High-resolution crystal structure of RNA kinase ArK1 from G. acetivorans.

U47 phosphorylation (Up47) is a novel tRNA modification discovered recently; it can confer thermal stability and nuclease resistance to tRNAs. U47 phosphorylation is catalyzed by Archaeal RNA kinase (Ark1) in an ATP-dependent manner. However, the structural basis for tRNA and/or ATP binding by Ark1 is unclear. Here, we report the expression, purification, and crystallization studies of Ark1 from G. acetivorans (GaArk1). In addition to the Apo-form structure, one GaArk1-ATP complex was also determined in atomic resolution and revealed the detailed basis for ATP binding by GaArk1. The GaArk1-ATP complex represents the only ATP-bound structure of the Ark1 protein. The majority of the ATP-binding residues are conserved, suggesting that GaArk1 and the homologous proteins share similar mechanism in ATP binding. Sequence and structural analysis further indicated that endogenous guanosine will only inhibit the activities of certain Ark1 proteins, such as Ark1 from T. kodakarensis.

Upgrade of wood sugar d-xylose to a value-added nutraceutical by in vitro metabolic engineering.

The upgrade of D-xylose, the most abundant pentose, to value-added biochemicals is economically important to next-generation biorefineries. myo-Inositol, as vitamin B8, has a six-carbon carbon-carbon ring. Here we designed an in vitro artificial NAD(P)-free 12-enzyme pathway that can effectively convert the five-carbon xylose to inositol involving xylose phosphorylation, carbon-carbon (C-C) rearrangement, C-C bond circulation, and dephosphorylation. The reaction conditions catalyzed by all thermostable enzymes from hyperthermophilic microorganisms Thermus thermophiles, Thermotoga maritima, and Archaeoglobus fulgidus were optimized in reaction temperature, buffer type and concentration, enzyme composition, Mg2+ concentration, and fed-batch addition of ATP. The 11-enzyme cocktail, whereas a fructose 1,6-bisphosphatase from T. maritima has another function of inositol monophosphatase, converted 20 mM xylose to 16.1 mM inositol with a conversion efficiency of 96.6% at 70 °C. Polyphosphate was found to replace ATP for xylulose phosphorylation due to broad substrate promiscuity of the T. maritima xylulokinase. The Tris-HCl buffer effectively mitigated the Maillard reaction at 70 °C or higher temperature. The co-production of value-added biochemicals, such as inositol, from wood sugar could greatly improve economics of new biorefineries, similar to oil refineries that make value-added plastic precursors to subsidize gasoline/diesel production.

Glycerol Phosphate Cytidylyltransferase Stereospecificity Is Key to Understanding the Distinct Stereochemical Compositions of Glycerophosphoinositol in Bacteria and Archaea.

Glycerophosphoinositol (GPI) is a compatible solute present in a few hyperthermophiles. Interestingly, different GPI stereoisomers accumulate in Bacteria and Archaea, and the basis for this domain-dependent specificity was investigated herein. The archaeon Archaeoglobus fulgidus and the bacterium Aquifex aeolicus were used as model organisms. The synthesis of GPI involves glycerol phosphate cytidylyltransferase (GCT), which catalyzes the production of CDP-glycerol from CTP and glycerol phosphate, and di-myo-inositol phosphate-phosphate synthase (DIPPS), catalyzing the formation of phosphorylated GPI from CDP-glycerol and l-myo-inositol 1-phosphate. DIPPS of A. fulgidus recognized the two CDP-glycerol stereoisomers similarly. This feature and the ability of 31P nuclear magnetic resonance (NMR) to distinguish the GPI diastereomers provided a means to study the stereospecificity of GCTs. The AF1418 gene and genes aq_185 and aq_1368 are annotated as putative GCT genes in the genomes of A. fulgidus and Aq. aeolicus, respectively. The functions of these genes were determined by assaying the activity of the respective recombinant proteins: AQ1368 and AQ185 are GCTs, while AF1418 has flavin adenine dinucleotide (FAD) synthetase activity. AQ185 is absolutely specific for sn-glycerol 3-phosphate, while AQ1368 recognizes the two enantiomers but has a 2:1 preference for sn-glycerol 3-phosphate. In contrast, the partially purified A. fulgidus GCT uses sn-glycerol 1-phosphate preferentially (4:1). Significantly, the predominant GPI stereoforms found in the bacterium and the archaeon reflect the distinct stereospecificities of the respective GCTs: i.e., A. fulgidus accumulates predominantly sn-glycero-1-phospho-3-l-myo-inositol, while Aq. aeolicus accumulates sn-glycero-3-phospho-3-l-myo-inositol. IMPORTANCE: Compatible solutes of hyperthermophiles show high efficacy in thermal protection of proteins in comparison with solutes typical of mesophiles; therefore, they are potentially useful in several biotechnological applications. Glycerophosphoinositol (GPI) is synthesized from CDP-glycerol and l-myo-inositol 1-phosphate in a few hyperthermophiles. In this study, the molecular configuration of the GPI stereoisomers accumulated by members of the Bacteria and Archaea was established. The stereospecificity of glycerol phosphate cytidylyltransferase (GCT), the enzyme catalyzing the synthesis of CDP-glycerol, is crucial to the stereochemistry of GPI. However, the stereospecific properties of GCTs have not been investigated thus far. We devised a method to characterize GCT stereospecificity which does not require sn-glycerol 1-phosphate, a commercially unavailable substrate. This led us to understand the biochemical basis for the distinct GPI stereoisomer composition observed in archaea and bacteria.

