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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.

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.

Although Pyrococcus furiosus is one of the best studied hyperthermophilic archaea, to date no experimental investigation of the extent of protein secretion has been performed. We describe experimental verification of the extracellular proteome of P. furiosus grown on starch. LC–MS/MS-based analysis of culture supernatants led to the identification of 58 proteins. Fifteen of these proteins had a putative N-terminal signal peptide (SP), tagging the proteins for translocation across the membrane. The detected proteins with predicted SPs and known function were almost exclusively involved in important extracellular functions, like substrate degradation or transport. Most of the 43 proteins without predicted N-terminal signal sequences are known to have intracellular functions, mainly (70 %) related to intracellular metabolism. In silico analyses indicated that the genome of P. furiosus encodes 145 proteins with N-terminal SPs, including 21 putative lipoproteins and 17 with a class III peptide. From these we identified 15 (10 %; 7 SPI, 3 SPIII and 5 lipoproteins) under the specific growth conditions of this study. The putative lipoprotein signal peptides have a unique sequence motif, distinct from the motifs in bacteria and other archaeal orders. Electronic supplementary material The online version of this article (doi:10.1007/s00792-013-0574-0) contains supplementary material, which is available to authorized users.

We present structural and biochemical evidence for a redox switch in the archaeal transcriptional regulator SurR of Pyrococcus furiosus, a hyperthermophilic anaerobe. P. furiosus produces H(2) during fermentation, but undergoes a metabolic shift to produce H(2) S when elemental sulfur (S(0) ) becomes available. Changes in gene expression occur within minutes of S(0) addition, and the majority of these S(0) -responsive genes are regulatory targets of SurR, a key regulator involved in primary S(0) response. SurR was shown in vitro to have dual functionality, activating transcription of some of these genes, notably the hydrogenase operons, and repressing others, including a gene-encoding sulfur reductase. This work demonstrates via biochemical and structural evidence that the activity of SurR is modulated by cysteine residues in a CxxC motif that constitutes a redox switch. Oxidation of the switch with S(0) inhibits sequence-specific DNA binding by SurR, leading to deactivation of genes related to H(2) production and derepression of genes involved in S(0) metabolism.

Pyrococcus furiosus is a model organism for analyses of molecular biology and biochemistry of archaea, but so far no useful genetic tools for this species have been described. We report here a genetic transformation system for P. furiosus based on the shuttle vector system pYS2 from Pyrococcus abyssi . In the redesigned vector, the pyrE gene from Sulfolobus was replaced as a selectable marker by the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene (HMG-CoA) conferring resistance of transformants to the antibiotic simvastatin. Use of this modified plasmid resulted in the overexpression of the HMG-CoA reductase in P. furiosus , allowing the selection of strains by growth in the presence of simvastatin. The modified shuttle vector replicated in P. furio s us , but the copy number was only one to two per chromosome. This system was used for overexpression of His 6 -tagged subunit D of the RNA polymerase (RNAP) in Pyrococcus cells. Functional RNAP was purified from transformed cells in two steps by Ni-NTA and gel filtration chromatography. Our data provide evidence that expression of transformed genes can be controlled from a regulated gluconeogenetic promoter.

The hyperthermophilic archaeon Pyrococcus furiosus grows optimally near 100°C and undergoes a heat shock response at 105°C, mediated at least in part by the heat shock regulator Phr. Genes encoding a small heat shock protein (HSP20) and a member of the AAA + ATPase are the only known targets of the regulator, but a genetic mutant of Phr has yet to be characterized. We describe here an alternative approach for the identification of the regulon of Phr based on cell-free transcription of fragmented chromosomal DNA in the presence or absence of the regulator and hybridization of in vitro RNA to P. furiosus whole-genome microarrays. Our results confirmed the phr , the hsp20 , and the aaa + ATPase genes as targets of Phr and also identified six additional open reading frames, PF0624, PF1042, PF1291, PF1292, PF1488, and PF1616, as Phr-responsive genes, which include that encoding di-myo-inositol phosphate synthase. Transcription of the identified novel genes was inhibited by Phr in standard transcription assays, and the novel consensus sequence 5′-TTTAnnnACnnnnnGTnAnnAAAA-3′ (uppercase letters denote a high conservation of the bases) was inferred from our data as the Phr recognition motif. Mutational evidence for the significance of this sequence as Phr recognition was provided in DNA-binding experiments.

This work describes the identification and characterization of SurR, Pyrococcus furiosus sulphur (S(0)) response regulator. SurR was captured from cell extract using promoter DNA of a hydrogenase operon that is downregulated in the primary response of P. furiosus to S(0), as revealed by DNA microarray experiments. SurR was validated as a sequence-specific DNA binding protein, and characterization of the SurR DNA binding motif GTTn(3)AAC led to the identification of several target genes that contain an extended motif in their promoters. A number of these were validated to contain upstream SurR binding sites. These SurR targets strongly correspond with open reading frames and operons both up- and downregulated in the primary response to S(0). In vitro transcription revealed that SurR is an activator for its own gene as well as for two hydrogenase operons whose expression is downregulated during the primary S(0) response; it is also a repressor for two genes upregulated during the primary S(0) response, one of which encodes the primary S(0)-reducing enzyme NAD(P)H sulphur reductase. Herein we give evidence for the role of SurR in both mediating the primary response to S(0) and controlling hydrogen production in P. furiosus.

