Transcriptional factor fur from Thermoplasma volcanium binds its own promoter DNA in a divalent cation-dependent manner.

Because archaea possess many respiratory enzymes or radical scavengers with catalytic domains that contain iron, the expression of the genes encoding these enzymes might be regulated by iron acquisition. The genome of an archaeon, Thermoplasma volcanium contains a gene that encodes Fur (TVN0292). The fur gene of T. volcanium was amplified by PCR, and cloned into plasmid pET28a. TvFur (T. volcanium Fur protein) was expressed in E. coli cells and then purified. EMSA revealed that TvFur binds to its own promoter DNA. The binding to its own promoter was in an Mn(2+)-, Zn(2+)-, and Ni(2+)-dependent manner. DNase I footprinting analysis revealed that the binding sequence of tvfur promoter was 5'-G TTATTAT G TTTATAT A TTAATTA G-3'. An analysis utilizing oligonucleotides in TvFur-binding sequences revealed that TvFur binds to the TATA-box or regions in the vicinity of the TATA-box in the promoter. These results indicated that TvFur regulates transcription depending on the availability of environmental divalent cations.

Culturable and molecular phylogenetic diversity of microorganisms in an open-dumped, extremely acidic Pb/Zn mine tailings.

A combination of cultivation-based and molecular-based approaches was used to reveal the culturable and molecular diversity of the microbes inhabiting an open-dumped Pb/Zn mine tailings that was undergoing intensive acid generation (pH 1.9). Culturable bacteria found in the extremely acidic mine tailings were Acidithiobacillus ferrooxidans, Leptospirillum ferriphilum, Sulfobacillus thermotolerans and Acidiphilium cryptum, where the number of acidophilic heterotrophs was ten times higher than that of the iron- and sulfur-oxidizing bacteria. Cloning and phylogenetic analysis revealed that, in contrast to the adjacent AMD, the mine tailings possessed a low microbial diversity with archaeal sequence types dominating the 16S rRNA gene library. Of the 141 clones examined, 132 were represented by two sequence types phylogenetically affiliated with the iron-oxidizing archaea Ferroplasma acidiphilum and three belonged to two tentative groups within the Thermoplasma lineage so far represented by only a few environmental sequences. Six clones in the library were represented by the only bacterial sequence type and were closely related to the well-described iron-oxidizer L. ferriphilum. The significant differences in the prokaryotic community structures of the extremely acidic mine tailings and the AMD associated with it highlights the importance of studying the microbial communities that are more directly involved in the iron and sulfur cycles of mine tailings.

Structural and mutational analysis of tRNA intron-splicing endonuclease from Thermoplasma acidophilum DSM 1728: catalytic mechanism of tRNA intron-splicing endonucleases.

In archaea, RNA endonucleases that act specifically on RNA with bulge-helix-bulge motifs play the main role in the recognition and excision of introns, while the eukaryal enzymes use a measuring mechanism to determine the positions of the universally positioned splice sites relative to the conserved domain of pre-tRNA. Two crystallographic structures of tRNA intron-splicing endonuclease from Thermoplasma acidophilum DSM 1728 (EndA(Ta)) have been solved to 2.5-A and 2.7-A resolution by molecular replacement, using the 2.7-A resolution data as the initial model and the single-wavelength anomalous-dispersion phasing method using selenomethionine as anomalous signals, respectively. The models show that EndA(Ta) is a homodimer and that it has overall folding similar to that of other archaeal tRNA endonucleases. From structural and mutational analyses of H236A, Y229F, and K265I in vitro, we have demonstrated that they play critical roles in recognizing the splice site and in cleaving the pre-tRNA substrate.

An actin homolog of the archaeon Thermoplasma acidophilum that retains the ancient characteristics of eukaryotic actin.

