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Summary Thaumarchaeota are globally distributed and abundant microorganisms occurring in diverse habitats and thus represent a major source of archaeal lipids. The scope of lipids as taxonomic markers in microbial ecological studies is limited by the scarcity of comparative data on the membrane lipid composition of cultivated representatives, including the phylum Thaumarchaeota. Here, we comprehensively describe the core and intact polar lipid (IPL) inventory of ten ammonia-oxidising thaumarchaeal cultures representing all four characterized phylogenetic clades. IPLs of these thaumarchaeal strains are generally similar and consist of membrane-spanning, glycerol dibiphytanyl glycerol tetraethers with monoglycosyl, diglycosyl, phosphohexose and hexose-phosphohexose headgroups. However, the relative abundances of these IPLs and their core lipid compositions differ systematically between the phylogenetic subgroups, indicating high potential for chemotaxonomic distinction of thaumarchaeal clades. Comparative lipidomic analyses of 19 euryarchaeal and crenarchaeal strains suggested that the lipid methoxy archaeol is synthesized exclusively by Thaumarchaeota and may thus represent a diagnostic lipid biomarker for this phylum. The unprecedented diversity of the thaumarchaeal lipidome with 118 different lipids suggests that membrane lipid composition and adaptation mechanisms in Thaumarchaeota are more complex than previously thought and include unique lipids with as yet unresolved properties.

The majority of cells in nature probably exist in a stationary-phase-like state, due to nutrient limitation in most environments. Studies on bacteria and yeast reveal morphological and physiological changes throughout the stationary phase, which lead to an increased ability to survive prolonged nutrient limitation. However, there is little information on archaeal stationary phase responses. We investigated protein- and lipid-level changes inThermococcus kodakarensiswith extended time in the stationary phase. Adaptations to time in stationary phase included increased proportion of membrane lipids with a tetraether backbone, synthesis of proteins that ensure translational fidelity, specific regulation of ABC transporters (upregulation of some, downregulation of others), and upregulation of proteins involved in coenzyme production. Given that the biological mechanism of tetraether synthesis is unknown, we also considered whether any of the protein-level changes inT. kodakarensismight shed light on the production of tetraether lipids across the same period. A putative carbon-nitrogen hydrolase, a TldE (a protease inEscherichia coli) homologue, and a membrane bound hydrogenase complex subunit were candidates for possible involvement in tetraether-related reactions, while upregulation of adenosylcobalamin synthesis proteins might lend support to a possible radical mechanism as a trigger for tetraether synthesis.

Transcription initiation by archaeal RNA polymerase (RNAP) and eukaryotic RNAP II requires the general transcription factor (TF) B/ IIB. Structural analyses of eukaryotic transcription initiation complexes locate the B-reader domain of TFIIB in close proximity to the active site of RNAP II. Here, we present the first crosslinking mapping data that describe the dynamic transitions of an archaeal TFB to provide evidence for structural rearrangements within the transcription complex during transition from initiation to early elongation phase of transcription. Using a highly specific UV-inducible crosslinking system based on the unnatural amino acid para-benzoyl-phenylalanine allowed us to analyze contacts of the Pyrococcus furiosus TFB B-reader domain with site-specific radiolabeled DNA templates in preinitiation and initially transcribing complexes. Crosslink reactions at different initiation steps demonstrate interactions of TFB with DNA at registers +6 to +14, and reduced contacts at +15, with structural transitions of the B-reader domain detected at register +10. Our data suggest that the B-reader domain of TFB interacts with nascent RNA at register +6 and +8 and it is displaced from the transcribed-strand during the transition from +9 to +10, followed by the collapse of the transcription bubble and release of TFB from register +15 onwards.

The distribution of respiratory quinone electron carriers among cultivated organisms provides clues on both the taxonomy of their producers and the redox processes these are mediating. Our study of the quinone inventories of 25 archaeal species belonging to the phyla Eury-, Cren- and Thaumarchaeota facilitates their use as chemotaxonomic markers for ecologically important archaeal clades. Saturated and monounsaturated menaquinones with six isoprenoid units forming the alkyl chain may serve as chemotaxonomic markers for Thaumarchaeota. Other diagnostic biomarkers are thiophene-bearing quinones for Sulfolobales and methanophenazines as functional quinone analogues of the Methanosarcinales. The ubiquity of saturated menaquinones in the Archaea in comparison to Bacteria suggests that these compounds may represent an ancestral and diagnostic feature of the Archaea. Overlap between quinone compositions of distinct thermophilic and halophilic archaea and bacteria may indicate lateral gene transfer. The biomarker potential of thaumarchaeal quinones was exemplarily demonstrated on a water column profile of the Black Sea. Both, thaumarchaeal quinones and membrane lipids showed similar distributions with maxima at the chemocline. Quinone distributions indicate that Thaumarchaeota dominate respiratory activity at a narrow interval in the chemocline, while they contribute only 9% to the microbial biomass at this depth, as determined by membrane lipid analysis.