Archaeoglobus sulfaticallidus sp. nov., a thermophilic and facultatively lithoautotrophic sulfate-reducer isolated from black rust exposed to hot ridge flank crustal fluids.

A novel thermophilic and lithoautotrophic sulfate-reducing archaeon was isolated from black rust formed on the steel surface of a borehole observatory (CORK 1026B) retrieved during IODP Expedition 301 on the eastern flank of Juan de Fuca Ridge, eastern Pacific Ocean. Cells of the strain were lobe-shaped or triangular. The optimum temperature, pH and NaCl concentration for growth were 75°C, pH 7 and 2 % (w/v), respectively. The isolate was strictly anaerobic, growing lithoautotrophically on H(2) and CO(2) using sulfate, sulfite or thiosulfate as electron acceptors. Lactate and pyruvate could serve as alternative energy and carbon sources. The G+C content of the genomic DNA was 42 mol%. Phylogenetic analyses of the 16S rRNA gene indicated that the isolate was closely related to members of the family Archaeoglobaceae, with sequence similarities of 90.3-94.4 %. Physiological and molecular properties showed that the isolate represents a novel species of the genus Archaeoglobus. The name Archaeoglobus sulfaticallidus sp. nov. is proposed; the type strain is PM70-1(T) (=DSM 19444(T)=JCM 14716(T)).

Metabolic capability and in situ activity of microorganisms in an oil reservoir.

BACKGROUND: Microorganisms have long been associated with oxic and anoxic degradation of hydrocarbons in oil reservoirs and oil production facilities. While we can readily determine the abundance of microorganisms in the reservoir and study their activity in the laboratory, it has been challenging to resolve what microbes are actively participating in crude oil degradation in situ and to gain insight into what metabolic pathways they deploy. RESULTS: Here, we describe the metabolic potential and in situ activity of microbial communities obtained from the Jiangsu Oil Reservoir (China) by an integrated metagenomics and metatranscriptomics approach. Almost complete genome sequences obtained by differential binning highlight the distinct capability of different community members to degrade hydrocarbons under oxic or anoxic condition. Transcriptomic data delineate active members of the community and give insights that Acinetobacter species completely oxidize alkanes into carbon dioxide with the involvement of oxygen, and Archaeoglobus species mainly ferment alkanes to generate acetate which could be consumed by Methanosaeta species. Furthermore, nutritional requirements based on amino acid and vitamin auxotrophies suggest a complex network of interactions and dependencies among active community members that go beyond classical syntrophic exchanges; this network defines community composition and microbial ecology in oil reservoirs undergoing secondary recovery. CONCLUSION: Our data expand current knowledge of the metabolic potential and role in hydrocarbon metabolism of individual members of thermophilic microbial communities from an oil reservoir. The study also reveals potential metabolic exchanges based on vitamin and amino acid auxotrophies indicating the presence of complex network of interactions between microbial taxa within the community.

Secondary Structure Determination by Means of ATR-FTIR Spectroscopy.

Specialized infrared spectroscopic techniques have been developed that allow studying the secondary structure of membrane proteins and the influence of crucial parameters like lipid content and detergent. Here, we focus on an ATR-FTIR spectroscopic study of Af-Amt1 and the influence of LDAO/glycerol on its structural integrity. Our results clearly indicate that infrared spectroscopy can be used to identify the adapted sample conditions.

Biochemical characterization of a novel hypoxanthine/xanthine dNTP pyrophosphatase from Methanococcus jannaschii.

A novel dNTP pyrophosphatase, Mj0226 from Methanococcus jannaschii, which catalyzes the hydrolysis of nucleoside triphosphates to the monophosphate and PPi, has been characterized. Mj0226 protein catalyzes hydrolysis of two major substrates, dITP and XTP, suggesting that the 6-keto group of hypoxanthine and xanthine is critical for interaction with the protein. Under optimal reaction conditions the k(ca)(t) /K(m) value for these substrates was approximately 10 000 times that with dATP. Neither endonuclease nor 3'-exonuclease activities were detected in this protein. Interestingly, dITP was efficiently inserted opposite a dC residue in a DNA template and four dNTPs were also incorporated opposite a hypoxanthine residue in template DNA by DNA polymerase I. Two protein homologs of Mj0226 from Escherichia coli and Archaeoglobus fulgidus were also cloned and purified. These have catalytic activities similar to Mj0226 protein under optimal conditions. The implications of these results have significance in understanding how homologous proteins, including Mj0226, act biologically in many organisms. It seems likely that Mj0226 and its homologs have a major role in preventing mutations caused by incorporation of dITP and XTP formed spontaneously in the nucleotide pool into DNA. This report is the first identification and functional characterization of an enzyme hydrolyzing non-canonical nucleotides, dITP and XTP.

