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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 cryoelectron 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., Recycling of ribosomes for a new round of translation initiation is an essential part of protein synthesis. In archaea and eukaryotes recycling has been shown to require the highly conserved [...]

The traditional tree of life shows eukaryotes as a distinct lineage of living things, but many studies have suggested that the first eukaryotic cells were chimeric, descended from both Eubacteria (through the mitochondrion) and Archaebacteria. Eukaryote nuclei thus contain genes of both eubacterial and archaebacterial origins, and these genes have different functions within eukaryotic cells. Here we report that archaebacterium-derived genes are significantly more likely to be essential to yeast viability, are more highly expressed, and are significantly more highly connected and more central in the yeast protein interaction network. These findings hold irrespective of whether the genes have an informational or operational function, so that many features of eukaryotic genes with prokaryotic homologs can be explained by their origin, rather than their function. Taken together, our results show that genes of archaebacterial origin are in some senses more important to yeast metabolism than genes of eubacterial origin. This importance reflects these genes' origin as the ancestral nuclear component of the eukaryotic genome. endosymbiosis | gene essentiality | eukaryote origin | protein interaction network doi/ 10.1073/pnas.1000265107

We report a long-term (i.e., 4.5 consecutive yr) monitoring of surface marine archaeal assemblages of the coastal Mediterranean Sea using quantitative polymerase chain reaction against specific phylogenetic and functional genes, and, for some specific samples, clone libraries of the 16S ribosomal ribonucleic acid gene. Archaea had a marked seasonal periodicity, with recurrent peaks of abundance in December and January and very low occurrence during summer, parallel to temporal changes in community composition. Group II.b Euryarchaeota sequences were mostly present during winter when water was nutrient-enriched, and phytoplankton were abundant. Group II.a sequences were, in turn, more abundant during summer when the water column is stratified, and nutrient concentrations and phytoplankton stocks were lower. Group I Crenarchaeota abundance was highest during winter and significantly correlated with that of archaeal ammonia monooxygenase (amoA) gene copies and nitrite concentrations, suggesting that Group I Crenarchaeota were ammonia oxidizers. The periodicity of archaeal assemblages matched the strong and predictable seasonality of the surface-water conditions in the northwestern Mediterranean Sea, and suggests a low degree of functional redundancy between archaeal groups. The distinct seasonal dynamics for Group II.a and II.b Euryarchaeota, and their close association with major ecosystem processes, indicate that they may play an important but as yet largely unknown role in the ocean. doi: 10.4319/lo.2010.55.5.2117

Metagenomics has provided access to genomes of as yet uncultivated microorganisms in natural environments, yet there are gaps in our knowledge--particularly for Archaea--that occur at relatively low abundance and in extreme environments. Ultrasmall cells ( acid mine drainage | archaeal Richmond Mine acidophilic nanoorganisms | metagenomics | phylogeny | microbial ecology doi/10.1073/pnas.0914470107

Modification of the cytidine in the first anticodon position of the AUA decoding [tRNA.sup.Ile] ([tRNA.sup.Ile.sub.2]) of bacteria and archaea is essential for this tRNA to read the isoleucine codon AUA and to differentiate between AUA and the methionine codon AUG. To identify the modified cytidine in archaea, we have purified this tRNA species from Haloarcula marismortui, established its codon reading properties, used liquid chromatography-mass spectrometry (LC-MS) to map RNase A and T1 digestion products onto the tRNA, and used LC-MS/MS to sequence the oligonucleotides in RNase A digests. These analyses revealed that the modification of cytidine in the anticodon of [tRNA.sup.Ile.sub.2] adds 112 mass units to its molecular mass and makes the glycosidic bond unusually labile during mass spectral analyses. Accurate mass LC-MS and LC-MS/MS analysis of total nucleoside digests of the [tRNA.sup.Ile.sub.2] demonstrated the absence in the modified cytidine of the C2-oxo group and its replacement by agmatine (decarboxy-arginine) through a secondary amine linkage. We propose the name agmatidine, abbreviation [C.sup.+], for this modified cytidine. Agmatidine is also present in Methanococcus maripaludis [tRNA.sup.Ile.sub.2] and in Sulfolobus solfataricus total tRNA, indicating its probable occurrence in the AUA decoding [tRNA.sup.Ile] of euryarchaea and crenarchaea. The identification of agmatidine shows that bacteria and archaea have developed very similar strategies for reading the isoleucine codon AUA while discriminating against the methionine codon AUG. agmatine | decoding | RNA modification | tRNA | wobble pairing www.pnas.org/cgi/doi/10.1073/pnas.0914869107

Microbes rely on diverse defense mechanisms that allow them to withstand viral predation and exposure to invading nucleic acid. In many Bacteria and most Archaea, clustered regularly interspaced short palindromic repeats (CRISPR) form peculiar genetic loci, which provide acquired immunity against viruses and plasmids by targeting nucleic acid in a sequence-specific manner. These hypervariable loci take up genetic material from invasive elements and build up inheritable DNA-encoded immunity over time. Conversely, viruses have devised mutational escape strategies that allow them to circumvent the CRISPR/Cas system, albeit at a cost. CRISPR features may be exploited for typing purposes, epidemiological studies, host-virus ecological surveys, building specific immunity against undesirable genetic elements, and enhancing viral resistance in domesticated microbes. 10.1126/science.1179555

Two new autotrophic carbon fixation cycles have been recently described in Crenarchaeota. The 3-hydroxypropionate/4-hydroxybutyrate cycle using acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the carboxylating enzyme has been identified for (micro)aerobic members of the Sulfolobales. The dicarboxylate/4-hydroxybutyrate cycle using oxygen-sensitive pyruvate synthase and phosphoenolpyruvate carboxylase as carboxylating enzymes has been found in members of the anaerobic Desulfurococcales and Thermoproteales. However, Sulfolobales include anaerobic and Desulfurococcales aerobic autotrophic representatives, raising the question of which of the two cycles they use. We studied the mechanisms of autotrophic C[O.sub.2] fixation in the strictly anaerobic Stygiolobus azoricus (Sulfolobales) and in the facultatively aerobic Pyrolobus fumarii (Desulfurococcales). The activities of all enzymes of the 3-hydroxypropionate/4-hydroxybutyrate cycle were found in the anaerobic S. azoricus. In contrast, the aerobic or denitrifying P. fumarii possesses all enzyme activities of the dicarboxylate/4-hydroxybutyrate cycle. We conclude that autotrophic Crenarchaeota use one of the two cycles, and that their distribution correlates with the 16S rRNA-based phylogeny of this group, rather than with the aerobic or anaerobic lifestyle. DOI 10.1099/mic.0.034298-0

