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The anaerobic formation and oxidation of methane involve unique enzymatic mechanisms and cofactors, all of which are believed to be specific for C[sub.1]-compounds. Here we show that an anaerobic thermophilic enrichment culture composed of dense consortia of archaea and bacteria apparently uses partly similar pathways to oxidize the C[sub.4] hydrocarbon butane. The archaea, proposed genus Candidatus Syntrophoarchaeum, show the characteristic autofluorescence of methanogens, and contain highly expressed genes encoding enzymes similar to methyl-coenzyme M reductase. We detect butyl-coenzyme M, indicating archaeal butane activation analogous to the first step in anaerobic methane oxidation. In addition, Ca. Syntrophoarchaeum expresses the genes encoding -oxidation enzymes, carbon monoxide dehydrogenase and reversible C[sub.1] methanogenesis enzymes. This allows for the complete oxidation of butane. Reducing equivalents are seemingly channelled to HotSeep-1, a thermophilic sulfate-reducing partner bacterium known from the anaerobic oxidation of methane. Genes encoding 16S rRNA and methyl-coenzyme M reductase similar to those identifying Ca. Syntrophoarchaeum were repeatedly retrieved from marine subsurface sediments, suggesting that the presented activation mechanism is naturally widespread in the anaerobic oxidation of short-chain hydrocarbons., Author(s): Rafael Laso-Prez [1, 2]; Gunter Wegener (corresponding author) [1, 2, 3]; Katrin Knittel [1]; Friedrich Widdel [1]; Katie J. Harding [1]; Viola Krukenberg [1, 2]; Dimitri V. Meier [1]; [...]

Tubulin is a major component of the eukaryotic cytoskeleton, controlling cell shape, structure and dynamics, whereas its bacterial homologue FtsZ establishes the cytokinetic ring that constricts during cell division (1,2). How such different roles of tubulin and FtsZ evolved is unknown. Studying Archaea may provide clues as these organisms share characteristics with Eukarya and Bacteria (3). Here we report the structure and function of proteins from a distinct family related to tubulin and FtsZ, named CetZ, which co-exists with FtsZ in many archaea. CetZ X-ray crystal structures showed the FtsZ/tubulin superfamily fold, and one crystal form contained sheets of protofilaments, suggesting a structural role. However, inactivation of CetZ proteins in Haloferax volcanii did not affect cell division. Instead, CetZ1 was required for differentiation of the irregular plate-shaped cells into a rod-shaped cell type that was essential for normal swimming motility. CetZ1 formed dynamic cytoskeletal structures in vivo, relating to its capacity to remodel the cell envelope and direct rod formation. CetZ2 was also implicated in H. volcanii cell shape control. Our findings expand the known roles of the FtsZ/ tubulin superfamily to include archaeal cell shape dynamics, suggesting that a cytoskeletal role might predate eukaryotic cell evolution, and they support the premise that a major function of the microbial rod shape is to facilitate swimming., Many archaea produce FtsZ that appears to function in cell division (4-8). However, unlike bacteria, archaeal genomes frequently contain additional genes belonging to the FtsZ/tubulin superfamily (9). These genes are [...]

Anaerobic oxidation of methane (AOM) is critical for controlling the flux of methane from anoxic environments. AOM coupled to iron (1),manganese (1) and sulphate (2) reduction have been demonstrated in [...]

Microbial rhodopsins, a diverse group of photoactive proteins found in Archaea, Bacteria, and Eukarya, function in photosensing and photoenergy harvesting and may have been present in the resource-limited early global environment. Four different physiological functions have been identified and characterized for nearly 5,000 retinal-binding photoreceptors, these being ion transporters that transport proton or chloride and sensory rhodopsins that mediate light-attractant and/or -repellent responses. The greatest number of rhodopsins previously observed in a single archaeon had been four. Here, we report a newly discovered six-rhodopsin system in a single archaeon, Haloarcula marismortui, which shows a more diverse absorbance spectral distribution than any previously known rhodopsin system, and, for the first time, two light-driven proton transporters that respond to the same wavelength. All six rhodopsins, the greatest number ever identified in a single archaeon, were first shown to be expressed in H. marismortui, and these were then overexpressed in Escherichia coli. The proteins were purified for absorption spectra and photocycle determination, followed by measurement of ion transportation and phototaxis. The results clearly indicate the existence of a proton transporter system with two isochromatic rhodopsins and a new type of sensory rhodopsin-like transducer in H. marismortui. doi: 10.1128/JB.00642-10