The genome of the hyperthermophile archaeon Pyrococcus furiosus encodes two transcription factor B (TFB) paralogs, one of which (TFB1) was previously characterized in transcription initiation. The second TFB (TFB2) is unusual in that it lacks recognizable homology to the archaeal TFB/eukaryotic TFIIB B-finger motif. TFB2 functions poorly in promoter-dependent transcription initiation, but photochemical cross-linking experiments indicated that the orientation and occupancy of transcription complexes formed with TFB2 at the strong gdh promoter are similar to the orientation and occupancy of transcription complexes formed with TFB1. Initiation complexes formed by TFB2 display a promoter opening defect that can be bypassed with a preformed transcription bubble, suggesting a mechanism to explain the low TFB2 transcription activity. Domain swaps between TFB1 and TFB2 showed that the low activity of TFB2 is determined mainly by its N terminus. The low activity of TFB2 in promoter opening and transcription can be partially relieved by transcription factor E (TFE). The results indicate that the TFB N-terminal region, containing conserved Zn ribbon and B-finger motifs, is important in promoter opening and that TFE can compensate for defects in the N terminus through enhancement of promoter opening.

The characterization of the transcriptional regulator TrmBL1 of the hyperthermophilic archaeon Pyrococcus furiosus, homologous to TrmB (transcriptional regulator of the maltose system), was studied. The genome of P. furiosus contains three TrmB paralogues. One of the TrmB-like proteins (TrmBL), PF0124 (TrmBL1), was analysed in more detail. It regulated the expression of the genes encoding enzymes of the glycolytic pathway as well as the maltodextrin (MD) ABC transporter. By molecular sieve chromatography, purified TrmBL1 behaved at ambient temperature as a tetramer of 148.8 kDa. In the presence of 1 mM maltotriose or 5 mM maltose TrmBL1 formed octamers. As shown by electrophoretic mobility shift assay (EMSA) TrmBL1 was found to bind the MD (maltodextrin ABC transport genes) promoter DNA with sixfold higher binding affinity (K(d) 0.2 microM) than to the trehalose/maltose ABC transporter (TM) promoter (K(d) 1.2 microM). Maltotriose and maltose interfered in these assays indicating inducer function. In vitro transcription assays using purified transcription components corroborated the data obtained with EMSA and showed inhibition of transcription of the MD promoter by TrmBL1. Recently, van de Werken et al. (FEMS Microbiol Lett 2006; 260: 69-76) identified TGM, a conserved sequence (Thermococcales-Glycolytic-Motif) upstream of genes encoding glycolytic enzymes and the MD ABC transporter. The position of TGM is invariably located downstream of the BRE-TATA box and overlapping the transcription start site on each promoter. By footprint analysis TrmBL1 was found to recognize the TGM sequence in several TGM-containing promoter sequences. We identified the recognition helix in TrmBL1 revealing tyrosine (Y49) to be essential for target DNA binding. However, the TGM motif was not essential for TrmBL1 binding. We conclude that TrmBL1 is a global sugar-sensing transcriptional regulator controlling the genes of transport systems and of sugar-metabolizing enzymes.

TrmB is a transcriptional repressor of the hyperthermophilic archaeon Pyrococcus furiosus serving at least two operons. TrmB represses genes encoding an ABC transporter for trehalose and maltose (the TM system) with trehalose and maltose as inducers. TrmB also represses genes encoding another ABC transporter for maltodextrins (the MD system) with maltotriose and sucrose as inducers. Here we report that glucose which was also bound by TrmB acted as a corepressor (causing stronger repression) for both the TM and the MD system. Binding of glucose by TrmB was increased in the presence of TM promoter DNA. Maltose which acted as inducer for the TM system acted as a corepressor for the MD system intensifying repression. We propose that the differential conformational changes of TrmB in response to binding the different sugars governs the ability of TrmB to interact with the promoter region and represents a simple mechanism for selecting the usage of one carbon source over the other, reminiscent of catabolite repression in bacteria.

TrmB is the transcriptional repressor for the gene cluster of the trehalose/maltose ABC transporter of the hyperthermophilic archaea Thermococcus litoralis and Pyrococcus furiosus (malE or TM operon), with maltose and trehalose acting as inducers. We found that TrmB (the protein is identical in both organisms) also regulated the transcription of genes encoding a separate maltodextrin ABC transporter in P. furiosus (mdxE or MD operon) with maltotriose, longer maltodextrins and sucrose acting as inducers, but not with maltose or trehalose. In vitro transcription of the malE and the mdxE operons was inhibited by TrmB binding to the different operator sequences. Inhibition of the TM operon was released by maltose and trehalose whereas inhibition of the MD operon was released by maltotriose and larger maltodextrins as well as by sucrose. Scanning mutagenesis of the TM operator revealed the role of the palindromic TACTNNNAGTA sequence for TrmB recognition. TrmB exhibits a broad spectrum of sugar-binding specificity, binding maltose, sucrose, maltotriose and trehalose in decreasing order of affinity, half-maximal binding occurring at 20, 60, 250 and 500 microM substrate concentration respectively. Of all substrates, only maltose shows sigmoidal binding characteristics with a Hill coefficient of 2. As measured by molecular sieve chromatography and cross-linking TrmB behaved as dimer in dilute buffer solution at room temperature. We conclude that TrmB acts as a bifunctional transcriptional regulator acting on two different promoters and being differentially controlled by binding to different sugars. We believe this to represent a novel strategy of prokaryotic transcription regulation.