Ignicoccus hospitalis forms many cell surface appendages, the Iho670 fibers (width, 14 nm; length, up to 20 [micro]m), which constitute up to 5% of cellular protein. They are composed mainly of protein Iho670, possessing no homology to archaeal flagellins or fimbrins. Their existence as structures different from archaeal flagella or fimbriae have gone unnoticed up to now because they are very brittle. doi: 10.1128/JB.00858-09

Archae possess unique biochemical systems quite distinct from the pathways present in eukaryotes and eubacteria. 7,8-Dimethyl-8-hydroxy-5deazaflavin ([F.sub.0]) and [F.sub.420] are unique deazaflavin-containing coenzyme and methanogenic signature molecules, essential for a variety of biochemical transformations associated with methane biosynthesis and light-dependent DNA repair. The deazaflavin cofactor system functions during methane biosynthesis as a low-potential hydrid shuttle [F.sub.420]/[F.sub.420][H.sub.2]. In DNA photolyase repair proteins, the deazaflavin cofactor is in the deprotonated state active as a light-collecting energy transfer pigment. As such, it converts blue sunlight into energy used by the proteins to drive an essential repair process. Analysis of a eukaryotic (6-4) DNA photolyase from Drosophila melanogaster revealed a binding pocket, which tightly binds [F.sub.0]. Residues in the pocket activate the cofactor by deprotonation so that light absorption and energy transfer are switched on. The crystal structure of [F.sub.0] in complex with the D. melanogaster protein shows the atomic details of [F.sub.0] binding and activation, allowing characterization of the residues involved in [F.sub.0] activation. The results show that the [F.sub.0]/[F.sub.420] coenzyme system, so far believed to be strictly limited to the archael kingdom of life, is far more widespread than anticipated. Analysis of a D. melanogaster extract and of a DNA photolyase from the primitive eukaryote Ostreococcus tauri provided direct proof for the presence of the [F.sub.0] cofactor also in higher eukaryotes. crystal structure | deazaflavin | DNA photolesion | DNA repair | photolyase

Proteasomes are energy-dependent proteolytic machines. We elaborate here on the previously observed [N.sup.[alpha]] acetylation of the initiator methionine of the [alpha]1 protein of 20S core particles (CPs) of Haloferax volcanii proteasomes. Quantitative mass spectrometry revealed this was the dominant N-terminal form of [alpha]1 in H. volcanii cells. To further examine this, [alpha]1 proteins with substitutions in the N-terminal penultimate residue as well as deletion of the CP 'gate' formed by the [alpha]l N terminus were examined for their [N.sup.[alpha]] acetylation. Both the 'gate' deletion and Q2A substitution completely altered the [N.sup.[alpha]]-acetylation pattern of [alpha]1, with the deletion rendering [alpha]1 unavailable for [N.sup.[alpha]] acetylation and the Q2A modification apparently enhancing cleavage of [alpha]1 by methionine aminopeptidase (MAP), resulting in acetylation of the N-terminal alanine. Cells expressing these two [alpha]1 variants were less tolerant of hypoosmotic stress than the wild type and produced CPs with enhanced peptidase activity. Although [alpha]1 proteins with Q2D, Q2P, and Q2T substitutions were [N.sup.[alpha]] acetylated in CPs similar to the wild type, cells expressing these variants accumulated unusually high levels of [alpha]1 as rings in [N.sup.[alpha]]-acetylated, unmodified, and/or MAP-cleaved forms. More detailed examination of this group revealed that while CP peptidase activity was not impaired, cells expressing these [alpha]1 variants displayed higher growth rates and were more tolerant of hypoosmotic and high-temperature stress than the wild type. Overall, these results suggest that [N.sup.[alpha]] acetylation of [alpha]1 is important in CP assembly and activity, high levels of [alpha]1 rings enhance cell proliferation and stress tolerance, and unregulated opening of the CP 'gate' impairs the ability of cells to overcome salt stress.

Transfer RNA (tRNA) is essential for decoding the genome sequence into proteins. In Archaea, previous studies have revealed unique multiple intron-containing tRNAs and tRNAs that are encoded on 2 separate genes, so-called split tRNAs. Here, we discovered 10 fragmented tRNA genes in the complete genome of the hyperthermoacidophilic Archaeon Caldivirga maquilingensis that are individually transcribed and further trans-spliced to generate all of the missing tRNAs encoding glycine, alanine, and glutamate. Notably, the 3 mature [tRNA.sup.Gly]'s with synonymous codons are created from 1 constitutive 3' half transcript and 4 alternatively switching transcripts, representing tRNA made from a total of 3 transcripts named a 'tri-split tRNA.' Expression and nucleotide sequences of 10 split tRNA genes and their joined tRNA products were experimentally verified. The intervening sequences of split tRNA have high identity to tRNA intron sequences located at the same positions in intron-containing tRNAs in related Thermoproteales species. This suggests that an evolutionary relationship between intron-containing and split tRNAs exists. Our findings demonstrate the first example of split tRNA genes in a free-living organism and a unique tri-split tRNA gene that provides further insight into the evolution of fragmented tRNAs. tRNA intron | RNA processing | molecular evolution | trans-splicing | Caldivirga maquilingensis