The ESCRT pathway helps mediate the final abscission step of cytokinesis in mammals and archaea. In mammals, two early acting proteins of the ESCRT pathway, ALIX and TSGI01, are recruited to the midbody through direct interactions with the phosphoprotein CEP55. CEP55 resides at the centrosome through most of the cell cycle but then migrates to the midbody at the start of cytokinesis, suggesting that the ESCRT pathway may also have centrosomal links. Here, we have systematically analyzed the requirements for late-acting mammalian ESCRT-III and VPS4 proteins at different stages of mitosis and cell division. We found that depletion of VPS4A, VPS4B, or any of the 11 different human ESCRT-III (CHMP) proteins inhibited abscission. Remarkably, depletion of individual ESCRT-III and VPS4 proteins also altered centrosome and spindle pole numbers, producing multipolar spindles (most ESCRT-III/VPS4 proteins) or monopolar spindles (CHMP2A or CHMP5) and causing defects in chromosome segregation and nuclear morphology. VPS4 proteins concentrated at spindle poles during mitosis and then at midbodies during cytokinesis, implying that these proteins function directly at both sites. We conclude that ESCRT-III/VPS4 proteins function at centrosomes to help regulate their maintenance or proliferation and then at midbodies during abscission, thereby helping ensure the ordered progression through the different stages of cell division. ESCRT | CHMP | mitosis | cytokinesis | abscission doi/ 10.1073/pnas.1005938107

The proteasome catalyzes the majority of protein degradation in the cell and prays an integral role in cellular homeostasis. Control over proteolysis by the 20S core-particle (CP) proteasome is achieved by gated access of substrate; thus, an understanding of the molecular mechanism by which these gates regulate substrate entry is critical We used methyl--transverse relaxation optimized nuclear magnetic resonance spectroscopy to show that the amino-terminal residues that compose the gates of the a subunits of the Thermoplasma acidophilum proteasome are highly dynamic over a broad spectrum of time scales and that gating termini are in conformations that extend either well inside (closed gate) or outside (open gate) of the antechamber. Interconversion between these conformers on a time scale of seconds leads to a dynamic regulation of 20S CP proteolysis activity. 10.1126/science.1184991

Protein phosphorylation plays an important role in cell signaling. However, in the Archaea, little is known about which proteins are phosphorylated and which kinases are involved. In this study, we identified, for the first time, a typical eukaryote-like Ser/Thr protein kinase and its protein partner, a forkhead-associated (FHA)-domain-containing protein, from the archaeon Sulfolobus tokodaii strain 7. This protein kinase, ST1565, physically interacted with the FHA-domain-containing protein, ST0829, both in vivo and in vitro. ST1565 preferred [Mn.sup.2+] as a cofactor for autophosphorylation and for substrate phosphorylation; the optimal temperature for this was 45[degrees]C, and the optimal pH was 5.5 to 7.5. The critical amino acid residues of the conserved FHA and kinase domain sites were identified by performing a series of mutation assays. Thr329 was part of a major activation site in the kinase, while Thr326 was a negative regulation site. Several mutants with amino acid substitutions in the conserved FHA domain sites of ST0829 did not physically interact with ST1565. A structural model for the FHA domain demonstrated that the mutation sites were located at the edge of the protein and thus were in the domain that potentially interacts with ST1565. This report describes pioneering work on the third domain of life, the Archaea, showing that a protein kinase interacts with and phosphorylates an FHA-domain-containing protein. Our data provide critical information on the structural or functional characteristics of archaeal proteins and could help increase our understanding of fundamental signaling mechanisms in all three domains of life. doi:10.1128/JB.01471-09

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

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates during autotrophic C[O.sub.2] fixation in various members of the Crenarchaea. In this cycle, as determined using Metallosphaera sedula, malonyl-coenzyme A (malonyl-CoA) and succinyl-CoA are reductively converted via their semialdehydes to the corresponding alcohols 3-hydroxypropionate and 4-hydroxybutyrate. Here three missing oxidoreductases of this cycle were purified from M. sedula and studied. Malonic semialdehyde reductase, a member of the 3-hydroxyacyl-CoA dehydrogenase family, reduces malonic semialdehyde with NADPH to 3-hydroxypropionate. The latter compound is converted via propionyl-CoA to succinyl-CoA. Succinyl-CoA reduction to succinic semialdehyde is catalyzed by malonyl-CoA/succinyl-CoA reductase, a promiscuous NADPH-dependent enzyme that is a paralogue of aspartate semialdehyde dehydrogenase. Succinic semialdehyde is then reduced with NADPH to 4-hydroxybutyrate by succinic semialdehyde reductase, an enzyme belonging to the Zn-dependent alcohol dehydrogenase family. Genes highly similar to the Metallosphaera genes were found in other members of the Sulfolobales. Only distantly related genes were found in the genomes of autotrophic marine Crenarchaeota that may use a similar cycle in autotrophic carbon fixation. doi: 10.1128/JB.00794-09