The posttranscriptional processing of tRNA produces a diverse wealth of modified nucleotides (29, 30, 41), most of which occur at conserved RNA sequence locations in all three phylogenetic domains (4, 45). Many of the functional roles of these modifications, in addition to other factors, such as G-C and metal ion content, are associated with their influence on secondary and tertiary structures in RNA (1, 14, 43). Thus, RNA modifications offer an important means of mediation of RNA structure across the entire temperature range of natural habitats for microorganisms: in low-temperature organisms, a degree of conformational flexibility in tRNA must be maintained during translation, while in the case of thermophiles, protection against environmental temperatures which may exceed the melting point of unmodified base-paired stems is required (27, 50). For example, it has been shown with increases in growth temperature for a single species (2, 27, 51) or through comparison of closely related organisms growing optimally at different temperatures (32) that selected stabilizing tRNA modifications are associated with increased culture temperature. By contrast, in bacterial psychrophiles, low levels of modification have been reported, with the exception of dihydrouridine (10), a modified tRNA nucleoside which is associated with enhancement and maintenance of molecular flexibility at low temperatures (12). From a phylogenetic perspective, it is interesting that the nucleoside structural motifs used for RNA stabilization at higher temperatures are known to be different in bacterial thermophiles (13, 24) and archaeal thermophiles (17), but tRNA modifications in low-temperature archaea have not previously been examined. We report here a detailed study of the identities and levels of nucleoside modifications in unfractionated tRNA from the psychrotolerant archaeon Methanococcoides burtonii (20). Cultures were grown at 23°C (the optimum growth temperature) and at 4°C (closer to the natural habitat temperature of 1 to 2°C [20]) in order to examine the overall level of tRNA modifications in comparison with that in bacteria, as well as recently studied mesophilic and thermophilic methanococci (32), and to assess the influence of a significant reduction in culture temperature upon modification. For contrast within the archaeal domain, we examined tRNA from the hyperthermophile Stetteria hydrogenophila (25) cultured at 93°C, near the growth optimum of 95°C. In both cases nucleoside modifications were measured by analysis of total enzymatic digests of tRNA using combined LC/MS, a definitive method for structural identification of RNA nucleoside modifications (7, 39).

A novel methanogen, Methanosarcina baltica GS1-AT, DSM 14042, JCM 11281, was isolated from sediment at a depth of 241 m in the Gotland Deep of the Baltic Sea. Cells were irregular, monopolar monotrichous flagellated cocci 1.5-3 microm in diameter often occurring in pairs or tetrads. The catabolic substrates used included methanol, methylated amines, and acetate, but not formate or H2/CO2. Growth was observed in a temperature range between 4 degrees and 27 degrees C with an optimum at 25 degrees C. The doubling time with methanol as substrate was 84 h at 25 degrees C, 120 h at 9 degrees C, and 167 h at 4 degrees C. The doubling time with acetate as substrate was 252 h at 25 degrees C and 425 h at 20 degrees C. After the transfer of methanol-grown cultures, long lag phases were observed that lasted 15-20 days at 25 degrees C and 25 days at 4 degrees -9 degrees C. The NaCl optimum for growth was 2%-4%, and the fastest growth occurred within a pH range of 6.5-7.5. Analysis of the 16S rDNA sequence revealed that the strain was phylogenetically related to Methanosarcina. The sequence similarity to described species of95.7% and its physiological properties distinguished strain GS1-A(T) from all described species of the genus Methanosarcina.

Microbes are organisms which are well adapted to their habitat. Their survival depends on the regulation of gene expression levels in response to environmental signals. The most important step in regulation of gene expression takes place at the transcriptional level. This regulation is intriguing in Archaea because the eu-karyotic-like transcription apparatus is modulated by bacterial-like transcription regulators. The transcriptional regulator of mal operon (TrmB) family is well known as a very large group of regulators in Archaea with more than 250 members to date. One special feature of these regulators is that some of them can act as repressor, some as activator and others as both repressor and activator. This review gives a short updated overview of the TrmB family and their regulatory patterns in different Archaea as a lot of new data have been published on this topic since the last review from 2008.

Transcription in Archaea is initiated by association of a TATA box binding protein (TBP) with a TATA box. This interaction is stabilized by the binding of the transcription factor IIB (TFIIB) orthologue TFB. We show here that the RNA polymerase of the archaeon Methanococcus , in contrast to polymerase II, does not require hydrolysis of the β-γ bond of ATP for initiation of transcription and open complex formation on linearized DNA. Permanganate probing revealed that the archaeal open complex spanned at least the DNA region from −11 to −1 at a tRNA Val promoter. The Methanococcus TBP-TFB promoter complex protected the DNA region from −40 to −14 on the noncoding DNA strand and the DNA segment from −36 to −17 on the coding DNA strand from DNase I digestion. This DNase I footprint was extended only to the downstream end by the addition of the RNA polymerase to position +17 on the noncoding strand and to position +13 on the coding DNA strand.

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).

The extreme sensitivity of many Archaea to oxygen is a major obstacle for their cultivation in the laboratory and the development of archaeal genetic exchange systems. The technique of Balch and Wolfe (1976) is suitable for the cultivation of anaerobic Archaea but involves time-consuming procedures such as the use of air locks and glove boxes. We describe here a procedure for the cultivation of anaerobic Archaea that is more convenient and faster and allows the preparation of liquid media without the use of an anaerobic chamber. When the reducing agent sodium sulfide (Na2S) was replaced by sodium sulfite (Na2SO3), anaerobic media could be prepared without protection from oxygen outside an anaerobic chamber. Exchange of the headspace of serum bottles by appropriate gases was sufficient to maintain anaerobic conditions in the culture media. Organisms that were unable to utilize sulfite as a source for cellular sulfur were supplemented with hydrogen sulfide. H2S was simply added to the headspace of serum bottles by a syringe. The use of H2S as a source for sulfur minimized the precipitation of cations by sulfide. Representatives of 12 genera of anaerobic Archaea studied here were able to grow in media prepared by this procedure. For the extremely oxygen-sensitive organism Methanococcus thermolithotrophicus, we show that plates could be prepared outside an anaerobic chamber when sulfite was used as reducing agent. The application of this method may faciliate the cultivation and handling of extreme anaerobic Archaea considerably.

