An enormous diversity of previously unknown bacteria and archaea has been discovered recently, yet their functional capacities and distributions in the terrestrial subsurface remain uncertain. Here, we continually sampled a CO-driven geyser (Colorado Plateau, Utah, USA) over its 5-day eruption cycle to test the hypothesis that stratified, sandstone-hosted aquifers sampled over three phases of the eruption cycle have microbial communities that differ both in membership and function. Genome-resolved metagenomics, single-cell genomics and geochemical analyses confirmed this hypothesis and linked microorganisms to groundwater compositions from different depths. Autotrophic Candidatus "Altiarchaeum sp." and phylogenetically deep-branching nanoarchaea dominate the deepest groundwater. A nanoarchaeon with limited metabolic capacity is inferred to be a potential symbiont of the Ca. "Altiarchaeum". Candidate Phyla Radiation bacteria are also present in the deepest groundwater and they are relatively abundant in water from intermediate depths. During the recovery phase of the geyser, microaerophilic Fe- and S-oxidizers have high in situ genome replication rates. Autotrophic Sulfurimonas sustained by aerobic sulfide oxidation and with the capacity for N fixation dominate the shallow aquifer. Overall, 104 different phylum-level lineages are present in water from these subsurface environments, with uncultivated archaea and bacteria partitioned to the deeper subsurface.

The recently discovered seventh order of methanogens, the Methanomassiliicoccales (previously referred to as "Methanoplasmatales"), so far consists exclusively of obligately hydrogen-dependent methylotrophs. We sequenced the complete genome of "Candidatus Methanoplasma termitum" from a highly enriched culture obtained from the intestinal tract of termites and compared it with the previously published genomes of three other strains from the human gut, including the first isolate of the order. Like all other strains, "Ca. Methanoplasma termitum" lacks the entire pathway for CO 2 reduction to methyl coenzyme M and produces methane by hydrogen-dependent reduction of methanol or methylamines, which is consistent with additional physiological data. However, the shared absence of cytochromes and an energy-converting hydrogenase for the reoxidation of the ferredoxin produced by the soluble heterodisulfide reductase indicates that Methanomassiliicoccales employ a new mode of energy metabolism, which differs from that proposed for the obligately methylotrophic Methanosphaera stadtmanae. Instead, all strains possess a novel complex that is related to the F 420 :methanophenazine oxidoreductase (Fpo) of Methanosarcinales but lacks an F 420 -oxidizing module, resembling the apparently ferredoxin-dependent Fpo-like homolog in Methanosaeta thermophila. Since all Methanomassiliicoccales also lack the subunit E of the membrane-bound heterodisulfide reductase (HdrDE), we propose that the Fpo-like complex interacts directly with subunit D, forming an energy-converting ferredoxin:heterodisulfide oxidoreductase. The dual function of heterodisulfide in Methanomassiliicoccales, which serves both in electron bifurcation and as terminal acceptor in a membrane-associated redox process, may be a unique characteristic of the novel order. Methanogenesis is catalyzed exclusively by members of the archaeal domain. Methanogenic archaea occur only in the phylum Euryarchaeota and are phylogenetically diverse. The species described to date fall into seven orders that differ both in the biochemistry of their catabolic pathways and in their ecological niches (1, 2).Methanogens from all basal orders (Methanopyrales, Methanococcales, and Methanobacteriales) are hydrogenotrophs. They reduce CO 2 to CH 4 via the C 1 pathway, using H 2 or sometimes formate as an electron donor (1, 2). The hydrogenotrophic pathway is found also in most of the derived lineages of methanogens (Methanomicrobiales and Methanocellales) and was most probably present already in the common ancestor of the Euryarchaeota (3). Hydrogenotrophic methanogens typically lack cytochromes and conserve energy with the methyltetrahydromethanopterin (methyl-H 4 MPT):coenzyme M methyltransferase complex (Mtr), which uses the free energy of methyl transfer to establish a Na ϩ -motive force across the membrane (4). The low-potential reducing equivalents for CO 2 reduction are provided by electron bifurcation at the cytoplasmic heterodisulfide reductase complex (HdrABC) (5, 6).Members of the orde...

SummaryThe ability to move towards favourable conditions provides fundamental advantages to organisms. Interestingly, flagella as motility structures evolved independently in the bacterial and the archaeal kingdom. Whereas bacterial flagella have been intensively studied, our knowledge regarding the archaeal counterpart is mostly restricted to Euryarchaeota rather than crenarchaeal flagella. We therefore investigated the flagellar assembly system of the crenarchaeal model organism Sulfolobus acidocaldarius in vivo. Promoter studies and qRT-PCR analyses of the flagella gene cluster provided evidence that the expression of the fla genes was induced by tryptone starvation. Moreover, we confirmed presence of a secondary fla promoter within the flaB gene that regulates the transcription of downstream genes flaX-J. Markerless in-frame deletions for all fla genes encoded in the fla gene cluster were constructed. Western blot analysis of all fla deletion strains suggested hierarchical protein interactions during the archaeal flagella assembly. Moreover, functional analysis by thermomicroscopy revealed non-motile cells for each of the mutant strains. Electron micrographs demonstrated that lack of motility coincided with the loss of flagellar assembly. Thus we demonstrated that all seven fla genes are essential for crenarchaeal flagellum assembly and function.