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

Nicotianamine (NA), a small molecule ubiquitous in plants, is an important divalent metal chelator and the main precursor of phytosiderophores. Nicotianamine synthase (NAS) is the enzyme catalyzing NA synthesis by the condensation of three aminopropyl moieties of S-adenosylmethionine (SAM) and the cyclization of one of them to form an azetidine ring. Here we report five crystal structures of an archaeal NAS from Methanothermobacter thermautotrophicus, either free or in complex with its product(s) and substrate(s). These structures reveal a two-domains fold arrangement of MtNAS, a small molecule related to NA (named here thermoNicotianamine or tNA), and an original mechanism of synthesis in a buried reaction chamber. This reaction chamber is open to the solvent through a small inlet, and a single active site allows the selective entrance of only one substrate at a time that is then processed and translocated stepwise. metal homeostasis | reaction mechanism

Methylation of ribosomal RNA (rRNA) is required for optimal protein synthesis. Multiple 2'-Oribose methylations are carried out by box C/D guide ribonucleoproteins [small ribonucleoproteins (sRNPs) and small nucleolar ribonucleoproteins (snoRNPs)], which are conserved from archaea to eukaryotes. Methylation is dictated by base pairing between the specific guide RNA component of the sRNP or snoRNP and the target rRNA. We determined the structure of a reconstituted and catalytically active box C/D sRNP from the archaeon Methanocaldococcus jannoschii by single-particle electron microscopy. We found that archaeal box C/D sRNPs unexpectedly formed a dimeric structure with an alternative organization of their RNA and protein components that challenges the conventional view of their architecture. Mutational analysis demonstrated that this di-sRNP structure was relevant for the enzymatic function of archaeal box C/D sRNPs.

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

Little is known about the infection cycles of viruses infecting cells from Archaea, the third domain of life. Here, we demonstrate that the virions of the archaeal Sulfolobus islandicus rod-shaped virus 2 (SIRV2) are released from the host cell through a mechanism, involving the formation of specific cellular structures. Large pyramidal virus-induced protrusions transect the cell envelope at several positions, rupturing the S-layer; they eventually open out, thus creating large apertures through which virions escape the cell. We also demonstrate that massive degradation of the host chromosomes occurs because of virus infection, and that virion assembly occurs in the cytoplasm. Furthermore, intracellular viral DNA is visualized by flow cytometry. The results show that SIRV2 is a lytic virus, and that the host cell dies as a consequence of elaborated mechanisms orchestrated by the virus. The generation of specific cellular structures for a distinct step of virus life cycle is known in eukaryal virus-host systems but is unprecedented in cells from other domains. lysis | virus factory | hyperthermophile | infection cycle

The tryptophan (W) repressor-binding proteins (WrbA) from Echerichia coli (EcWrbA) and Archaeoglobus fulgidus (AfWrbA) were investigated for possible use in 1,4-dihydronicotinamide adenine dinucleotide (NADH) dependent amperometric biosensors and biofuel cells. EcWrbA and AfWrbA are oligomeric flavoproteins binding one flavin mononucleotide (FMN) per monomer and belonging to a new family of NAD(P)H:quinone oxidoreductases (NQOs). The enzymes were covalently linked to a low potential Os redox polymer onto graphite in the presence of single-walled carbon nanotube (SWCNT) preparations of varying average lengths. The performance of the enzyme modified electrodes for NADH oxidation was strongly depending on the average length of the applied SWCNTs. By blending the Os redox polymer with SWCNTs, the electrocatalytic current could be increased up to a factor of 5. Results obtained for AfWrbA modified electrodes were better than those for EcWrbA. For NADH detection, a linear range between 5 [micro]M and 1 mM, a lower limit of detection of 3 [micro]M, and a sensitivity of 56.5 nA [micro][M.sup.-1] [cm.sup.-2] could be reached. Additionally spectroelectrochemical measurements were carded out in order to determine the midpoint potentials of the enzymes (-115 mV vs NHE for EcWrbA and -100 mV vs NHE for AfWrbA pH 7.0). Furthermore, an AfWrbA modified electrode was used as an anode in combination with a Pt black cathode as a biofuel cell prototype.

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 minichromosome maintenance (MCM) helicase is the presumptive replicative helicase in archaea and eukaryotes. The archaeal homomultimeric MCM has a two-tier structure. One tier contains the AAA+ motor domains of the proteins, and these are the minimal functional helicase domains. The second tier is formed by the N-terminal domains. These domains are not essential for MCM helicase activity but act to enhance the processivity of the helicase. We reveal that a conserved loop facilitates communication between processivity and motor tiers. Interestingly, this allostery seems to be mediated by interactions between, rather than within, individual protomers in the MCM ring. archaea | DNA replication | AAA+ protein | Sulfolobus

In contrast to the cell division machineries of bacteria, euryarchaea, and eukaryotes, no division components have been identified in the second main archaeal phylum, Crenarchaeota. Here, we demonstrate that a three-gene operon, cdv, in the crenarchaeon Sulfolobus acidocaldarius, forms part of a unique cell division machinery. The operon is induced at the onset of genome segregation and division, and the Cdv proteins then polymerize between segregating nucleoids and persist throughout cell division, forming a successively smaller structure during constriction. The cdv operon is dramatically down-regulated after UV irradiation, indicating division inhibition in response to DNA damage, reminiscent of eukaryotic checkpoint systems. The cdv genes exhibit a complementary phylogenetic range relative to FtsZ-based archaeal division systems such that, in most archaeal lineages, either one or the other system is present. Two of the Cdv proteins, CdvB and CdvC, display homology to components of the eukaryotic ESCRT-III sorting complex involved in budding of luminal vesicles and HIV-1 virion release, suggesting mechanistic similarities and a common evolutionary origin. cdv | Crenarchaeota | cytokinesis | ftsZ | Sulfolobus

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.