The fermentative conversion of glucose in anaerobic hyperthermophilic Archaea is a variant of the classical Embden-Meyerhof pathway found in Bacteria and Eukarya. A major difference of the archaeal glycolytic pathway concerns the conversion of glyceraldehyde-3-phosphate. In the hyperthermophilic archaeon Pyrococcus furiosus, this reaction is catalyzed by an unique enzyme, glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR). Here, we report the isolation, characterization, and transcriptional analysis of the GAPOR-encoding gene. GAPOR is related to a family of ferredoxin-dependent tungsten enzymes in (hyper)thermophilic Archaea and, in addition, to a hypothetical protein in Escherichia coli. Electron paramagnetic resonance analysis of the purified P. furiosus GAPOR protein confirms the anticipated involvement of tungsten in catalysis. During glycolysis in P. furiosus, GAPOR gene expression is induced, whereas the activity of glyceraldehyde-3-phosphate dehydrogenase is repressed. It is discussed that this unprecedented unidirectional reaction couple in the pyrococcal glycolysis and gluconeogenesis gives rise to a novel site of glycolytic regulation that might be widespread among Archaea.

A new hyperthermophilic, strictly anaerobic crenarchaeote, Stetteria hydrogenophila DSM11227 representing a new genus within the family of Desulfurococcaceae, was isolated from the sediment of a marine hydrothermal system at Paleohori Bay in Milos, Greece. Cells are gram-negative irregular and disc-shaped cocci, 0.5-1.5 microm in diameter, which are flagellate and can form cytoplasmatic protrusions up to 2 microm in length. The strain grew optimally at 95 degrees C at pH 6.0 and at a NaCl concentration of 3%. The organism grew mixotrophically on peptide substrates. It required elemental sulfur as an external electron acceptor, and in addition, its growth was completely dependent on the presence of molecular hydrogen. Sulfur could be replaced by thiosulfate. H2S, CO2, acetate, and ethanol were identified as products of metabolism. The G + C content of DNA was 65 mol%. Analysis of its phylogenetic position by sequence analysis of 16S rRNA placed this organism in the family of Desulfurococcaceae. The dependence of this organism on both hydrogen and sulfur during growth on peptide substrates distinguishes Stetteria from all previously described species of Crenarchaeota.

The uncultured miscellaneous crenarchaeotic group (MCG) archaea comprise one of the most abundant microbial groups in the Earth's subsurface environment. However, very little information is available regarding the lifestyle, physiology, and factors controlling the distribution of members of this group. We established a novel method using both cultivation and molecular techniques, including a pre-PCR propidium monoazide treatment, to investigate viable members of the MCG in vitro . Enrichment cultures prepared from estuarine sediment were provided with one of a variety of carbon substrates or cultivation conditions and incubated for 3 weeks. Compared with the samples from time zero, there was an order-of-magnitude increase in the number of MCG 16S rRNA genes in almost all cultures, indicating that MCG archaea are amenable to in vitro cultivation. None of the tested substrates or conditions significantly stimulated growth of MCG archaea more than the basal medium alone; however, glycerol (0.02%) had a significantly inhibitory effect ( P < 0.05). Diversity analysis of populations resulting from four culture treatments (basal medium, addition of amino acids, H 2 -CO 2 as the gas phase, or initial aerobic conditions) revealed that the majority of viable MCG archaea were affiliated with the MCG-8 and MCG-4 clusters. There were no significant differences in MCG diversity between these treatments, also indicating that some members of MCG-4 and MCG-8 are tolerant of initially oxic conditions. The methods outlined here will be useful for further investigation of MCG archaea and comparison of substrates and cultivation conditions that influence their growth in vitro .

Summary We have recently shown that specific transcription in cell extracts of Methanococcus thermolithotrophicus depends upon a transcription factor that was separated from the RNA polymerase by phosphocellulose chromatography. Here, we provide evidence for a second transcription factor. This factor copurified with RNA polymerase during initial Chromatographic steps, but it was resolved as a distinct activity required for cell-free transcription after centrifugation in sucrose density gradients. The native molecular weight of this factor was estimated by gel filtration to be 56 000. After electrophoresis under denaturing conditions, a 28 kDa polypeptide was correlated with factor activity. Thus, the native transcription factor appeared to be a dimer composed of two polypeptides of identical molecular mass. Oligo-dT sequences at the 3′-end of a tRNA Val gene and internal sequences of this tRNA were modified by DNA deletions in order to investigate the nature of archaeal transcription terminators. Deletion of two residues of the decameric sequence 5′-TTTTAATTTT-3′ reduced termination efficiency to about 27 percent of wild-type levels. Deletion of two additional residues from the 3′-end completely abolished terminator function. Deletions in the DNA region encoding the TΨC stem and loop of tRNA also significantly reduced termination efficiency. In addition a Rho-independent terminator of Escherichia coli perfectly replaced the decameric Methanococcus terminator sequence. These findings suggested that transcription termination sequences in archaea are similar to the terminator sequences in bacteria.