The origin of the eukaryotic genetic apparatus is thought to be central to understanding the evolution of the eukaryotic cell. Disagreement about the source of the relevant genes has spawned competing hypotheses for the origins of the eukaryote nuclear lineage. The iconic rooted 3-domains tree of life shows eukaryotes and archaebacteria as separate groups that share a common ancestor to the exclusion of eubacteria. By contrast, the eocyte hypothesis has eukaryotes originating within the archaebacteria and sharing a common ancestor with a particular group called the Crenarchaeota or eocytes. Here, we have investigated the relative support for each hypothesis from analysis of 53 genes spanning the 3 domains, including essential components of the eukaryotic nucleic acid replication, transcription, and translation apparatus. As an important component of our analysis, we investigated the fit between model and data with respect to composition. Compositional heterogeneity is a pervasive problem for reconstruction of ancient relationships, which, if ignored, can produce an incorrect tree with strong support. To mitigate its effects, we used phylogenetic models that allow for changing nucleotide or amino acid compositions over the tree and data. Our analyses favor a topology that supports the eocyte hypothesis rather than archaebacterial monophyly and the 3-domains tree of life. archaebacteria | evolution | eocyte | nucleus | phylogenetics

Members of the genus Thermococcus, sulfur-reducing hyperthermophilic archaea, are ubiquitously present in various deep-sea hydrothermal vent systems and are considered to play a significant role in the microbial consortia. We present the complete genome sequence and feature analysis of Thermococcus onnurineus NA1 isolated from a deep-sea hydrothermal vent area, which reveal clues to its physiology. Based on results of genomic analysis, T. onnurineus NA1 possesses the metabolic pathways for organotrophic growth on peptides, amino acids, or sugars. More interesting was the discovery that the genome encoded unique proteins that are involved in carboxydotrophy to generate energy by oxidation of CO to C[O.sub.2] thereby providing a mechanistic basis for growth with CO as a substrate. This lithotrophic feature in combination with carbon fixation via RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) introduces a new strategy with a complementing energy supply for T. onnurineus NA1 potentially allowing it to cope with nutrient stress in the surrounding of hydrothermal vents, providing the first genomic evidence for the carboxydotrophy in Thermococcus.

Deep drilling into the marine sea floor has uncovered a vast sedimentary ecosystem of microbial cells (1,2). Extrapolation of direct counts of stained microbial cells to the total volume of [...]

Archaea are abundant and drive critical microbial processes in the Earth's cold biosphere. Despite this, not enough is known about the molecular mechanisms of cold adaptation and no biochemical studies have been performed on stenopsychrophilic archaea (e.g., Methanogenium frigidum). This study examined the structural and functional properties of cold shock proteins (Csps) from archaea, including biochemical analysis of the Csp from M. frigidum. csp genes are present in most bacteria and some eucarya but absent from most archaeal genome sequences, most notably, those of all archaeal thermophiles and hyperthermophiles. In bacteria, Csps are small, nucleic acid binding proteins involved in a variety of cellular processes, such as transcription. In this study, archaeal Csp function was assessed by examining the ability of csp genes from psychrophilic and mesophilic Euryarchaeota and Crenarchaeota to complement a cold-sensitive growth defect in Escherichia coli. In addition, an archaeal gene with a cold shock domain (CSD) fold but little sequence identity to Csps was also examined. Genes encoding Csps or a CSD structural analog from three psychrophilic archaea rescued the E. coli growth defect. The three proteins were predicted to have a higher content of solvent-exposed basic residues than the noncomplementing proteins, and the basic residues were located on the nucleic acid binding surface, similar to their arrangement in E. coli CspA. The M. frigidum Csp was purified and found to be a single-domain protein that folds by a reversible two-state mechanism and to exhibit a low conformational stability typical of cold-adapted proteins. Moreover, M. frigidum Csp was characterized as binding E. coli single-stranded RNA, consistent with its ability to complement function in E. coli. The studies show that some Csp and CSD fold proteins have retained sufficient similarity throughout evolution in the Archaea to be able to function effectively in the Bacteria and that the function of the archaeal proteins relates to cold adaptation. The initial biochemical analysis of M. frigidum Csp has developed a platform for further characterization and demonstrates the potential for expanding molecular studies of proteins from this important archaeal stenopsychrophile.

The use of molecular hydrogen as electron donor for energy generation is a defining characteristic of the hydrogenotrophic methanogens, an ancient group that dominates the phylum Euryarchaeota. We present here a global study of changes in mRNA abundance in response to hydrogen availability for a hydrogenotrophic methanogen. Cells of Methanococcus maripaludis were grown by using continuous culture to deconvolute the effects of hydrogen limitation and growth rate, and microarray analyses were conducted. Hydrogen limitation markedly increased mRNA levels for genes encoding enzymes of the methanogenic pathway that reduce or oxidize the electron-carrying deazaflavin, coenzyme [F.sub.420]. [F.sub.420]-dependent redox functions in energy-generating metabolism are characteristic of the methanogenic Archaea, and the results show that their regulation is distinct from other redox processes in the cell. Rapid growth increased mRNA levels of the gene for an unusual hydrogenase, the hydrogen-dependent methylenetetrahydromethanopterin dehydrogenase. coenzyme [F.sub.420] | microarrays | Methanococcus maripaludis

In all three domains of life, DNA replication begins at specialized loci termed replication origins. In bacteria, replication initiates from a single, clearly defined site. In contrast, eukaryotic organisms exploit a multitude of replication origins, dividing their genomes into an array of short contiguous units. Recently, the multiple replication origin paradigm has also been demonstrated within the archaeal domain of life, with the discovery that the hyperthermophilic archaeon Sulfolobus has three replication origins. However, the evolutionary mechanism driving the progression from single to multiple origin usage remains unclear. Here, we demonstrate that Aeropyrum pernix, a distant relative of Sulfolobus, has two origins. Comparison with the Sulfolobus origins provides evidence for evolution of replicon complexity by capture of extrachromosomal genetic elements. We additionally identify a previously unrecognized candidate archaeal initiator protein that is distantly related to eukaryotic Cdt1. Our data thus provide evidence that horizontal gene transfer, in addition to its well-established role in contributing to the information content of chromosomes, may fundamentally alter the manner in which the host chromosome is replicated. Archaea | DNA replication | viral integration