Halocin C8 (HaiC8) is a stable microhalocin exhibiting strong antimicrobial activity against a wide range of haloarchaea. HalI, a 207-amino-acid peptide derived from the N terminus of the HalC8 preproprotein, is the immunity protein of HalC8. In this study, the molecular mechanism of the immunity function of HalI was investigated. Both pull-down and surface plasmon resonance assays revealed that HalI directly interacted with HalC8, and a mixture of purified HalI and HalC8 readily formed a heterocomplex, which was verified by gel filtration. Interestingly, HalC8 tended to form a self-associated complex, and one immunity protein likely sequestered multiple halocins. Significantly, the helix-loop-helix (HLH) motif containing a 4-amino-acid repeat (RELA) at the N terminus of HalI played a key role in its immunity activity. Disruption of the HLH motif or mutagenesis of the key residues of the RELA repeat resulted in loss of both the immunity function and the ability of HalI to bind to HalC8. These results demonstrated that HalI sequestered the activity of HalC8 through specific and direct binding.

The precise nick site in the double-strand origin (DSO) of pZMX201, a 1,668-bp roiling-circle replication (RCR) plasmid from the haloarchaeon Natrinema sp. CX2021, was determined by electron microscopy and DSO mapping. In this plasmid, DSO nicking occurred between residues C404 and G405 within a heptanucleotide sequence (TCTC/GGC) located in the stem region of an imperfect hairpin structure. This nick site sequence was conserved among the haloarchaeal RCR plasmids, including pNB101, suggesting that the DSO nick site might be the same for all members of this plasmid family. Interestingly, the DSOs of pZMX201 and pNB101 were found to be cross-recognized in RCR initiation and termination in a hybrid plasmid system. Mutation analysis of the DSO from pZMX201 ([DSO.sub.Z]) in this hybrid plasmid system revealed that: (i) the nucleotides in the middle of the conserved TCTCGGC sequence play more-important roles in the initiation and termination process; (ii) the left half of the hairpin structure is required for initiation but not for termination; and (iii) a 36-bp sequence containing TCTCGGC and the downstream sequence is essential and sufficient for termination. In conclusion, these haloarchaeal plasmids, with novel features that are different from the characteristics of both single-stranded DNA phages and bacterial RCR plasmids, might serve as a good model for studying the evolution of RCR replicons.

The hyperthermophilic archaea Acidianus hospitalis, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrobaculum calidifontis, and Sulfolobus tokodaii representing three different orders in the phylum Crenarchaeota were analyzed by flow cytometry and combined phase-contrast and epifluorescence microscopy. The overall organization of the cell cycle was found to be similar in all species, with a short prereplicative period and a dominant postreplicative period that accounted for 64 to 77% of the generation time. Thus, in all Crenarchaeota analyzed to date, cell division and initiation of chromosome replication occur in close succession, and a long time interval separates termination of replication from cell division. In Pyrobaculum, chromosome segregation overlapped with or closely followed DNA replication, and further genome separation appeared to occur cOncomitant with cellular growth. Cell division in P. aerophilum took place without visible constriction.

Ammonia-oxidizing archaea (AOA) have recently been found to be potentially important in nitrogen cycling in a variety of environments, such as terrestrial soils, wastewater treatment reactors, marine waters and sediments, and especially in estuaries, where high input of anthropogenic nitrogen is often experienced. The sedimentary AOA diversity, community structure and spatial distribution in the Changjiang Estuary and the adjacent East China Sea were studied. Multivariate statistical analysis indicated that the archaeal amoA genotype communities could be clustered according to sampling transects, and the station located in an estuarine mixing zone harboured a distinct AOA community. The distribution of AOA communities correlated significantly with the gradients of surface-water salinity and sediment sorting coefficient. The spatial distribution of putative soil-related AOA in certain sampling stations indicated a strong impact of the Changjiang freshwater discharge on the marine benthic microbial ecosystem. Besides freshwater, nutrients, organic matter and suspended particles, the Changjiang Diluted Water might also contribute to the transport of terrestrial archaea into the seawater and sediments along its flow path.