Two strains of rod-shaped, pyrite-oxidizing acidithiobacilli, their cell envelope structure and their interaction with pyrite were investigated in this study. Cells of both strains, Acidithiobacillus ferrooxidans strain SP5/1 and the moderately thermophilic Acidithiobacillus sp. strain HV2/2, were similar in size, with slight variations in length and diameter. Two kinds of cell appendages were observed: flagella and pili. Besides a typical Gram-negative cell architecture with inner and outer membrane, enclosing a periplasm, both strains were covered by a hitherto undescribed, regularly arranged 2-D protein crystal with p2-symmetry. In A. ferrooxidans, this protein forms a stripe-like structure on the surface. A similar surface pattern with almost identical lattice vectors was also seen on the cells of strain HV2/2. For the surface layer of both bacteria, a direct contact to pyrite crystals was observed in ultrathin sections, indicating that the S-layer is involved in maintaining this contact site. Observations on an S-layer-deficient strain show, however, that cell adhesion does not strictly depend on the presence of the S-layer and that this surface protein has an influence on cell shape. Furthermore, the presented data suggest the ability of the S-layer protein to complex Fe3+ ions, suggesting a role in the physiology of the microorganisms.

The gene encoding the archaeal 7S RNA in the hyperthermophile Methanothermus fervidus is linked to a tRNASer and rRNA operon in the arrangement 5′-7S RNA-14nt-tRNASer-196nt-16S rRNA and the promoter directing transcription of this 7S RNA gene has now been identified. Initiation of transcription of the 7S RNA gene has been shown to occur both in vivo in M. fervidus and in vitro, using a Methanococcus thermolithotrophicus derived cell-free transcription system, at the first G residue within the initiator sequence ATGG, located 6 bp upstream of the 5′ end of the 7S RNA coding region. Cotranscription of the 7S RNA and tRNASer has been demonstrated in vitro.

The general subunit of all three eukaryotic RNA polymerases, Rpb12, and subunit P of the archaeal enzyme show sequence similarities in their N-terminal zinc ribbon and some highly conserved residues in the C-terminus. We report here that archaeal subunit P under the control of a strong yeast promoter could complement the lethal phenotype of a RPB12 deletion mutant and that subunit Rpb12 from yeast can functionally replace subunit P during reconstitution of the archaeal RNA polymerase. The DeltaP enzyme is unable to form stable open complexes, but can efficiently extend a dinucleotide on a premelted template or RNA on an elongation scaffold. This suggests that subunit P is directly or indirectly involved in promoter opening. The activity of the DeltaP enzyme can be rescued by the addition of Rpb12 or subunit P to transcription reactions. Mutation of cysteine residues in the zinc ribbon impair the activity of the enzyme in several assays and this mutated form of P is rapidly replaced by wild-type P in transcription reactions. The conserved zinc ribbon in the N-terminus seems to be important for proper interaction of the complete subunit with other RNA polymerase subunits and a 17-amino-acid C-terminal peptide is sufficient to support all basic RNA polymerase functions in vitro.

An archaeal histone gene from the hyperthermophile Pyrococcus furiosus containing four consecutive putative oligo-dT terminator sequences was used as a model system to investigate termination signals and the mechanism of termination in vitro. The archaeal RNA polymerase terminated with high efficiency at the first terminator at 90 degrees C when it contained five to six T residues, at 80 degrees C readthrough was significantly increased. A putative hairpin structure upstream of the first terminator had no effect on termination efficiency. Template competition experiments starting with RNA polymerase molecules engaged in ternary complexes revealed recycling of RNA polymerase from the terminator to the promoter of the same template. This facilitated reinitiation was dependent upon the presence of a terminator sequence suggesting that pausing at the terminator is required for recycling as in the RNA polymerase III system. Replacement of the sequences immediately downstream of the oligo-dT terminator by an AT-rich segment improved termination efficiency. Both AT-rich and GC-rich downstream sequences seemed to impair the facilitated reinitiation pathway. Our data suggest that recycling is dependent on a subtle interplay of pausing of RNA polymerase at the terminator and RNA polymerase translocation beyond the oligo-dT termination signal that is dramatically affected by downstream sequences.

Summary The immediate neighborhood of the active center of the DNA-directed RNA polymerase from the archaebacterium Methanobacterium thermoautotrophicum is radioactively labeled upon incubation of the enzyme with one of 15 different chemically reactive nucleotide analogs which function as initiating substrate and with [α- 33 PJUTP as elongating substrate in presence of poly[d(A-T)J as template. All analogs lead to the labeling of subunit B′. With analogs containing an alkyl chloride as the chemically reactive group, subunit B″ also becomes labeled. This suggests that regions of subunit B″ must also be located close to the active center. In the case of subunit B′ the label is attached between tyr 273 and met 383 . Comparison of the amino acid sequence of the labeled region with that of labeled regions in RNA polymerase from other sources indicates a strong conservation of the active center during evolution.

A specific 16S rRNA-targeted oligonucleotide probe (PIR1223) for the genus Pirellula and a species-specific probe (RB454) for Pirellula sp. strain SH1 have been designed and optimized. Together with the already existing order-specific probe PLA886, the two newly designed probes were used to detect and identify planctomycetes, pirellulae, and close relatives of Pirellula sp. strain SH1 in different habitats. With the help of these probes for detection and identification, bacteria of the genus Pirellula were detected and cultivated from tissue of the Mediterranean sponge Aplysina aerophoba and from the water column of the Kiel Fjord. An unexpected result was the close phylogenetic relationship of the isolate from the sponge and the brackish water habitat Kiel Fjord as revealed by DNA/DNA hybridization.