The two proteins involved in the regulation of gas vesicle formation in Haloferax mediterranei, mcGvpE (activator) and mcGvpD (repressive function), are able to interact in vitro. It was also found that the respective proteins cGvpE and cGvpD of Halobacterium salinarum and the heterologous pairs mcGvpD-cGvpE and cGvpD-mcGvpE were able to interact. Previously constructed mcGvpD mutants with alterations in regions affecting the repressive function of GvpD (p-loop motif or the two arginine-rich regions bR1 and bR2) were tested for their ability to interact with GvpE, and all still bound GvpE. Even a deletion of or near the p-loop motif in GvpD did not affect this ability to interact. Further deletion variants lacking larger N- or C-terminal portions of mcGvpD yielded that neither the N-terminal region with the p-loop motif nor the C-terminal portion were important for the binding of GvpE, and suggested that the central portion is involved in GvpE binding. The GvpD protein also induces a reduction in the amount of GvpE in Haloferax volcanii transformants expressing both genes under fdx promoter control on a single plasmid. Such D[E.sup.ex] transformants contain GvpD, but no detectable GvpE, whereas large amounts of GvpE are found in AD[E.sup.ex] transformants that have incurred a deletion within the gvpD gene. A similar reduction was observed in Dex 4- Eex transformants harbouring both reading frames under fdx promoter control on two different plasmids. GvpD wild-type and also GvpD mutants were tested, and a significant reduction in the amount of GvpE was obtained in the case of GvpD wild-type and the super-repressor mutant Gvp[D.sub.3-AAA]. In contrast, transformants harbouring GvpD mutants with alterations in the p-loop motif or the bR1 region still contained GvpE. Since the amount of gvpE transcript was not reduced, the reduction occurred at the protein level. These results underlined that a functional p-loop and the arginine-rich region bR1 of GvpD were required for the GvpD-mediated reduction in the amount of GvpE.

Cells responding to dramatic environmental changes or undergoing a developmental switch typically change the expression of numerous genes. In bacteria, [sigma] factors regulate much of this process, whereas in eukaryotes, four RNA polymerases and a multiplicity of generalized transcription factors (GTFs) are required. Here, by using a systems approach, we provide experimental evidence (including protein-coimmunoprecipitation, ChIP-Chip, GTF perturbation and knockout, and measurement of transcriptional changes in these genetically perturbed strains) for how archaea likely accomplish similar large-scale transcriptional segregation and modulation of physiological functions. We are able to associate GTFs to nearly half of all putative promoters and show evidence for at least 7 of the possible 42 functional GTF pairs. This report represents a significant contribution toward closing the gap in our understanding of gene regulation by GTFs for all three domains of life and provides an example for how to use various experimental techniques to rapidly learn significant portions of a global gene regulatory network of organisms for which little has been previously known. archaea | regulatory networks | systems biology

The survey conducted about the microbial diversity in the sediments of seven springs in the Canadian Arctic region by denaturing gradient gel electrophoresis (DGGE) and analyzing clone libraries of 16S rRNA genes amplified with Bacteria and Archaea-specific primers is presented. Results showed utilization and cycling of sulfur compounds responsible for energy production and maintenance of microbial communities in Canadian cold environments.

Relative RNA abundance was measured at different cell-cycle stages in synchronized cultures of the hyperthermophilic archaeon Sulfolobus acidocaldarius. Cyclic induction was observed for >160 genes, demonstrating central roles for transcriptional regulation and cell-cycle-specific gene expression in archaeal cell-cycle progression. Many replication genes were induced in a cell-cyclespecific manner, and novel replisome components are likely to be among the genes of unknown function with similar induction patterns. Candidate genes for the unknown genome segregation and cell division machineries were also identified, as well as seven transcription factors likely to be involved in cell-cycle control. Two serine-threonine protein kinases showed distinct cell-cycle-specific induction, suggesting regulation of the archaeal cell cycle also through protein modification. Two candidate recognition elements, CCR boxes, for transcription factors in control of cell-cycle regulons were identified among gene sets with similar induction kinetics. The results allow detailed characterization of the genome segregation, division, and replication processes and may, because of the extensive homologies between the archaeal and eukaryotic information machineries, also be applicable to core features of the eukaryotic cell cycle. Archaea | chromosome replication | microarrays | mitosis l Sulfolobus

Evolutionary processes that give rise to, and limit, diversification within strain populations can be deduced from the form and distribution of genomic heterogeneity. The extent of genomic change that distinguishes the acidophilic archaeon Ferroplasma acidarmanus fer1 from an environmental population of the same species from the same site, fer1(env), was determined by comparing the 1.94-megabase (Mb) genome sequence of the isolate with that reconstructed from 8 Mb of environmental sequence data. The fer1(env) composite sequence sampled [approximately or equal to] 92% of the isolate genome. Environmental sequence data were also analyzed to reveal genomic heterogeneity within the coexisting, coevolving fer1(env) population. Analyses revealed that transposase movement and the insertion and loss of blocks of novel genes of probable phage origin occur rapidly enough to give rise to heterogeneity in gene content within the local population. Because the environmental DNA was derived from many closely related individuals, it was possible to quantify gene sequence variability within the population. All but a few gene variants show evidence of strong purifying selection. Based on the small number of distinct sequence types and their distribution, we infer that the population is undergoing frequent genetic recombination, resulting in a mosaic genome pool that is shaped by selection. The larger genetic potential of the population relative to individuals within it and the combinatorial process that results in many closely related genome types may provide the basis for adaptation to environmental fluctuations. Archaea | Ferroplasrna | population genomics | recombination

Pyrrolysine has entered natural genetic codes by the translation of UAG, a canonical stop codon. UAG translation as pyrrolysine requires the pylT gene product, an amber-decoding [tRNA.sup.Pyl] that is aminoacylated with pyrrolysine by the pyrrolysyl-tRNA synthetase produced from the pylS gene. The pylTS genes form a gene cluster with pylBCD, whose functions have not been investigated. The pylTSBCD gene order is maintained not only in methanogenic Archaea but also in a distantly related Gram-positive Bacterium, indicating past horizontal gene transfer of all five genes. Here we show that lateral transfer of pylTSBCD introduces biosynthesis and genetic encoding of pyrrolysine into a naive organism. PylS-based assays demonstrated that pyrrolysine was biosynthesized in Escherichia coli expressing pylBCD from Methanosarcina acetivorans. Production of pyrrolysine did not require [tRNA.sup.Pyl] or PylS. However, when pylTSBCD were coexpressed with mtmB1, encoding the methanogen monomethylamine methyltransferase, UAG was translated as pyrrolysine to produce recombinant monomethylamine methyltransferase. Expression of pylTSBCD also suppressed an amber codon introduced into the E. coli uidA gene. Strains lacking one of the pylBCD genes did not produce pyrrolysine or translate UAG as pyrrolysine. These results indicated that pylBCD gene products biosynthesize pyrrolysine using metabolites common to Bacteria and Archaea and, furthermore, that the pyl gene cluster represents a 'genetic code expansion cassette,' previously unprecedented in natural organisms, whose transfer allows an existing codon to be translated as a novel endogenously synthesized free amino acid. Analogous cassettes may have served similar functions for other amino acids during the evolutionary expansion of the canonical genetic code. Archaea | methanogenesis | methanosarcina | pyrrolysyl-tRNA | [tRNA.sup.Pyl]

The methanogenic archaea found in the ceca of leghorn chicken were quantitatively studied for phylogeny based on the 16S rRNA gene studies. Methanobrevibacter woesei is the major species found in the chicken ceca.