A proteomic survey of the halophilic archaeon Haloferax volcanii was performed by comparative two-dimensional gel electrophoresis in order to determine the molecular effects of salt stress on the organism. Cells were grown under optimal (2.1 M) and high (3.5 M) NaCl conditions. From this analysis, over 44 protein spots responsive to these conditions were detected. These spots were excised, digested in-gel with trypsin, subjected to QSTAR tandem mass spectrometry (LC/MS/MS) analysis, and identified by comparing the MS/MS-derived peptide sequence to that deduced from the H. volcanii genome. Approximately 40% of the proteins detected (18 in total) displayed differential abundance based on the detection of at least two peptide fragments per protein and overall MOWSE scores of [less than or equal to] 75 per protein. All of these identified proteins were either uniquely present or 2.3- to 26-fold higher in abundance under one condition compared to the other. The majority of proteins identified in this study were preferentially displayed under optimal salinity and primarily involved in translation, transport and metabolism. However, one protein of interest whose transcript levels were confirmed in these studies to be upregulated under high salt conditions was identified as a homologue of the phage shock protein PspA. The pspA gene belongs to the psp stress-responsive regulon commonly found among Gram-negative bacteria where its transcription is stimulated by a wide variety of stressors, including heat shock, osmotic shock and prolonged stationary-phase incubation. Homologues of PspA are also found among the genomes of cyanobacteria, higher plants and other Archaea, suggesting that this protein may retain some aspects of functional conservation across the three domains of life. Given its integral role in sensing a variety of membrane stressors in bacteria, these results suggest that PspA may play an important role in hypersaline adaptation in H. volcanii.

Archaea, like Eukarya and Bacteria, are able to N glycosylate select protein targets. However, in contrast to relatively advanced understanding of the eukaryal N glycosylation process and the information being amassed on the bacterial process, little is known of this posttranslational modification in Archaea. Toward remedying this situation, the present report continues ongoing efforts to identify components involved in the N glycosylation of the Haloferax volcanii S-layer glycoprotein. By combining gene deletion together with mass spectrometry, AglE, originally identified as a homologue of murine Dpm1, was shown to play a role in the addition of the 190-Da sugar subunit of the novel pentasaccharide decorating the S-layer glycoprotein. Topological analysis of an AglE-based chimeric reporter assigns AglE as an integral membrane protein, with its N terminus and putative active site facing the cytoplasm. These finding, therefore, contribute to the developing picture of the N glycosylation pathway in Archaea.

Archaea and eukaryotes share a dolichol phosphate-dependent system for protein N-glycosylation. In both domains, the acetamido sugar N-acetylglucosamine (GlcNAc) forms part of the core oligosaccharide. However, the archaeal Methanococcales produce GlcNAc using the bacterial biosynthetic pathway. Key enzymes in this pathway belong to large families of proteins with diverse functions; therefore, the archaeal enzymes could not be identified solely using comparative sequence analysis. Genes encoding acetamido sugar-biosynthetic proteins were identified in Methanococcus maripaludis using phylogenetic and gene cluster analyses. Proteins expressed in Escherichia coli were purified and assayed for the predicted activities. The MMP1680 protein encodes a universally conserved glucosamine-6-phosphate synthase. The MMP1077 phosphomutase converted [alpha]-D-glucosamine-6-phosphate to [alpha]-D-glucosamine-1-phosphate, although this protein is more closely related to archaeal pentose and glucose phosphomutases than to bacterial glucosamine phosphomutases. The thermostable MJ1101 protein catalyzed both the acetylation of glucosamine-1-phosphate and the uridylyl-transferase reaction with UTP to produce UDP-GlcNAc. The MMP0705 protein catalyzed the C-2 epimerization of UDP-GlcNAc, and the MMP0706 protein used [NAD.sup.+] to oxidize UDP-N-acetylmannosamine, forming UDPN-acetylmannosaminuronate (ManNAcA). These two proteins are similar to enzymes used for proteobacterial lipopolysaccharide biosynthesis and gram-positive bacterial capsule production, suggesting a common evolutionary origin and a widespread distribution of ManNAcA. UDP-GlcNAc and UDP-ManNAcA biosynthesis evolved early in the euryarchaeal lineage, because most of their genomes contain orthologs of the five genes characterized here. These UDP-acetamido sugars are predicted to be precursors for flagellin and S-layer protein modifications and for the biosynthesis of methanogenic coenzyme B.

We report the complete genome of Thermofilum pendens, a deeply branching, hyperthermophilic member of the order Thermoproteales in the archaeal kingdom Crenarchaeota. T. pendens is a sulfur-dependent, anaerobic heterotroph isolated from a solfatara in Iceland. It is an extracellular commensal, requiring an extract of Thermoproteus tenax for growth, and the genome sequence reveals that biosynthetic pathways for purines, most amino acids, and most cofactors are absent. In fact, T. pendens has fewer biosynthetic enzymes than obligate intracellular parasites, although it does not display other features that are common among obligate parasites and thus does not appear to be in the process of becoming a parasite. It appears that T. pendens has adapted to life in an environment rich in nutrients. T. pendens was known previously to utilize peptides as an energy source, but the genome revealed a substantial ability to grow on carbohydrates. T. pendens is the first crenarchaeote and only the second archaeon found to have a transporter of the phosphotransferase system. In addition to fermentation, T. pendens may obtain energy from sulfur reduction with hydrogen and formate as electron donors. It may also be capable of sulfur-independent growth on formate with formate hydrogen lyase. Additional novel features are the presence of a monomethylamine:corrinoid methyltransferase, the first time that this enzyme has been found outside the Methanosarcinales, and the presence of a presenilin-related protein. The predicted highly expressed proteins do not include proteins encoded by housekeeping genes and instead include ABC transporters for carbohydrates and peptides and clustered regularly interspaced short palindromic repeat-associated proteins.