A novel thermophilic, hydrogen-oxidizing bacterium, VM1T, has been isolated from a marine hydrothermal area of Vulcano Island, Italy. Cells of the strain were gram-negative rods, 2-4 microm long and 1-1.5 microm wide with four to seven monopolarly inserted flagella. Cells grew chemolithoautotrophically under an atmosphere of H2/CO2 (80:20) in the presence of low concentrations of O2 (optimum 1-2%). Carbohydrates and peptide substrates were not utilized, neither for energy generation nor as a source of cellular carbon. Growth of VM1T occurred between 45 and 80 degrees C with an optimum at 65 degrees C. Growth was observed between pH 5 and 7. NaCl stimulated growth in the range 0.5-6% with an optimum at 2-3%. Hydrogen could not be replaced by elemental sulfur or thiosulfate as electron donors. Nitrate and sulfate were not used as electron acceptors. The major respiratory lipoquinone was a new menathioquinone. Analysis of the fatty acids of VM1T revealed straight-chain saturated C18:0 and the unsaturated C18:1omega9c and C20:1omega9c as major components. The G+C content of the total DNA was 43 mol%. Phylogenetic analysis placed strain VM1T near the members of the genera Hydrogenobacter, Thermocrinis and Aquifex on a separate deep-branching phylogenetic lineage. Therefore, it is proposed that strain VM1T (= DSM 12046T = JCM 10974T) represents a novel species within a new genus, for which the name Hydrogenothermus marinus gen. nov., sp. nov., is proposed. In addition, it is shown that Calderobacterium hydrogenophilum should be transferred to the genus Hydrogenobacter; the name Hydrogenobacter hydrogenophilus comb. nov. (DSM 2913T = JCM 8158T) is proposed for this organism. Furthermore, on the basis of 16S rRNA sequence analysis, Hydrogenobacter acidophilus is only distantly related to Hydrogenobacter species. Owing to this finding and its growth at low pH, the name Hydrogenobaculum acidophilum gen. nov., comb. nov., is proposed for Hydrogenobacter acidophilus. The type strain is JCM 8795T (= DSM 11251T).

A novel thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus hirschii Yel5aT (= DSM 11420T = JCM 10831T) has been isolated from the Angel Terrace Spring, Yellowstone National Park. The isolate was rod-shaped (1.0-1.5 x 0.8 microm) with a polarly inserted flagellum. Cells grew chemolithoautotrophically under an atmosphere of H2 and CO2 (80:20) in the presence of low concentrations of O2 (optimum 2.5%). Organotrophic growth occurred on complex organic substrates such as yeast extract and peptone and on organic acids. Carbohydrates and amino acids were not utilized. The strain grew between 50 and 67 degrees C; optimal growth occurred at a temperature of 63 degrees C. The pH optimum was 6.5. NaCl inhibited growth at concentrations higher than 1.5%. The major respiratory lipoquinone was ubiquinone-8. Analysis of fatty acids of Yel5aT revealed a straight-chain saturated C16:0 as the major component followed by cyclo C17:0 and cyclo C19:0. The G+C content of total DNA was 61 mol%. Phylogenetic analysis placed the strain in the beta-proteobacteria. The 16S rDNA sequence of strain Yel5aT was related to that of Hydrogenophilus thermoluteolus. To our knowledge, Hydrogenophilus hirschii is the most thermophilic micro-organism found within the proteobacteria that grows in the temperature range 50-68 degrees C.

Two novel, hyperthermophilic, anaerobic, heterotrophic archaea were isolated from shallow hydrothermal vents off Palaeochori Bay, Milos, Greece. Strain P5T (BK17S6-3-b2T) is an irregular coccus, with a single polar flagellum, growing optimally at 90 degrees C, pH 6 and 2% NaCl. The DNA G+C content was 45 mol%. Due to its morphology, phylogenetic analyses based on 16S rRNA gene sequencing, DNA-DNA hybridization experiments, physiological properties and nutritional features, this strain represents a new species within the genus Thermococcus for which the name Thermococcus aegaeicus is proposed. The type strain is P5T (= DSM 12767T = JCM 10828T). Strain p8T (BK20S6-10-b1T) is a coccus that forms aggregates. It grew optimally at 85 degrees C, pH 6 and 3% NaCl. The DNA G+C content was 38 mol%. Physiological properties and sequence analysis of the 165 rRNA gene, as well as DNA-DNA hybridization experiments, indicate that this strain is a new species belonging to the genus Staphylothermus for which the name Staphylothermus hellenicus is proposed. The type strain is P8T (= DSM 12710T = JCM 10830T).

RNA polymerase (RNAP) purified from Methanobacterium thermoautotrophicum ΔH has been shown to initiate transcription accurately in vitro from the hmtB archaeal histone promoter with either native or recombinant forms of the M. thermoautotrophicum TATA-binding protein and transcription factor TFB. Efforts to obtain transcription initiation from hydrogen-regulated methane gene promoters were, however, unsuccessful. Two previously unrecognized archaeal RNAP subunits have been identified, and complex formation by the M. thermoautotrophicum RNAP and TFB has been demonstrated.

The genetic organization, expression, and regulation of the celB locus of the hyperthermophilic archaeon Pyrococcus furiosus were analyzed. This locus includes the celB gene, which codes for an intracellular β-glucosidase, and a divergently orientated gene cluster, adhA-adhB-lamA , which codes for two alcohol dehydrogenases and an extracellular β-1,3-endoglucanase that is transcribed as a polycistronic messenger (the lamA operon). During growth of P. furiosus on either the β-1,4-linked glucose dimer cellobiose or the β-1,3-linked glucose polymer laminarin, the activities of both β-glucosidase and endoglucanase were increased at least fivefold compared with levels during growth on maltose or pyruvate. Northern blot analysis revealed an enhanced transcription of both the celB gene and the lamA operon in the presence of these glucose-containing substrates. The in vivo and in vitro transcription initiation sites of both the celB gene and the lamA operon were identified 25 nucleotides downstream of conserved TATA box motifs. A number of repeating sequences have been recognized in the celB-adhA intergenic region, some of which might be part of a transcriptional regulator-binding site.