The genomes of the related crenarchaea Pyrobaculum aerophilum and Thermoproteus tenax lack any obvious gene encoding a single-stranded DNA binding protein (SSB). SSBs are essential for DNA replication, recombination, and repair and are found in all other genomes across the three domains of life. These two archaeal genomes also have only one identifiable gene encoding a chromatin protein (the Alba protein), while most other archaea have at least two different abundant chromatin proteins. We performed a biochemical screen for novel nucleic acid binding proteins present in cell extracts of T. tenax. An assay for proteins capable of binding to a single-stranded DNA oligonucleotide resulted in identification of three proteins. The first protein, Alba, has been shown previously to bind single-stranded DNA as well as duplex DNA. The two other proteins, which we designated CC1 (for crenarchaeal chromatin protein 1), are very closely related to one another, and homologs are restricted to the P. aerophilum and Aeropyrum pernix genomes. CC1 is a 6-kDa, monomeric, basic protein that is expressed at a high level in T. tenax. This protein binds single- and double-stranded DNAs with similar affinities. These properties are consistent with a role for CC1 as a crenarchaeal chromatin protein.

In vitro, archaeal SRP54 binds SRP RNA in the absence of SRP19, suggesting the latter to be expendable in Archaea. Accordingly, the Haloferax volcanii SRP19 gene was deleted. Although normally transcribed at a level comparable to that of the essential SRP54 gene, SRP19 deletion had no effect on cell growth, membrane protein insertion, protein secretion, or ribosome levels. The absence of SRP19 did, however, increase membrane bacterioruberin levels.

Various structures for individual components and higher-order assemblies of archaea replication factors are being designed and obtained to present a better understanding of the various mechanisms involved in DNA replication of archaea, as well as eukaryotes. The findings reveal that the DNA replication machinery is a simplified and presumably ancestral form of that found in eukaryotes and is evolutionarily very different and distinct from that usually found in bacteria.

Crenarchaeota are ubiquitous and abundant microbial constituents of soils, sediments, lakes, and ocean waters. To further describe the cosmopolitan nonthermophilic Crenarchaeota, we analyzed the genome sequence of one representative, the uncultivated sponge symbiont Cenarchaeum symbiosum. C. symbiosum genotypes coinhabiting the same host partitioned into two dominant populations, corresponding to previously described a- and b-type ribosomal RNA variants. Although they were syntenic, overlapping a- and b-type ribotype genomes harbored significant variability. A single tiling path comprising the dominant a-type genotype was assembled and used to explore the genomic properties of C. symbiosum and its planktonic relatives. Of 2,066 ORFs, 55.6% matched genes with predicted function from previously sequenced genomes. The remaining genes partitioned between functional RNAs (2.4%) and hypotheticals (42%) with limited homology to known functional genes. The latter category included some genes likely involved in the archaeal-sponge symbiotic association. Conversely, 525 C. symbiosum ORFs were most highly similar to sequences from marine environmental genomic surveys, and they apparently represent orthologous genes from free-living planktonic Crenarchaeota. In total, the C. symbiosum genome was remarkably distinct from those of other known Archaea and shared many core metabolic features in common with its free-living planktonic relatives. Archaea | Crenarchaea | environmental genomics | marine microbiology | population genomics

Sso7d is a chromosomal protein of hyperthermophilic Archaea. The crystal structure of Sso7d-d[([GTAAT.sub.1]UAC).sub.2] has been clarified at high resolution, showing that the protein binds in the minor groove of DNA, causing a sharp kink of ~60[degrees]. Recently, we found that photoirradiation of Sso7d and 5-iodouracil-([sup.1]U)-containing 5'-d([GTAAT.sup.1]UAC)-3' efficiently induced the abstraction of hydrogen from the methyl group of [T.sub.5] at the kink. In the present study, we found that the photoreactivity of 5-bromouracil ([sup.Br]U)-containing 5'-d([GTAAT.sup.Br]UAC)-3' was enhanced in the presence of Sso7d. Using hypoxanthine (I)-containing 5'-d([ITAAT.sup.Br]UAC)-3', we demonstrated that electron transfer occurs efficiently from Sso7d to DNA. Product analysis showed that Trp-24 of Sso7d, located at the surface of the DNA, is consumed to produce N'-formylkynurenine during photoirradiation, indicating that Trp-24 acts as an electron source. To explore the possibility of electron transfer between Sso7d and other DNA substrates, we examined the photochemical repair of the thymine dimer 5'-d(GTAAT< >TAC)-3' by Sso7d. Sso7d effectively repaired 5'-d(GTAAT< >TAC)-3' to 5'-d(GTAATTAC)-3' under irradiation conditions. During this reaction, Trp-24 was not oxidized significantly, indicating that the anion radical of the repaired TT sequence is oxidized by the cation radical of Trp-24, and that a so-called 'circular electron transfer' mechanism is operating in this system. Archaea | DNA repair | electron transfer | thymine dimer

20S proteasomes are large, multicatalytic proteases that play an important role in intracellular protein degradation. The barrel-like architecture of 20S proteasomes, formed by the stacking of four heptameric protein rings, is highly conserved from archaea to eukaryotes. The outer two rings are composed of [alpha]-type subunits, and the inner two rings are composed of [beta]-type subunits. The halophilic archaeon Haloferax volcanii synthesizes two different [alpha]-type proteins, [alpha]1 and [alpha]2, and one [beta]-type protein that assemble into at least two 20S proteasome subtypes. In this study, we demonstrate that all three of these 20S proteasomal proteins ([alpha]1, [alpha]2, and [beta]) are modified either post- or cotranslationally. Using electrospray ionization quadrupole time-of-flight mass spectrometry, a phosphorylation site of the [beta] subunit was identified at Ser129 of the deduced protein sequence. In addition, [alpha]1 and [alpha]2 contained N-terminal acetyl groups. These findings represent the first evidence of acetylation and phosphorylation of archaeal proteasomes and are one of the limited examples of post- and/or cotranslational modification of proteins in this unusual group of organisms.