The TON_0887 gene was identified as the missing histidinol-phosphate phosphatase (HolPase) in the hyperthermophilic archaeon 'Thermococcus onnurineus' NA1. The protein contained conserved motifs of the DDDD superfamily of phosphohydrolase, and the recombinantly expressed protein exhibited strong HolPase activity. In this study, we functionally assessed for the first time the monofunctional DDDD-type HolPase, which is organized in the gene cluster.

Nanoarchaeum equitans and lgnicoccus hospitalis represent a unique, intimate association of two archaea. Both form a stable coculture which is mandatory for N. equitans but not for the host L hospitalis. Here, we investigated interactions and mutual influence between these microorganisms. Fermentation studies revealed that during exponential growth only about 25% of L hospitalis cells are occupied by N. equitans cells (one to three cells). The latter strongly proliferate in the stationary phase of L hospitalis, until 80 to 90% of the L hospitalis cells carry around 10 N. equitans cells. Furthermore, the expulsion of [H.sub.2]S, the major metabolic end product of I. hospitalis, by strong gas stripping yields huge amounts of free N. equitans cells. N. equitans had no influence on the doubling times, final cell concentrations, and growth temperature, pH, or salt concentration ranges or optima of I. hospitalis. However, isolation studies using optical tweezers revealed that infection with N. equitans inhibited the proliferation of individual L hospitalis cells. This inhibition might be caused by deprivation of the host of cell components like amino acids, as demonstrated by [.sup.13]C-labeling studies. The strong dependence of N. equitans on L hospitalis was affirmed by live-dead staining and electron microscopic analyses, which indicated a tight physiological and structural connection between the two microorganisms. No alternative hosts, including other Ignicoccus species, were accepted by N. equitans. In summary, the data show a highly specialized association of N. equitans and L hospitalis which so far cannot be assigned to a classical symbiosis, commensalism, or parasitism.

Pyrococcus furiosus has two types of NiFe-hydrogenases: a heterotetrameric soluble hydrogenase and a multimeric transmembrane hydrogenase. Originally, the soluble hydrogenase was proposed to be a new type of [H.sub.2] evolution hydrogenase, because, in contrast to all of the then known NiFe-hydrogenases, the hydrogen production activity at 80[degree]C was found to be higher than the hydrogen consumption activity and CO inhibition appeared to be absent. NADPH was proposed to be the electron donor. Later, it was found that the membrane-bound hydrogenase exhibits very high hydrogen production activity sufficient to explain cellular [H.sub.2] production levels, and this seems to eliminate the need for a soluble hydrogen production activity and therefore leave the soluble hydrogenase without a physiological function. Therefore, the steady-state kinetics of the soluble hydrogenase were reinvestigated. In contrast to previous reports, a low [K.sub.m] for [H.sub.2] (-20 [micro]M) was found, which suggests a relatively high affinity for hydrogen. Also, the hydrogen consumption activity was 1 order of magnitude higher than the hydrogen production activity, and CO inhibition was significant (50% inhibition with 20 [micro]M dissolved CO). Since the [K.sub.m] for NAD[P.sup.+] is ~37 [micro]M, we concluded that the soluble hydrogenase from P. furiosus is likely to function in the regeneration of NADPH and thus reuses the hydrogen produced by the membrane-bound hydrogenase in proton respiration.

The recent discovery of ammonia-oxidizing archaea (AOA) dramatically changed our perception of the diversity and evolutionary history of microbes involved in nitrification. In this study, a moderately thermophilic (46[degrees]C) ammonia-oxidizing enrichment culture, which had been seeded with biomass from a hot spring, was screened for ammonia oxidizers. Although gene sequences for crenarchaeota116S rRNA and two subunits of the ammonia monooxygenase (amoA and amoB) were detected via PCR, no hints for known ammonia-oxidizing bacteria were obtained. Comparative sequence analyses of these gene fragments demonstrated the presence of a single operational taxonomic unit and thus enabled the assignment of the amoA and amoB sequences to the respective 16S rRNA phylotype, which belongs to the widely distributed group 1.1b (soil group) of the Crenarchaeota. Catalyzed reporter deposition (CARD)-FISH combined with microautoradiography (MAR) demonstrated metabolic activity of this archaeon in the presence of ammonium. This finding was corroborated by the detection of amoA gene transcripts in the enrichment. CARD-FISH/ MAR showed that the moderately thermophilic AOA is highly active at 0.14 and 0.79 mM ammonium and is partially inhibited by a concentration of 3.08 mM. The enriched AOA, which is provisionally classified as ''Candidatus Nitrososphaera gargensis,' is the first described thermophilic ammonia oxidizer and the first member of the crenarchaeotal group 1.1b for which ammonium oxidation has been verified on a cellular level. Its preference for thermophilic conditions reinvigorates the debate on the thermophilic ancestry of AOA. ammonia oxidation | archaea | nitrification | thermophile | amoA