To obtain new insights into community compositions of hyperthermophilic microorganisms, defined as having optimal growth temperatures of 80 degrees C and above, sediment and water samples were taken from two shallow marine hydrothermal vents (I and II) with temperatures of 100 degrees C at Vulcano Island, Italy. A combinatorial approach of denaturant gradient gel electrophoresis (DGGE) and metagenomic sequencing was used for microbial community analyses of the samples. In addition, enrichment cultures, growing anaerobically on selected polysaccharides such as starch and cellulose, were also analyzed by the combinatorial approach. Our results showed a high abundance of hyperthermophilic archaea, especially in sample II, and a comparable diverse archaeal community composition in both samples. In particular, the strains of the hyperthermophilic anaerobic genera Staphylothermus and Thermococcus, and strains of the aerobic hyperthermophilic genus Aeropyrum, were abundant. Regarding the bacterial community, e-Proteobacteria, especially the genera Sulfurimonas and Sulfurovum, were highly abundant. The microbial diversity of the enrichment cultures changed significantly by showing a high dominance of archaea, particularly the genera Thermococcus and Palaeococcus, depending on the carbon source and the selected temperature.

The archaeal transcription apparatus is closely related to the eukaryotic RNA polymerase (RNAP) II system, while\ud archaeal genomes are more similar to bacteria with densely packed genes organized in operons. This makes understanding\ud transcription in archaea vital, both in terms of molecular mechanisms and evolution. Very little is known about how\ud archaeal cells orchestrate transcription on a systems level. We have characterized the genome-wide occupancy of the\ud Methanocaldococcus jannaschii transcription machinery and its transcriptome. Our data reveal how the TATA and BRE\ud promoter elements facilitate recruitment of the essential initiation factors TATA-binding protein and transcription factor B,\ud respectively, which in turn are responsible for the loading of RNAP into the transcription units. The occupancies of\ud RNAP and Spt4/5 strongly correlate with each other and with RNA levels. Our results show that Spt4/5 is a general\ud elongation factor in archaea as its presence on all genes matches RNAP. Spt4/5 is recruited proximal to the transcription\ud start site on the majority of transcription units, while on a subset of genes, including rRNA and CRISPR loci, Spt4/5 is\ud recruited to the transcription elongation complex during early elongation within 500 base pairs of the transcription start\ud site and akin to its bacterial homologue NusG.

Thaumarchaeota are globally distributed and abundant microorganisms occurring in diverse habitats and thus represent a major source of archaeal lipids. The scope of lipids as taxonomic markers in microbial ecological studies is limited by the scarcity of comparative data on the membrane lipid composition of cultivated representatives, including the phylum Thaumarchaeota. Here, we comprehensively describe the core and intact polar lipid (IPL) inventory of ten ammonia-oxidising thaumarchaeal cultures representing all four characterized phylogenetic clades. IPLs of these thaumarchaeal strains are generally similar and consist of membrane-spanning, glycerol dibiphytanyl glycerol tetraethers with monoglycosyl, diglycosyl, phosphohexose and hexose-phosphohexose headgroups. However, the relative abundances of these IPLs and their core lipid compositions differ systematically between the phylogenetic subgroups, indicating high potential for chemotaxonomic distinction of thaumarchaeal clades. Comparative lipidomic analyses of 19 euryarchaeal and crenarchaeal strains suggested that the lipid methoxy archaeol is synthesized exclusively by Thaumarchaeota and may thus represent a diagnostic lipid biomarker for this phylum. The unprecedented diversity of the thaumarchaeal lipidome with 118 different lipids suggests that membrane lipid composition and adaptation mechanisms in Thaumarchaeota are more complex than previously thought and include unique lipids with as yet unresolved properties.

The crystal structure of TrmBL2 from the archaeon Pyrococcus furiosus shows an association of two pseudosymmetric dimers. The dimers follow the prototypical design of known bacterial repressors with two helix–turn–helix (HTH) domains binding to successive major grooves of the DNA. However, in TrmBL2, the two dimers are arranged at a mutual displacement of approximately 2 bp so that they associate with the DNA along the double-helical axis at an angle of approximately 80°.While the deoxyribose phosphate groups of the double-stranded DNA (dsDNA) used for co-crystallization are clearly seen in the electron density map, most of the nucleobases are averaged out. Refinement required to assume a superposition of at least three mutually displaced dsDNAs. The HTH domains interact primarily with the deoxyribose phosphate groups and polar interactions with the nucleobases are almost absent.This hitherto unseen mode of DNA binding by TrmBL2 seems to arise from nonoptimal protein–DNA contacts made by its four HTH domains resulting in a low-affinity, nonspecific binding to DNA.

The distribution of respiratory quinone electron carriers among cultivated organisms provides clues on both the taxonomy of their producers and the redox processes these are mediating. Our study of the quinone inventories of 25 archaeal species belonging to the phyla Eury-, Cren- and Thaumarchaeota facilitates their use as chemotaxonomic markers for ecologically important archaeal clades. Saturated and monounsaturated menaquinones with six isoprenoid units forming the alkyl chain may serve as chemotaxonomic markers for Thaumarchaeota. Other diagnostic biomarkers are thiophene-bearing quinones for Sulfolobales and methanophenazines as functional quinone analogues of the Methanosarcinales. The ubiquity of saturated menaquinones in the Archaea in comparison to Bacteria suggests that these compounds may represent an ancestral and diagnostic feature of the Archaea. Overlap between quinone compositions of distinct thermophilic and halophilic archaea and bacteria may indicate lateral gene transfer. The biomarker potential of thaumarchaeal quinones was exemplarily demonstrated on a water column profile of the Black Sea. Both, thaumarchaeal quinones and membrane lipids showed similar distributions with maxima at the chemocline. Quinone distributions indicate that Thaumarchaeota dominate respiratory activity at a narrow interval in the chemocline, while they contribute only 9% to the microbial biomass at this depth, as determined by membrane lipid analysis.