Actin, a central component of the eukaryotic cytoskeleton, plays a crucial role in determining cell shape in addition to several other functions. Recently, the structure of the archaeal actin homolog Ta0583, isolated from the archaeon Thermoplasma acidophilum, which lacks a cell wall, was reported by Roeben et al. (J. Mol. Biol. 358:145-156, 2006). Here we show that Ta0583 assembles into bundles of filaments similar to those formed by eukaryotic actin. Specifically, Ta0583 forms a helix with a filament width of 5.5 nm and an axial repeating unit of 5.5 nm, both of which are comparable to those of eukaryotic actin. Eukaryotic actin shows a greater resemblance to Ta0583 than to bacterial MreB and ParM in terms of polymerization characteristics, such as the requirement for Mg(2+), critical concentration, and repeating unit size. Furthermore, phylogenetic analysis also showed a closer relationship between Ta0583 and eukaryotic actin than between MreB or ParM and actin. However, the low specificity of Ta0583 for nucleotide triphosphates indicates that Ta0583 is more primitive than eukaryotic actin. Taken together, our results suggest that Ta0583 retains the ancient characteristics of eukaryotic actin.

Ferroplasma cupricumulans sp. nov., a novel moderately thermophilic, acidophilic archaeon isolated from an industrial-scale chalcocite bioleach heap.

A new species of Archaea was isolated from an industrial mineral sulphide bioleach heap. Strain BH2, a non-motile pleomorphic coccus, was capable of chemomixotrophic growth on ferrous sulphate and yeast extract. Growth was not supported in the absence of yeast extract. Phylogenetic analysis based on the 16S rRNA gene showed that strain BH2 was most closely related to the species Ferroplasma acidiphilum; however, it showed only 95% sequence similarity with this species. Strain BH2 had a temperature optimum of 53.6 degrees C and a temperature range for growth between 22 and 63 degrees C. Thus, it is the first moderately thermophilic member of the genus Ferroplasma. The optimum pH for the growth of the strain occurred between pH 1.0 and 1.2 and the lowest pH at which growth was observed was 0.4. Based on 16S rRNA gene sequence analysis and other physiological characteristics, strain BH2 constitutes a new species within the genus Ferroplasma. The name Ferroplasma cupricumulans is proposed for the new species and strain BH2 (DSM 16651) is proposed as the type strain.

Thermoplasma acidophilum TAA43 is an archaeal member of the eukaryotic meiotic branch of AAA ATPases.

Sequencing of the Thermoplasma acidophilum genome revealed a new gene, taa43 , which codes for a 43-kDa protein containing one AAA domain; we therefore termed it Thermoplasma AAA ATPase of 43 kDa (TAA43). Close homologs of TAA43 are found only in related Thermoplasmales, e.g. T. volcanium and Ferroplasma acidarmanus , but not in other Archaea. A detailed phylogenetic analysis showed that TAA43 and its homologs belong to the 'meiotic' branch of the AAA family. Although AAA proteins usually assemble into high-molecular-weight complexes, native TAA43 is predominantly dimeric except for a minor fraction eluting in the void volume of a sizing column. Wild-type and mutant TAA43 proteins were overexpressed in Escherichia coli , purified as dimers and characterized functionally. Since the canonical proteasome activating nucleotidase is not present in Thermoplasmales, TAA43 was tested for stimulation of proteasome activity, which was, however, not detected. Interestingly, immunoprecipitation analysis with TAA43 specific antibodies found a fraction of native TAA43 associated with Thermoplasma ribosomal proteins.

PhyloGenie: automated phylome generation and analysis.