Phototaxis allows archaea to adjust flagellar motion in response to light. In the photophobic response of Natronobacterium pharaonis, light-activated sensory rhodopsin II causes conformational changes in the transducer II protein (pHtrII), initiating the two-component signaling system analogous to bacterial chemotaxis. pHtrII's cytoplasmic domain (pHtrII-cyt) is homologous to the cytoplasmic domains of eubacterial chemotaxis receptors. Chemotaxis receptors require dimerization for activity and are in vivo-organized in large clusters. In this study we investigated the oligomerization and aggregation states of pHtrII-cyt by using chemical cross-linking, analytical gel-filtration chromatography, and small-angle neutron scattering. We show that pHtrII-cyt is monomeric in dilute buffers, but forms dimers in 4 M KCl, the physiological salt concentration for halophilic archaea. At high ammonium sulfate concentration, the protein forms higher-order aggregates. The monomeric protein has a rod-like shape, 202 [Angstrom] in length and 14.4 [Angstrom] in diameter; upon dimerization the length increases to 248 [Angstrom] and the diameter to 18.2 [Angstrom]. These results suggest that under high salt concentration the shape and oligomerization state of pHtrII-cyt are comparable to those of chemotaxis receptors. archaebacteria | dynamics | halophilic | small-angle neutron scattering

The structure of glucose dehydrogenase from the extreme halophile Haloferax mediterranei has been solved at 1.6-[Angstrom] resolution under crystallization conditions which closely mimic the 'in vivo' intracellular environment. The decoration of the enzyme's surface with acidic residues is only partially neutralized by bound potassium counterions, which also appear to play a role in substrate binding. The surface shows the expected reduction in hydrophobic character, surprisingly not from changes associated with the loss of exposed hydrophobic residues but rather arising from a loss of lysines consistent with the genome wide-reduction of this residue in extreme halophiles. The structure reveals a highly ordered, multilayered solvation shell that can be seen to be organized into one dominant network covering much of the exposed surface accessible area to an extent not seen in almost any other protein structure solved. This finding is consistent with the requirement of the enzyme to form a protective shell in a dehydrating environment. Archaea | x-ray structure | water structure | hydrophobic surface | surface lysines

To explore the structural basis of the unique selectivity spectrum and conductance of the transmembrane channel protein AqpM from the archaeon Methanothermobacter marburgensis, we determined the structure of AqpM to 1.68-[Angstrom] resolution by x-ray crystallography. The structure establishes AqpM as being in a unique subdivision between the two major subdivisions of aqua-porins, the water-selective aquaporins, and the water-plus-glycerol-conducting aquaglyceroporins. In AqpM, isoleucine replaces a key histidine residue found in the lumen of water channels, which becomes a glycine residue in aquaglyceroporins. As a result of this and other side-chain substituents in the walls of the channel, the channel is intermediate in size and exhibits differentially tuned electrostatics when compared with the other subfamilies. integral membrane protein | x-ray crystallography | water channel | gas

Saturated thalassic brines are among the most physically demanding habitats on Earth: few microbes survive in them. Salinibacter ruber is among these organisms and has been found repeatedly in significant numbers in climax saltern crystallizer communities. The phenotype of this bacterium is remarkably similar to that of the hyperhalophilic Archaea (Haloarchaea). The genome sequence suggests that this resemblance has arisen through convergence at the physiological level (different genes producing similar overall phenotype) and the molecular level (independent mutations yielding similar sequences or structures). Several genes and gene clusters also derive by lateral transfer from (or may have been laterally transferred to) haloarchaea. S. ruber encodes four rhodopsins. One resembles bacterial proteorhodopsins and three are of the haloarchaeal type, previously uncharacterized in a bacterial genome. The impact of these modular adaptive elements on the cell biology and ecology of S. ruber is substantial, affecting salt adaptation, bioenergetics, and photobiology. halophile | lateral gene transfer | convergence | prokaryotic evolution | rhodopsins

Among the tRNA population of the archaeal parasite Nanoarchaeum equitans are five species assembled from separate 5' and 3' tRNA halves and four species derived from tRNA precursors containing introns. In both groups an intervening sequence element must be removed during tRNA maturation. A bulge-helix-bulge (BHB) motif is the hallmark structure required by the archaeal splicing endonuclease for recognition and excision of all introns. BHB motifs are recognizable at the joining sites of all five noncontinuous tRNA species, although deviations from the canonical BHB motif are clearly present in at least two of them. Here, we show that the N. equitans splicing endonuclease cleaves tRNA precursors containing normal introns, as well as all five noncontinuous precursor tRNAs, at the predicted splice sites, indicating the enzyme's dual role in the removal of tRNA introns and processing of tRNA halves to be joined in trans. The cleavage activity on a set of synthetic canonical and noncanonical BHB constructs showed that the N. equitans splicing endonuclease accepts a broader range of substrates than the homodimeric Archaeoglobus fulgidus enzyme. In contrast to the A. fulgidus endonuclease, the N. equitans splicing enzyme possesses two different subunits. This heteromeric endonuclease type, found in N. equitans, in all Crenarchaeota, and in Methanopyrus kandleri, is able to act on the noncanonical tRNA introns present only in these organisms, which suggests coevolution of enzyme and substrate. tRNA processing

