Michael Thomm

Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90°C

A novel type of bacterium has been isolated from various geothermally heated locales on the sea floor. The organisms are strictly anaerobic, rod-shaped, fermentative, extremely thermophilic and grow between 55 and 90°C with an optimum of around 80°C. Cells show a unique sheath-like structure and monotrichous flagellation. By 16S rRNA sequencing they clearly belong to the eubacteria, although no close relationship to any known group could be detected. The majority of their lipids appear to be unique in structure among the eubacteria. Isolate MSB8 is described as Thermotoga maritima, representing the new genus Thermotoga.

Isolation of Extremely Thermophilic Sulfate Reducers: Evidence for a Novel Branch of Archaebacteria

Extremely thermophilic archaebacteria are known to be metabolizers of elemental sulfur and the methanogens. A novel group of extremely thermophilic archaebacteria is described, which consists of sulfate-respiring organisms that contain pure factor 420 and that have been isolated from marine hydrothermal systems in Italy. They possess a third type of archaebacterial RNA polymerase structure previously unknown, indicating an exceptional phylogenetic position. Most likely, this group represents a third major branch within the archaebacteria. The existence of sulfate reducers at extremely high temperatures could explain hydrogen sulfide formation in hot sulfate-containing environments, such as submarine hydrothermal systems and deep oil wells.

RNA polymerase II–TFIIB structure and mechanism of transcription initiation

To initiate gene transcription, RNA polymerase II (Pol II) requires the transcription factor IIB (B). Here we present the crystal structure of the complete Pol II–B complex at 4.3 Å resolution, and complementary functional data. The results indicate the mechanism of transcription initiation, including the transition to RNA elongation. Promoter DNA is positioned over the Pol II active centre cleft with the ‘B-core’ domain that binds the wall at the end of the cleft. DNA is then opened with the help of the ‘B-linker’ that binds the Pol II rudder and clamp coiled-coil at the edge of the cleft. The DNA template strand slips into the cleft and is scanned for the transcription start site with the help of the ‘B-reader’ that approaches the active site. Synthesis of the RNA chain and rewinding of upstream DNA displace the B-reader and B-linker, respectively, to trigger B release and elongation complex formation.

Methanococcus thermolithotrophicus, a novel thermophilic lithotrophic methanogen

An autotrophic thermophilic motile coccoid methanogen was isolated from geothermally heated sea sediments close to Naples, Italy. Growth occurs on H2/CO2 and on formate between 30 and 70°C with an optimum at 65°C. The optimal doubling time is only 55 min. The NaCl-concentration ranges from 1.3% to 8.3% with an optimum around 4%. By its G+C-content of 31.3 mol%, its subunit envelope, and by DNA-RNA hybridization the new isolate is clearly defined to be a member of the genusMethanococcus. We name itMethanococcus thermolithotrophicus.

Structural basis of highly conserved ribosome recycling in eukaryotes and archaea

Ribosome-driven protein biosynthesis is comprised of four phases: initiation, elongation, termination and recycling. In bacteria, ribosome recycling requires ribosome recycling factor and elongation factor G, and several structures of bacterial recycling complexes have been determined. In the eukaryotic and archaeal kingdoms, however, recycling involves the ABC-type ATPase ABCE1 and little is known about its structural basis. Here we present cryo-electron microscopy reconstructions of eukaryotic and archaeal ribosome recycling complexes containing ABCE1 and the termination factor paralogue Pelota. These structures reveal the overall binding mode of ABCE1 to be similar to canonical translation factors. Moreover, the iron–sulphur cluster domain of ABCE1 interacts with and stabilizes Pelota in a conformation that reaches towards the peptidyl transferase centre, thus explaining how ABCE1 may stimulate peptide-release activity of canonical termination factors. Using the mechanochemical properties of ABCE1, a conserved mechanism in archaea and eukaryotes is suggested that couples translation termination to recycling, and eventually to re-initiation.

Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-Å resolution

Protein biosynthesis, the translation of the genetic code into polypeptides, occurs on ribonucleoprotein particles called ribosomes. Although X-ray structures of bacterial ribosomes are available, high-resolution structures of eukaryotic 80S ribosomes are lacking. Using cryoelectron microscopy and single-particle reconstruction, we have determined the structure of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution. This map, together with a 6.1-Å map of a Saccharomyces cerevisiae 80S ribosome, has enabled us to model ∼98% of the rRNA. Accurate assignment of the rRNA expansion segments (ES) and variable regions has revealed unique ES–ES and r-protein–ES interactions, providing insight into the structure and evolution of the eukaryotic ribosome.

Methanothermus fervidus, sp. nov., a novel extremely thermophilic methanogen isolated from an Icelandic hot spring

Summary A rod-shaped extremely thermophilic methanogen is described, growing between 65 and 97 °C with an optimal temperature around 83 °C and a doubling time of 170 min. The GC-content of its DNA is 33 mol %. The isolated cell wall sacculus contains pseudomurein. The complex cell envelope exhibits two layers, each about 12 nm thick; the inner represents the pseudomurein sacculus and the outer a protein envelope. An enriched fraction of RNA polymerase does not react with antiserum against RNA polymerase from Metbanobacterium thermoautotrophicum, indicating that the isolate belongs to a new family, the Methanothermaceae, within the order Methanobacteriales. The new organism is named Methanothermus fervidus.

Activation of archaeal transcription by recruitment of the TATA-binding protein

The hyperthermophilic archaeon Methanococcus jannaschii encodes two putative transcription regulators, Ptr1 and Ptr2, that are members of the Lrp/AsnC family of bacterial transcription regulators. In contrast, this archaeons RNA polymerase and core transcription factors are of eukaryotic type. Using the M. jannaschii high-temperature in vitro transcription system, we show that Ptr2 is a potent transcriptional activator, and that it conveys its stimulatory effects on its cognate eukaryal-type transcription machinery from an upstream activating region composed of two Ptr2-binding sites. Transcriptional activation is generated, at least in part, by Ptr2-mediated recruitment of the TATA-binding protein to the promoter.

Carbon isotope fractionation during bacterial methanogenesis by CO2 reduction

The carbon isotope fractionation between CO2 and CH4 was studied during open system (related to gas flow) culture experiments with CO2-reducing methanogenic Archaea. To study the temperature dependence of isotope fractionation during biological methanogenesis, three representatives of the order Methanococcales were cultivated in the temperature range 35-85°C. In the stationary growth phase, the carbon isotope fractionation factor between CO2 and CH4 was found to range between 1.048 and 1.079, depending on the growth temperature and on the type of fermentor. In contrast to published data derived from culture experiments, our results fall in the range of naturally occurring carbon isotope fractionations of coexisting CO2---CH4 pairs in marine sediments. Moreover, the fractionation closely approached the thermodynamic equilibrium between both gases, although thermal isotope exchange processes are unlikely to occur below 200°C. Our findings suggest that flow-through culture experiments are useful when studying biological methanogenesis and associated (carbon-) isotope fractionation as a means of deciphering complex methanogenic processes in sediments.

Filobacillus milensis gen. nov., sp. nov., a new halophilic spore-forming bacterium with Orn-D-Glu-type peptidoglycan

A spore-forming, halophilic bacterium was isolated from surface sediment located on the beach of Palaeochori Bay near to a shallow water hydrothermal vent area, Milos, Greece. The bacterium, designated SH 714T, consisted of motile, strictly aerobic rods which contained an Orn-D-Glu type murein and a G+C content of 35 mol%. Thin sections showed a cell wall typical for Gram-positive bacteria; the peptidoglycan layer, however, was very thin. The Gram-reaction of the organism was negative. Comparative 16S rRNA gene sequencing demonstrated that the isolate represents a new line of descent within the spore-forming rods branching at the periphery of the rRNA group 1 Bacillus (Bacillus sensu stricto). The nearest phylogenetic neighbours of the unknown bacterium were Bacillus haloalkaliphilus, Marinococcus albus and Halobacillus species. Based on phylogenetic and phenotypic evidence it is proposed that the unknown bacterium be classified as Filobacillus milensis gen. nov., sp. nov. The type strain is SH 714T (= DSM 13259T = ATCC 700960T).