A modified 3-hydroxypropionate cycle has been proposed as the autotrophic C[O.sub.2] fixation pathway for the thermoacidophilic crenarchaeon Metallosphaera sedula. The cycle requires the reductive conversion of 3-hydroxypropionate to propionyl-coenzyme A (propionyl-CoA). The specific activity of the 3-hydroxypropionate-, CoA-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.023 [micro]mol [min.sup.-1]mg [protein.sup.-1]. The reaction sequence is catalyzed by at least two enzymes. The first enzyme, 3-hydroxypropionylCoA synthetase, catalyzes the following reaction: 3-hydroxypropionate + ATP + CoA [right arrow] 3-hydroxypropionylCoA + AMP + P[P.sub.i]. The enzyme was purified 95-fold to a specific activity of 18 [micro]mol [min.sup.-1] mg [protein.sup.-1] from autotrophically grown M. sedula cells. An internal peptide sequence was determined and a gene encoding a homologous protein identified in the genome of Sulfolobus tokodaii; similar genes were found in S. solfataricus and S. acidocaldarius. The gene was heterologously expressed in Escherichia coli, and the His-tagged protein was purified. Both the native enzyme from M. sedula and the recombinant enzyme from S. tokodaii not only activated 3-hydroxypropionate to its CoA ester but also activated propionate, acrylate, acetate, and butyrate; however, with the exception of propionate, the affinities for these substrates were reduced. 3-Hydroxypropionyl-CoA synthetase is up-regulated eightfold in autotrophically versus heterotrophically grown M. sedula, supporting its proposed role during C[O.sub.2] fixation in this archaeon and possibly other members of the Sulfolobaceae family.

Initiation of translation in eukaryotes and in archaea involves eukaryotic/archaeal initiation factor (e/alF)1 and the heterotrimeric initiation factor e/alF2. In its GTP-bound form, e/alF2 provides the initiation complex with [Met-tRNA.sup.Met.sub.i]. After recognition of the start codon by initiator tRNA, e/alF1 leaves the complex. Finally, e/alF2, now in a GDP-bound form, loses affinity for [Met-tRNA.sup.Met.sub.i] and dissociates from the ribosome. Here, we report a 3D structure of an alF2 heterotrimer from the archeon Sulfolobus solfataricus obtained in the presence of GDP. Our report highlights how the two-switch regions involved in formation of the tRNA-binding site on subunit [gamma] exchange conformational information with ([alpha] and [beta]. The zinc-binding domain of [beta] lies close to the guanine nucleotide and directly contacts the switch 1 region. As a result, switch 1 adopts a not yet described conformation. Moreover, unexpectedly for a GDP-bound state, switch 2 has the 'ON' conformation. The stability of these conformations is accounted for by a ligand, most probably a phosphate ion, bound near the nucleotide binding site. The structure suggests that this GDPinorganic phosphate (Pi) bound state of alF2 may be proficient for tRNA binding. Recently, it has been proposed that dissociation of elF2 from the initiation complex is closely coupled to that of Pi from elF2[gamma] upon start codon recognition. The nucleotide state of alF2 shown here is indicative of a similar mechanism in archaea. Finally, we consider the possibility that release of Pi takes place after e/alF2[gamma] has been informed of e/alF1 dissociation by e/alF2[beta]. e/alF2 | G protein | initiation of translation | tRNA | start codon

Type IV secretion systems are common bacterial macromolecule transporters that have been adapted to various functions, such as effector protein translocation to eukaryotic cells, nucleoprotein transfer to bacterial or eukaryotic cells, and DNA transport into and out of bacterial cells. Helicobacter pylori, the causative agent of bacterial gastritis, peptic ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma, uses the Cag type IV secretion system to inject the CagA protein into host cells, thereby altering gene expression profiles and the host cell cytoskeleton. The molecular mechanism of CagA recognition as a type IV substrate is only poorly understood, but seems to be more complex than that of other type IV secretion systems. Apart from 14 essential components of the secretion apparatus, CagA translocation specifically requires the presence of four additional Cag proteins. Here we show that the CagA-binding protein CagF is a secretion chaperone-like protein that interacts with a 100 aa region that is adjacent to the C-terminal secretion signal of CagA. The interaction between CagA and CagF takes place at the bacterial cytoplasmic membrane, and is independent of a functional type IV secretion apparatus and other cag-encoded factors. Our data indicate that CagF binding precedes recognition of the C-terminal CagA translocation signal, and that both steps are required to recruit CagA to the type IV translocation channel.