A mesophilic, neutrophilic and aerobic, ammonia-oxidizing archaeon, strain EN76T, was isolated from garden soil in Vienna (Austria). Cells were irregular cocci with a diameter of 0.6–0.9 µm and possessed archaella and archaeal pili as cell appendages. Electron microscopy also indicated clearly discernible areas of high and low electron density, as well as tubule-like structures. Strain EN76T had an S-layer with p3 symmetry, so far only reported for members of the Sulfolobales. Crenarchaeol was the major core lipid. The organism gained energy by oxidizing ammonia to nitrite aerobically, thereby fixing CO2, but growth depended on the addition of small amounts of organic acids. The optimal growth temperature was 42 °C and the optimal pH was 7.5, with ammonium and pyruvate concentrations of 2.6 and 1 mM, respectively. The genome of strain EN76T had a DNA G+C content of 52.7 mol%. Phylogenetic analyses of 16S rRNA genes showed that strain EN76T is affiliated with the recently proposed phylum Thaumarchaeota, sharing 85 % 16S rRNA gene sequence identity with the closest cultivated relative ‘Candidatus Nitrosopumilus maritimus’ SCM1, a marine ammonia-oxidizing archaeon, and a maximum of 81 % 16S rRNA gene sequence identity with members of the phyla Crenarchaeota and Euryarchaeota and any of the other recently proposed phyla (e.g. ‘Korarchaeota’ and ‘Aigarchaeota’). We propose the name Nitrososphaera viennensis gen. nov., sp. nov. to accommodate strain EN76T. The type strain of Nitrososphaera viennensis is strain EN76T ( = DSM 26422T = JMC 19564T). Additionally, we propose the family Nitrososphaeraceae fam. nov., the order Nitrososphaerales ord. nov. and the class Nitrososphaeria classis nov.

Subsurface microbial life contributes significantly to biogeochemical cycling, yet it remains largely uncharacterized, especially its archaeal members. This 'microbial dark matter' has been explored by recent studies that were, however, mostly based on DNA sequence information only. Here, we use diverse techniques including ultrastuctural analyses to link genomics to biology for the SM1 Euryarchaeon lineage, an uncultivated group of subsurface archaea. Phylogenomic analyses reveal this lineage to belong to a widespread group of archaea that we propose to classify as a new euryarchaeal order ('Candidatus Altiarchaeales'). The representative, double-membraned species 'Candidatus Altiarchaeum hamiconexum' has an autotrophic metabolism that uses a not-yet-reported Factor 420 -free reductive acetyl-CoA pathway, confirmed by stable carbon isotopic measurements of archaeal lipids. Our results indicate that this lineage has evolved specific metabolic and structural features like nanograppling hooks empowering this widely distributed archaeon to predominate anaerobic groundwater, where it may represent an important carbon dioxide sink.

Prokaryotic Argonaute proteins acquire guide strands derived from invading or mobile genetic elements, via an unknown pathway, to direct guide-dependent cleavage of foreign DNA. Here, we report that Argonaute from the archaeal organism Methanocaldococcus jannaschii (MjAgo) possesses two modes of action: the canonical guide-dependent endonuclease activity and a non-guided DNA endonuclease activity. The latter allows MjAgo to process long double-stranded DNAs, including circular plasmid DNAs and genomic DNAs. Degradation of substrates in a guide-independent fashion primes MjAgo for subsequent rounds of DNA cleavage. Chromatinized genomic DNA is resistant to MjAgo degradation, and recombinant histones protect DNA from cleavage in vitro. Mutational analysis shows that key residues important for guide-dependent target processing are also involved in guide-independent MjAgo function. This is the first characterization of guide-independent cleavage activity for an Argonaute protein potentially serving as a guide biogenesis pathway in a prokaryotic system.

The number and location of flagella, bacterial organelles of locomotion, are species specific and appear in regular patterns that represent one of the earliest taxonomic criteria in microbiology. However, the mechanisms that reproducibly establish these patterns during each round of cell division are poorly understood. FlhG (previously YlxH) is a major determinant for a variety of flagellation patterns. Here, we show that FlhG is a structural homolog of the ATPase MinD, which serves in cell-division site determination. Like MinD, FlhG forms homodimers that are dependent on ATP and lipids. It interacts with a complex of the flagellar C-ring proteins FliM and FliY (also FliN) in the Gram-positive, peritrichousflagellated Bacillus subtilis and the Gram-negative, polar-flagellated Shewanella putrefaciens. FlhG interacts with FliM/FliY in a nucleotide-independent manner and activates FliM/FliY to assemble with the C-ring protein FliG in vitro. FlhG-driven assembly of the FliM/FliY/FliG complex is strongly enhanced by ATP and lipids. The protein shows a highly dynamic subcellular distribution between cytoplasm and flagellar basal bodies, suggesting that FlhG effects flagellar location and number during assembly of the C-ring. We describe the molecular evolution of a MinD-like ATPase into a flagellation pattern effector and suggest that the underappreciated structural diversity of the C-ring proteins might contribute to the formation of different flagellation patterns.flagellum | FlhG | C-ring | Bacillus | Shewanella

Microalgae are thought to offer great potential as expression system for various industrial, therapeutic and diagnostic recombinant proteins as they combine high growth rates with all benefits of eukaryotic expression systems. Moreover, microalgae exhibit a phototrophic lifestyle like land plants, hence protein expression is fuelled by photosynthesis, which is CO2-neutral and involves only low production costs. So far, however, research on algal bioreactors for recombinant protein expression is very rare calling for further investigations in this highly promising field. In this study, we present data on the expression of a monoclonal human IgG antibody against the Hepatitis B surface protein and the respective antigen in the diatom Phaeodactylum tricornutum. Antibodies are fully-assembled and functional and accumulate to 8.7% of total soluble protein, which complies with 21 mg antibody per gram algal dry weight. The Hepatitis B surface protein is functional as well and is recognized by algae-produced and commercial antibodies.