transfer of mcm genes between viruses, plasmids, and cells, the topology of the MCM tree is strikingly congruent with the consensus archaeal phylogeny, indicating that mobile elements encoding mcm have coevolved with their hosts and that DNA replication proteins can be also useful to reconstruct the history of the archaeal domain.

Methane production by methanogens in wetland is recognized as a significant contributor to global warming. Spartina alterniflora ( S. alterniflora ), which is an invasion plant in China’s wetland, was reported to have enormous effects on methane production. But studies on shifts in the methanogen community in response to S. alterniflora invasion at temporal and spatial scales in the initial invasion years are rare. Sediments derived from the invasive species S. alterniflora and the native species Phragmites australis ( P. australis ) in pairwise sites and an invasion chronosequence patch (4 years) were analyzed to investigate the abundance and community structure of methanogens using quantitative real-time PCR (qPCR) and Denaturing gradient gel electrophoresis (DGGE) cloning of the methyl-coenzyme M reductase A ( mcrA ) gene. For the pairwise sites, the abundance of methanogens in S. alterniflora soils was lower than that of P. australis soils. For the chronosequence patch, the abundance and diversity of methanogens was highest in the soil subjected to two years invasion, in which we detected some rare groups including Methanocellales and Methanococcales . These results indicated a priming effect at the initial invasion stages of S. alterniflora for microorganisms in the soil, which was also supported by the diverse root exudates. The shifts of methanogen communities after S. alterniflora invasion were due to changes in pH, salinity and sulfate. The results indicate that root exudates from S. alterniflora have a priming effect on methanogens in the initial years after invasion, and the predominate methylotrophic groups (Methanosarcinales) may adapt to the availability of diverse substrates and reflects the potential for high methane production after invasion by S. alterniflora .

The EKC/KEOPS yeast complex is involved in telomere maintenance and transcription. The Bud32p and kinase‐associated endopeptidase 1 (Kae1p) components of the complex are totally conserved in eukarya and archaea. Their genes are fused in several archaeal genomes, suggesting that they physically interact. We report here the structure of the Methanocaldococcus jannaschii Kae1/Bud32 fusion protein MJ1130. Kae1 is an iron protein with an ASKHA fold and Bud32 is an atypical small RIO‐type kinase. The structure MJ1130 suggests that association with Kae1 maintains the Bud32 kinase in an inactive state. We indeed show that yeast Kae1p represses the kinase activity of yeast Bud32p. Extensive conserved interactions between MjKae1 and MjBud32 suggest that Kae1p and Bud32p directly interact in both yeast and archaea. Mutations that disrupt the Kae1p/Bud32p interaction in the context of the yeast complex have dramatic effects in vivo and in vitro , similar to those observed with deletion mutations of the respective components. Direct interaction between Kae1p and Bud32p in yeast is required both for the transcription and the telomere homeostasis function of EKC/KEOPS.

The contemporary proteinogenic repertoire contains 20 amino acids with diverse functional groups and side chain geometries. Primordial proteins, in contrast, were presumably constructed from a subset of these building blocks. Subsequent expansion of the proteinogenic alphabet would have enhanced their capabilities, fostering the metabolic prowess and organismal fitness of early living systems. While the addition of amino acids bearing innovative functional groups directly enhances the chemical repertoire of proteomes, the inclusion of chemically redundant monomers is difficult to rationalize. Here, we studied how a simplified chorismate mutase evolves upon expanding its amino acid alphabet from nine to potentially 20 letters. Continuous evolution provided an enhanced enzyme variant that has only two point mutations, both of which extend the alphabet and jointly improve protein stability by >4 kcal/mol and catalytic activity tenfold. The same, seemingly innocuous substitutions (Ile→Thr, Leu→Val) occurred in several independent evolutionary trajectories. The increase in fitness they confer indicates that building blocks with very similar side chain structures are highly beneficial for fine-tuning protein structure and function.

The site-selective encoding of noncanonical amino acids (NAAs) is a powerful technique for the installation of novel chemical functional groups in proteins. This is often achieved by recoding a stop codon and requires two additional components: an evolved aminoacyl tRNA synthetase (AARS) and a cognate tRNA. Analysis of the most successful AARSs reveals common characteristics. The highest fidelity NAA systems derived from the Methanocaldococcus jannaschii tyrosyl AARS feature specific mutations to two residues reported to interact with the hydroxyl group of the substrate tyrosine. We demonstrate that the restoration of just one of these determinants for amino acid specificity results in the loss of fidelity as the evolved AARSs become noticeably promiscuous. These results offer a partial explanation of a recently retracted strategy for the synthesis of glycoproteins. Similarly, we reinvestigated a tryptophanyl AARS reported to allow the site-selective incorporation of 5-hydroxy tryptophan within mammalian cells. In multiple experiments, the enzyme displayed elements of promiscuity despite its previous characterization as a high fidelity enzyme. Given the many similarities of the TyrRSs and TrpRSs reevaluated here, our findings can be largely combined, and in doing so they reinforce the long-established central dogma regarding the molecular basis by which these enzymes contribute to the fidelity of translation. Thus, our view is that the central claims of fidelity reported in several NAA systems remain unproven and unprecedented.

The diversity of an archaeal community was analyzed in the water from a continental high-temperature, long-term water-flooded petroleum reservoir in Huabei Oilfield in China. The archaea were characterized by their 16S rRNA genes. An archaeal 16S rDNA clone library was constructed from the DNA isolated from the formation water, and 237 randomly selected positive clones were clustered in 28 phylotypes by sequencing analyses. Phylogenetic analysis of these sequences indicated that the dominant members of the archaeal phylotypes were affiliated with the order Methanomicrobiales. Totally, the archaeal community was composed of methanogens belonging to four orders: Methanobacteriales, Methanococcales, Methanomicrobiales, and Methanosarcinales. Most of the clones clustered with sequences previously described for methanogens, but there was a difference in the relative distribution of sequences detected here as compared to that of previous studies. Some thermophilic methanogens detected had been previously isolated from a number of high-temperature petroleum reservoirs worldwide; thus, they might exhibit adaptations to the environments and be the common habitants of geothermally heated subsurface environments.