The DNA binding and compacting activities of the recombinant (r) archaeal histones rHMfA and rHMfB from Methanothermus fervidus, and rHPyA1 from Pyrococcus species GB-3a, synthesized in Escherichia coli, have been shown to be completely resistant to incubation for 4h at 95 degrees C in the presence of 1M KCl. Continued incubation of rHMfA and rHMfB at 95 degrees C resulted in a gradual loss of these activities, and rHMfA and rHMfB lost activity more rapidly at 95 degrees C when the salt environment was reduced to 200 mM K Cl. rHPyA1, in contrast, retained full activity even after a 60-h incubation at 95 degrees C in 1 M KCl, and reducing the salt concentration did not affect the heat resistance of rHPyA1. rHPya1-DNA complexes remained intact at 100 degrees C, and rHPyA1 bound to the template DNA in in vitro transcription reaction mixtures assembled using Pyrococcus furiosus components at 90 degrees C. Transcription in vitro from the P. furiosus gdh promoter was reduced by rHPyA1 binding, in a manner that was dependent on the histone-to-DNA ratio and on the topology of the DNA template. Transcription from circular templates was more sensitive to rHPyA1 binding than transcription from a linear template, consistent with rHPyA1 binding introducing physical barriers to transcription and causing changes in the topology of circular templates that also reduced transcription.

Archaeal RNA polymerases show a weak ability in vitro to bind to promoter DNA and/or to initiate transcription with low activity independent of upstream regulatory DNA sequences. Active transcription in vitro and in vivo, however, depends strictly on a TATA box resembling the TATA box of eucaryal polII promoters. This TATA box is recognized by a polypeptide related to eucaryal TATA-binding protein (TBP) that was formerly designated aTFB. Template competition studies showed that this archaeal TATA-binding protein (aTBP) is stably sequestered at the promoter by interaction with the second archaeal transcription factor, aTFA, which is related to eucaryal transcription factor IIB (TFIIB). The association of archaeal TFIIB (aTFIIB) with the aTBP-promoter complex leads to template commitment, indicating that aTFIIB recruits archaeal RNA polymerase to the preinitiation complex. These analyses suggest the following order for assembly of transcription factors on the archaeal promoter: aTBP, aTFIIB, RNA polymerase, and provide evidence for a common molecular mechanism of transcription initiation by eucaryal RNA polymerase II and archaeal RNA polymerases. The sequence of the genes encoding aTBP and aTFIIB (TFB) showed all the characteristics conserved in their eucaryal counterparts. The degree of sequence similarity between archaeal and eucaryal transcription factors is between 27 to 35% for TFIIB and between 36 to 41% for TBP. The findings discussed here indicate that TBP and TFIIB perform analogous functions in Archaea and Eucarya and show that four essential components of archaeal and eucaryal transcriptional machineries. RNA polymerase, TATA box, TBP and TFIIB are homologous.

Immediately upstream of the hmfB gene, in a DNA fragment cloned from Methanothermus fervidus, are two identical tandemly repeated copies of a 73-bp sequence that contain the sequence 5'TTTATATA, which conforms precisely to the consensus TATA box element proposed for methanogen promoters. By using this duplicated region as the template DNA and a cell-free transcription system derived from Methanococcus thermolithotrophicus, transcription in vitro was found to initiate at two identical sites 73 bp apart, each 25 bp downstream from a TATA box, thus providing strong evidence for the functional conservation of this transcriptional signal in two phylogenetically very diverse methanogens. Transcription of the hmfB gene in vivo in M. fervidus was found to occur at only one of these sites, and consistent with this observation, recloning and sequencing of this intergenic region after its amplification by the polymerase chain reaction demonstrated that the genome of M. fervidus contains only one copy of the 73-bp sequence upstream of the hmfB gene. Since the second copy of the 73-bp sequence, presumably generated artifactually during the original hmfB cloning, functioned equally well as a promoter in the M. thermolithotrophicus transcription system, all information needed by the heterologous RNA polymerase to initiate transcription accurately in vitro must be present within this sequence. The hmfB gene encodes HMf-2, one of the two subunits of HMf, an abundant DNA binding protein in M. fervidus which binds to DNA molecules in vitro, forming nucleosomelike structures. Cell-free transcription was inhibited by adding HMf or eucaryotic core histones at protein-to-DNA mass ratios of 0.3:1 and 1:1, respectively, whereas the archael histonelike protein HTa from Thermoplasma acidophilum inhibited transcription in vitro only at much higher protein-to-DNA mass ratios and the bacterial histonelike protein HU from Escherichia coli had no detectable effect on transcription.

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.

Summary Using an immunochemical approach homologies between single components of DNA-dependent RNA polymerases from eubacteria, archaebacteria and eukaryotes were investigated. The largest components of all RNA polymerases included in this study are homologous to one another indicating a monophyletic origin of these proteins. Immunological crossreactions show that one of the large subunits present in the enzymes of sulfur-dependent archaebacteria is split into two smaller components in methanogens and halophiles. One of these smaller components roughly corresponds to the second largest subunit of the three eukaryotic enzymes whereas the other one shares antigenic determinants with subunit s of eubacterial RNA polymerases. Semi-quantitative evaluation of the data suggests that the three nuclear RNA polymerases of eukaryotes have evolved from an ancestral enzyme of the type that is found in sulfur-dependent archaebacteria.