We describe a CCCH type of zinc finger domain in a replication protein A (RPA) homolog found in members of different lineages of the Euryarchaeota, a subdomain of Archaea. The zinc finger is characterized by C[X.sub.2]C[X.sub.8]C[X.sub.2]H, where X is any amino acid. Using MacRPA3, a representative of this new group of RPA in Methanosarcina acetivorans, we made two deletion mutants: a C-terminal deletion mutant lacking the zinc finger and an N-terminal deletion mutant containing the zinc finger domain. Whereas the N-terminal deletion mutant contained zinc at a level comparable to the wild-type protein level, the C-terminal deletion mutant was devoid of zinc. We further created four different mutants of MacRPA3 by replacing each of the four invariable amino acids in the zinc finger with alanine. Each single mutation at an invariable position resulted in a protein containing less than 35% of the zinc found in the wild-type protein. Circular dichroism spectra suggested that although the mutation at the first cysteine resulted in minor perturbation of protein structure, mutations at the other invariable positions led to larger structural changes. All proteins harboring a mutation at one of the invariable positions bound to single-stranded DNA weakly, and this translated into reduced capacity to stimulate DNA synthesis by M. acetivorans DNA polymerase BI. By subjecting the protein and its mutants to oxidizing and reducing conditions, we demonstrated that ssDNA binding by MacRPA3 may be regulated by redox through the zinc finger. Thus, the zinc finger modules in euryarchaeal RPA proteins may serve as a means by which the function of these proteins is regulated in the cell.

Transcriptional regulation in the archaea involves a mosaic of DNA-binding proteins frequently (although not exclusively) of bacterial type, modulating a eukaryal-type core transcription apparatus. Methanocaldococcus jannaschii (Mja) Ptr2, a homologue of the Lrp/AsnC family of bacterial transcription regulators that are among the most widely disseminated archaeal DNA-binding proteins, has been shown to activate transcription by its conjugate hyperthermophilic RNA polymerase. Here, two in vitro systems have been exploited to show that Ptr2 and a Lrp homologue from the thermophile Methanothermococcus thermolithotrophicus (Mth) activate transcription over a [approximately equal to] 40[degrees]C range, in conjunction with their cognate TATA-binding proteins (TBPs) and with heterologous TBPs. A closely related homologue from the mesophile Methanococcus maripaludis (Mma) is nearly inert as a transcriptional activator, but a cluster of mutations that converts a surface patch of Mma Lrp to identity with Ptr2 confers transcriptional activity. Mja, Mth, and Mma TBPs are interchangeable for basal transcription, but their ability to support Lrp-mediated transcriptional activation varies widely, with Mja TBP the most active and Mth TBP the least active partner. The implications of this finding for understanding the roles of TBP paralogues in supporting the gene-regulatory repertoires of archaeal genomes are briefly noted. promoter | Ptr2 | RNA polymerase | TATA-binding protein | upstream activating site

Ammonium transporters (Amts) are integral membrane proteins found in all kingdoms of life that fulfill an essential function in the uptake of reduced nitrogen for biosynthetic purposes. Amt-1 is one of three Amts encoded in the genome of the hyperthermophilic archaeon Archaeoglobus fulgidus. The crystal structure of Amt-1 shows a compact trimer with 11 transmembrane helices per monomer and a central channel for substrate conduction in each monomer, similar to the known crystal structure of AmtB from Escherichia coli. Xenon derivatization has been used to identify apolar regions of Amt-1, emphasizing not only the hydrophobicity of the substrate channel but also the unexpected presence of extensive internal cavities that should be detrimental for protein stability. The substrates ammonium and methylammonium have been used for cocrystallization experiments with Amt-1, but the identification of binding sites that are distinct from water positions is not unambiguous. The well ordered cytoplasmic C terminus of the protein in the Amt-1 structure has allowed for the construction of a docking model between Amt-1 and a homology model for its physiological interaction partner, the [P.sub.II] protein GInB-1. In this model, GInB-1 binds tightly to the cytoplasmic face of the transporter, effectively blocking conduction through the three individual substrate channels. archaebacteria | membrane proteins | nitrogen metabolism

Archaeal systems are examined in order to study the natural pathways and to underscore the similarities between archaeal, eucaryal, and bacterial biology. Archaea has proven to be a valuable resource in the search for new information on posttransitional protein modification.

Based primarily on 16S rRNA sequence comparisons, life has been broadly divided into the three domains of Bacteria, Archaea, and Eukarya. Archaea is further classified into Crenarchaea and Euryarchaea. Archaea generally thrive in extreme environments as assessed by temperature, pH, and salinity. For many prokaryotic organisms, ribosomal proteins (RP), transcription/translation factors, and chaperone genes tend to be highly expressed. A gene is predicted highly expressed (PHX) if its codon usage is rather similar to the average codon usage of at least one of the RP, transcription/ translation factors, and chaperone gene classes and deviates strongly from the average gene of the genome. The thermosome (Ths) chaperonin family represents the most salient PHX genes among Archaea. The chaperones Trigger factor and HSP70 have overlapping functions in the folding process, but both of these proteins are lacking in most archaea where they may be substituted by the chaperone prefoldin. Other distinctive PHX proteins of Archaea, absent from Bacteria, include the proliferating cell nuclear antigen PCNA, a replication auxiliary factor responsible for tethering the catalytic unit of DNA polymerase to DNA during high-speed replication, and the acidic RP [P.sub.0], which helps to initiate mRNA translation at the ribosome. Other PHX genes feature Cell division control protein 48 (Cdc48), whereas the bacterial septation proteins FtsZ and minD are lacking in Crenarchaea. RadA is a major DNA repair and recombination protein of Archaea. Archaeal genomes feature a strong Shine-Dalgarno ribosome-binding motif more pronounced in Euryarchaea compared with Crenarchaea. acidic ribosomal proteins | Archaea | highly expressed proteins | thermosome

The characterization of enzymatic activities and crystal structure of an archael acylphosphatase is reported. The data suggests that, while putative acylphosphotase is less efficient than other mammalian homologues at 25 degree celsius, the thermophilic enzyme is fully active at the optimal growth temperature of Pyrococcus hirokoshii.