Phylogenetic reconstruction is the method of choice to determine the homologous relationships between sequences. Difficulties in producing high-quality alignments, which are the basis of good trees, and in automating the analysis of trees have unfortunately limited the use of phylogenetic reconstruction methods to individual genes or gene families. Due to the large number of sequences involved, phylogenetic analyses of proteomes preclude manual steps and therefore require a high degree of automation in sequence selection, alignment, phylogenetic inference and analysis of the resulting set of trees. We present a set of programs that automates the steps from seed sequence to phylogeny and a utility to extract all phylogenies that match specific topological constraints from a database of trees. Two example applications that show the type of questions that can be answered by phylome analysis are provided. The generation and analysis of the Thermoplasma acidophilum phylome with regard to lateral gene transfer between Thermoplasmata and Sulfolobus, showed best BLAST hits to be far less reliable indicators of lateral transfer than the corresponding protein phylogenies. The generation and analysis of the Danio rerio phylome provided more than twice as many proteins as described previously, supporting the hypothesis of an additional round of genome duplication in the actinopterygian lineage.

The evolution of lipids.

Living organisms on the Earth which are divided into three major domains--Archaea, Bacteria, and Eucarya, probably came from a common ancestral cell. Because there are many thermophilic microorganisms near the root of the universal phylogenetic tree, the common ancestral cell should be considered to be a thermophilic microorganism. The existence of a cell is necessary for the living organisms; the cell membrane is the essential structural component of a cell, so its amphiphilic property is vital for the molecule of lipids for cell membranes. Tetraether type glycerophospholipids with C40 isoprenoid chains are major membrane lipids widely distributed in archaeal cells. Cyclization number of C40 isoprenoid chains in thermophilic archaea influences the fluidity of lipids whereas the number of carbons and degree of unsaturation in fatty acids do so in bacteria and eucarya. In addition to the cyclization of the tetraether lipids, covalent bonding of two C40 isoprenoid chains was found in hyperthermophiles. These characteristic structures of the lipids seem to contribute to their fundamental physiological roles in hyperthermophiles. Stereochemical differences between G-1-P archaeal lipids and G-3-P bacterial and eucaryal lipids might have occurred by the function of some proteins long after the first cell was developed by the reactions of small organic molecules. We propose that the structure of lipids of the common ancestral cell may have been similar to those of hyperthermophilic archaea.

Archaeal-eubacterial mergers in the origin of Eukarya: phylogenetic classification of life.

A symbiosis-based phylogeny leads to a consistent, useful classification system for all life. "Kingdoms" and "Domains" are replaced by biological names for the most inclusive taxa: Prokarya (bacteria) and Eukarya (symbiosis-derived nucleated organisms). The earliest Eukarya, anaerobic mastigotes, hypothetically originated from permanent whole-cell fusion between members of Archaea (e.g., Thermoplasma-like organisms) and of Eubacteria (e.g., Spirochaeta-like organisms). Molecular biology, life-history, and fossil record evidence support the reunification of bacteria as Prokarya while subdividing Eukarya into uniquely defined subtaxa: Protoctista, Animalia, Fungi, and Plantae.

[A new species of Thermoplasma].

An extremely thermoacidophilic strain, ES-23, was isolated from a self-heating coal spoil pile, Sichuan, China. The new isolate grows between 50 and 75 degrees C with optimum 70 degrees C and pH 1.0-5.0 with optimum 2.0. Strain ES-23 is aerobic and facultative autotrophic depending both heterotrophically on organic compounds and autotrophically on elemental sulfur as energy source and CO2 as carbon source. G-, spherical or oval with 0.9-1.5 microns in diameter. The autotrophic growth showed peri pilus-like structure. Cells Lack a rigid cell wall and surrounded only by a single triple layed membrane containing ether lipids. GC content of DNA is 38 mol%(Tm). It has been determined as a new species of Thermoplasma genus and named Thermoplasma thiooxidans sp. nov.

Archaebacteria: their phylogenetic relationship with the eubacterial and eukaryotic kingdoms.

In microbiology the discovery of archaebacteria ten years ago has wrought a profound change in the concepts of physiology, taxonomy, ecology, biochemistry, molecular biology, genetics and phylogeny. This review offers a concise summary of the state of the art in this field with special reference to taxonomy and ecology as well as to the different methodologies used to study the phylogeny of this unusual group of microorganisms that question many well established biological concepts.