An angular plate-shaped weakly motile mesophilic methanogen was isolated from a swamp of drilling waste in Italy. Growth occurs on H2/CO2 or on formate. Acetate is required in addition. The optimal doubling time is 7 h at 40° C. The cell envelope is composed most likely of glycoprotein subunits in hexagonal arrangement. The GC-content of its DNA is 47.5 mol%. On the basis of DNA-RNA hybridization it was found to represent a new family, the Methanoplanaceae within the order Methanomicrobiales.

The methanogenic archaebacerium Methanococcus vannielii contains a type II restriction endonuclease. The enzyme was purified by a simple three-step procedure resulting in enzyme preparations free of contaminating unspecific nucleases. The restriction enzyme recognizes and cleaves the sequence 5′-CG ↓ CG-3′ ( Fnu DII and Tha I isoschizomer) and generates DNA fragments with blunt ends. Due to its purity and activity at moderate temperatures, Mvn I might be a useful alternative to Fnu DII and Tha I active at 60°C.

Summary The super-selective affinity labelling procedure for the active center of RNA polymerase from Escherichia coli ( Grachev et al, 1987a) was successfully applied to the enzyme from archaebacteria. Using adenosine-5′-trimetaphosphate or the p-hydroxybenzaldehyde ester of ATP, the second largest subunit B′ of the RNA polymerase from methanogenic/halophilic and sulphate reducing archaebacteria, and the largest subunit B of the non-methanogenic thermophilic archaebacterium Sulfolobus sp. strain B 12 are labelled specifically. The labelling reaction is strictly template-dependent and blocked by the transcription inhibitor heparin. We present evidence that adenosine-5′-trimetaphosphate is attached close to the catalytic center of the RNA polymerase via a phosphoamide bond to lys or his residues. The specific affinity labelling of subunit B′ from methanogens/halophiles together with the earlier observed imrnunological cross-reactivity ( Gropp et al., 1986) indicates that this subunit as well as subunit B″ contains amino acid sequences which are homologous to sequences in subunit B of the enzyme from non-methanogenic thermophilic archaebacteria and in subunit β from E. coli RNA polymerase.

The aim of the expedition to Tangkuban Perahu, West Java was to obtain archaeal samples from the solfatara fields located in Domas crater. This was one of the places, where scientists from the University of Regensburg Germany had formerly isolated Indonesian archaea, especially Thermoplasma and Sulfolobus species but not fully characterized. We collected five samples from mud holes with temperatures from 57 to 88 °C and pH of 1.5-2. A portion of each sample was grown at the University of Regensburg in modified Allen's medium at 80 °C. From four out of five samples enrichment cultures were obtained, autotrophically on elemental sulphur and heterotrophically on sulfur and yeast extract; electron micrographs are presented. In the laboratories of Universitas Indonesia the isolates were cultured at 55-60 °C in order to grow tetraetherlipid synthesizing archaea, both Thermoplasmatales and Sulfolobales. Here, we succeeded to culture the same type of archaeal cells, which had been cultured in Regensburg, probably a Sulfolobus species and in Freundt's medium, Thermoplasma species. The harvested cells are documented by phase contrast microscope equipped with a digital camera. Our next steps will be to further characterize genetically the cultured cells from Tangkuban Perahu isolates.

To identify an archaebacterial promoter sequence, nuclease protection studies with the purified RNA polymerase of Methanococcus vannielii were performed. The enzyme binds specifically both at protein-encoding (hisA and methyl CoM reductase, component C) and tRNA–rRNA genes. The binding region of the RNA polymerase extends from 30 base pairs (bp) upstream (−30) to 20 bp downstream (+20) from the in vivo transcription start site. This finding indicates that the archaebacterial enzyme recognizes promoters without transacting traascription factors. The DNA segment protected from nuclease digestion by bound RNA polymerase contains an octanucleotide sequence centered at −25, which is conserved between the protein-encoding and the stable RNA genes. According to the specific binding of the enzyme to only DNA-fragments harbouring this motif, we propose the sequence TTTATATA as the major recognition signal of the Methanococcus RNA polymerase. Comparison of this motif with published archaebacterial DNA sequences revealed the presence of homologous sequences at the same location upstream of 36 genes. We therefore consider the overall consensus [Formula: see text] as a general element of promoters in archaebacteria. In spite of the specific binding of the enzyme, most preparations of the Methanococcus vannielii RNA polymerase are unable to initiate transcription at the correct sites in vitro. Here we present first evidence for the possible existence of a transcription factor conferring the ability to the enzyme to initiate and terminate transcription specifically in vitro.Key words: promoter, footprint, TATA box, RNA polymerase, transcription.

The structural genes encoding the four largest subunits of RNA polymerase, A, B', B", and C, were physically mapped in Methanobacterium thermoautotrophicum Winter. The genes formed a cluster in the order B", B', A, C and had a common orientation. DNA hybridization experiments yielded different degrees of homology between RNA polymerase gene sequences of different species of Methanobacterium and Methanococcus voltae. No homology was detectable between Methanobacterium thermoautotrophicum and Methanosarcina barkeri. From Southern hybridization experiments in which probes of the four genes from Methanobacterium thermoautotrophicum Winter and restriction digests of the genomic DNAs of the different methanogens were used, a common gene order of the RNA polymerase genes could be deduced.

RNA polymerase, purified from Methanococcus vannielii, was shown by exonuclease III footprinting to bind to a 49-base-pair (bp) region of DNA in the intergenic region upstream of mcrB. S1 nuclease protection experiments demonstrated that transcription initiation in vivo occurs within this region at 32 or 33 bp 5' to the ATG translation initiation codon of mcrB and 19 or 20 bp 3' to a TATA box.

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 sheathlike 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.