Across evolution, type I signal peptidases are responsible for the cleavage of secretory signal peptides from proteins following their translocation across membranes. In Archaea, type I signal peptidases combine domain-specific features with traits found in either their eukaryal or bacterial counterparts. Eukaryal and bacterial type I signal peptidases differ in terms of catalytic mechanism, pharmacological profile, and oligomeric status. In this study, genes encoding Sec11a and Sec11b, two type I signal peptidases of the halophilic archaeon Haloferax volcanii, were cloned. Although both genes are expressed in cells grown in rich medium, gene deletion approaches suggest that Sec11b, but not Sec11a, is essential. For purification purposes, tagged versions of the protein products of both genes were expressed in transformed Haloferax volcanii, with Sec11a and Sec11b being fused to a cellulose-binding domain capable of interaction with cellulose in hypersaline surroundings. By employing an in vitro signal peptidase assay designed for use with high salt concentrations such as those encountered by halophilic archaea such as Haloferax volcanii, the signal peptide-cleaving activities of both isolated membranes and purified Sec11a and Sec11b were addressed. The results show that the two enzymes differentially cleave the assay substrate, raising the possibility that the Sec11a and Sec11b serve distinct physiological functions.

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

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 ability of archaea to salvage cobinamide has been under question because archaeal genomes lack orthologs to the bacterial nucleoside triphosphate:5'-deoxycobinamide kinase enzyme (cobU in Salmonella enterica). The latter activity is required for cobinamide salvaging in bacteria. This paper reports evidence that archaea salvage cobinamide from the environment by using a pathway different from the one used by bacteria. These studies demanded the functional characterization of two genes whose putative function had been annotated based solely on their homology to the bacterial genes encoding adenosylcobyric acid and adenosylcobinamide-phosphate synthases (cbiP and cbiB, respectively) of S. enterica. A cbiP mutant strain of the archaeon Halobacterium sp. strain NRC-1 was auxotrophic for adenosylcobyric acid, a known intermediate of the de novo cobamide biosynthesis pathway, but efficiently salvaged cobinamide from the environment, suggesting the existence of a salvaging pathway in this archaeon. A cbiB mutant strain of Halobacterium was auxotrophic for adenosylcobinamide-GDP, a known de novo intermediate, and did not salvage cobinamide. The results of the nutritional analyses of the cbiP and cbiB mutants suggested that the entry point for cobinamide salvaging is adenosylcobyric acid. The data are consistent with a salvaging pathway for cobinamide in which an amidohydrolase enzyme cleaves off the aminopropanol moiety of adenosylcobinamide to yield adenosylcobyric acid, which is converted by the adenosylcobinamide-phosphate synthase enzyme to adenosylcobinamide-phosphate, a known intermediate of the de novo biosynthetic pathway. The existence of an adenosylcobinamide amidohydrolase enzyme would explain the lack of an adenosylcobinamide kinase in archaea.

Archaea are best known in their capacities as extremophiles, i.e. micro-organisms able to thrive in some of the most drastic environments on Earth. The protein-based surface layer that envelopes many archaeal strains must thus correctly assemble and maintain its structural integrity in the face of the physical challenges associated with, for instance, life in high salinity, at elevated temperatures or in acidic surroundings. Study of archaeal surface-layer (glyco) proteins has thus offered insight into the strategies employed by these proteins to survive direct contact with extreme environments, yet has also served to elucidate other aspects of archaeal protein biosynthesis, including glycosylation, lipid modification and protein export. In this mini-review, recent advances in the study of archaeal surface-layer (glyco)proteins are discussed.

[V.sub.o][V.sub.1]-ATPase is responsible for acidification of eukaryotic intracellular compartments and ATP synthesis of Archaea and some eubacteria. From the similarity to [F.sub.o][F.sub.1]-ATP synthase, [V.sub.o][V.sub.1]-ATPase has been assumed to be a rotary motor, but to date there are no experimental data to support this. Here we visualized the rotation of single molecules of [V.sub.1]-ATPase, a catalytic subcomplex of [V.sub.o][V.sub.1]-ATPase. [V.sub.1]-ATPase from Thermus thermophilus was immobilized onto a glass surface, and a bead was attached to the D or F subunit through the biotin-streptavidin linkage. In both cases we observed ATP-dependent rotations of beads, the direction of which was always counterclockwise viewed from the membrane side. Given that three ATP molecules are hydrolyzed per one revolution, rates of rotation agree consistently with rates of ATP hydrolysis at saturating ATP concentrations. This study provides experimental evidence that [V.sub.o][V.sub.1]-ATPase is a rotary motor and that both D and F subunits constitute a rotor shaft.