A recently discovered DNA repair protein of 303 aa from the archaeal organism Ferroplasma acidarmanus was studied. This protein (AGTendoV) consists of a fusion of the C-terminal active site domain of [O.sup.6]-alkylguanine-DNA alkyltransferase (AGT) with an endonuclease V domain. The AGTendoV recombinant protein expressed in Escherichia coli and purified to homogeneity repaired [O.sup.6]-methylguanine lesions in DNA via alkyl transfer action despite the complete absence of the N-terminal domain and some differences in key active site residues present in known AGTs. The AGTendoV recombinant protein also cleaved DNA substrates that contained the deaminated bases uracil, hypoxanthine, or xanthine in a similar manner to E. coli endonuclease V. Expression of AGTendoV in E. coli GWR109, a strain that lacks endogenous AGT activity, protected against both the killing and mutagenic activity of N-methyl-N'-nitro-N-nitrosoguanidine and was more effective in preventing mutations than human alkyltransferase, suggesting that the endonuclease V activity may also repair a promutagenic lesion produced by this alkylating agent. Expression of AGTendoV in a DNA repair-deficient E. coli [nfi.sup.-] alk[A.sup.-] strain protected from spontaneous mutations arising in saturated cultures and restored the mutation frequency to that found in the [nfi.sup.+] alk[A.sup.+] strain. These results demonstrate the physiological occurrence of two completely different but functional DNA repair activities in a single polypeptide chain. deamination | genotoxicity | alkylation

Gram-negative bacteria use a wide variety of complex mechanisms to secrete proteins across their membranes or to assemble secreted proteins into surface structures. As most archaea only possess a cytoplasmic membrane surrounded by a membrane-anchored S-layer, the organization of such complexes might be significantly different from that in Gram-negative bacteria. Five proteins of Sulfolobus solfataricus, SSO0120, SSO0572, SSO2316, SSO2387 and SSO2680, which are homologous to secretion ATPases of bacterial type II, type IV secretion systems and the type IV pili assembly machinery, were identified. The operon structures of these putative secretion systems encoding gene clusters and the expression patterns of the ATPases under different growth conditions were determined, and it was established that all five putative ATPases do show a divalent cation-dependent ATPase activity at high temperature. These results show that the archaeal secretion systems are related to the bacterial secretion systems and might be powered in a similar way.

The structure and diversity of the Archaea collected from prawn farm sediment were investigated for the first time. A partial 16S ribosomal DNA library was constructed with Archaea-specific primers. Subsequently, 80 randomly selected archaeal clones from the library were analyzed by restriction fragment length polymorphism (RFLP), and resulted in 50 different RFLP patterns. Sequence analysis of representatives from each unique RFLP type revealed high diversity in the archaeal populations, and the majority of archaeal clones were either members of novel lineages or most closely related to uncultured clones. In the phylogenetic analysis, the archaeal clones could be grouped into discrete phylogenetic lineages within the two kingdoms Crenarchaeota and Euryarchaeota. Euryarchaeota dominated in our archaeal library, with up to 72.2% of the total clones, and Crenarchaeota represented 27.8%. Of all the Euryarchaeota clones, three clones (5.6%) were affiliated with Methanosarcinales, four clones (7.4%) were related to Methanomicrobiales, three clones (5.6%) were related to Halobacterium (with 93% similarity), and the remaining clones (81.5%) were related to those uncultured Euryarchaeota in the aquatic sediment ecosystem. None of the crenarchaeal clones were associated with any known cultured lineages. The selective dispersal of the archaeal population indicates that their ecological niches are associated with environmental characteristics. Novel phylotypes of Archaea would expand our understanding of the genetic diversity of Archaea in aquatic sediment systems and would be significant in the phylogenetic study of Archaea.

All methanogenic Archaea examined to date rely on methanogenesis as their sole means of energy conservation. Among these are ones that use carbon monoxide as a growth substrate, producing methane via a pathway that involves hydrogen as an intermediate. To further examine the role of hydrogen in this process, we tested the ability of Methanosarcina acetivorans C2A, a metabolically versatile methanogen devoid of significant hydrogen metabolism, to use CO as a growth substrate. M. acetivorans grew on CO to high cell densities ([approximately equal to]1 x [10.sup.8] per ml) with a doubling time of [approximately equal to]24 h. Surprisingly, acetate and formate, rather than methane, were the major metabolic end products as shown by [sup.13]C NMR studies and enzymatic analysis of culture supernatants. Methane formation surpassed acetate/formate formation only when the cultures entered stationary growth phase, strongly suggesting that M. acetivorans conserves energy by means of this acetogenic and formigenic process. Resting cell experiments showed that methane production decreased linearly with increasing CO partial pressures, consistent with inhibition of methanogenesis by CO. Transposon-induced M. acetivorans mutants with lesions in the operon encoding phosphotransacetylase and acetate kinase failed to use either acetate or CO as growth substrates, indicating that these enzymes are required for both aceticlastic methanogenesis and carboxidotrophic acetogenesis. These findings greatly extend our concept of energy conservation and metabolic versatility in the methanogenic Archaea. acetogenesis | methanogenesis

The halophilic archaeon Haloferax volcanii produces three different proteins ([alpha]1, [alpha]2, and [beta]) that assemble into at least two 20S proteasome isoforms. This work reports the cloning and sequencing of two H. volcanii proteasome-activating nucleotidase (PAN) genes (panA and panB). The deduced PAN proteins were 60% identical with Walker A and B motifs and a second region of homology typical of AAA ATPases. The most significant region of divergence was the N terminus predicted to adopt a coiled-coil conformation involved in substrate recognition. Of the five proteasomal proteins, the [alpha]1, [beta], and PanA proteins were the most abundant. Differential regulation of all five genes was observed, with a four- to eightfold increase in mRNA levels as cells entered stationary phase. In parallel with this mRNA increase, the protein levels of PanB and [alpha]2 increased severalfold during the transition from exponential growth to stationary phase, suggesting that these protein levels are regulated at least in part by mechanisms that control transcript levels. In contrast, the [beta] and PanA protein levels remained relatively constant, while the [alpha]1 protein levels exhibited only a modest increase. This lack of correlation between the mRNA and protein levels for [alpha]1, [beta], and PanA suggests posttranscriptional mechanisms are involved in regulating the levels of these major proteasomal proteins. Together these results support a model in which the cell regulates the ratio of the different 20S proteasome and PAN proteins to modulate the structure and ultimately the function of this central energy-dependent proteolytic system.