Optimization of Expression and Purification of Recombinant Archeoglobus fulgidus F420H2:NADP+ Oxidoreductase, an F420 Cofactor Dependent Enzyme.

Methanogens play a critical role in carbon cycling and contain a number of intriguing biosynthetic pathways. One unusual cofactor found in methanogenic and sulfate reducing archaea is Factor 420 (F420), which can be interconverted between its reduced and oxidized forms by the F420H2:NADP(+) oxidoreductase (Fno) through hydride transfer mechanisms. Here, we report an optimized expression and purification method for recombinant Fno derived from the extreme thermophile Archeoglobus fulgidus. An expression vector that is codon-optimized for heterologous expression in Escherichia coli, modified growth conditions, and a modified purification protocol involving a key polyethyleneimine precipitation step results in a highly purified, homogeneous preparation of Fno that displays high catalytic activity with a truncated F420 analog. This method should accelerate studies on how Fno uses the unusual F420 cofactor during catalysis.

Dual coenzyme specificity of Archaeoglobus fulgidus HMG-CoA reductase.

Comparison of the inferred amino acid sequence of orf AF1736 of Archaeoglobus fulgidus to that of Pseudomonas mevalonii HMG-CoA reductase suggested that AF1736 might encode a Class II HMG-CoA reductase. Following polymerase chain reaction-based cloning of AF1736 from A. fulgidus genomic DNA and expression in Escherichia coli, the encoded enzyme was purified to apparent homogeneity and its enzymic properties were determined. Activity was optimal at 85 degrees C, deltaHa was 54 kJ/mol, and the statin drug mevinolin inhibited competitively with HMG-CoA (Ki 180 microM). Protonated forms of His390 and Lys277, the apparent cognates of the active site histidine and lysine of the P. mevalonii enzyme, appear essential for activity. The mechanism proposed for catalysis of P. mevalonii HMG-CoA reductase thus appears valid for A. fulgidus HMG-CoA reductase. Unlike any other HMG-CoA reductase, the A. fulgidus enzyme exhibits dual coenzyme specificity. pH-activity profiles for all four reactions revealed that optimal activity using NADP(H) occurred at a pH from 1 to 3 units more acidic than that observed using NAD(H). Kinetic parameters were therefore determined for all substrates for all four catalyzed reactions using either NAD(H) or NADP(H). NADPH and NADH compete for occupancy of a common site. k(cat)[NAD(H)]/k(cat)[NADP(H)] varied from unity to under 70 for the four reactions, indicative of slight preference for NAD(H). The results indicate the importance of the protonated status of active site residues His390 and Lys277, shown by altered K(M) and k(cat) values, and indicate that NAD(H) and NADP(H) have comparable affinity for the same site.

Comparing function and structure between entire proteomes.

More than 30 organisms have been sequenced entirely. Here, we applied a variety of simple bioinformatics tools to analyze 29 proteomes for representatives from all three kingdoms: eukaryotes, prokaryotes, and archaebacteria. We confirmed that eukaryotes have relatively more long proteins than prokaryotes and archaes, and that the overall amino acid composition is similar among the three. We predicted that approximately 15%-30% of all proteins contained transmembrane helices. We could not find a correlation between the content of membrane proteins and the complexity of the organism. In particular, we did not find significantly higher percentages of helical membrane proteins in eukaryotes than in prokaryotes or archae. However, we found more proteins with seven transmembrane helices in eukaryotes and more with six and 12 transmembrane helices in prokaryotes. We found twice as many coiled-coil proteins in eukaryotes (10%) as in prokaryotes and archaes (4%-5%), and we predicted approximately 15%-25% of all proteins to be secreted by most eukaryotes and prokaryotes. Every tenth protein had no known homolog in current databases, and 30%-40% of the proteins fell into structural families with >100 members. A classification by cellular function verified that eukaryotes have a higher proportion of proteins for communication with the environment. Finally, we found at least one homolog of experimentally known structure for approximately 20%-45% of all proteins; the regions with structural homology covered 20%-30% of all residues. These numbers may or may not suggest that there are 1200-2600 folds in the universe of protein structures. All predictions are available at http://cubic.bioc.columbia.edu/genomes.

The ribosomal gene spacer region in archaebacteria.

Sequences for the spacer regions that separate the 16S and 23S ribosomal RNA genes have been determined for four more (strategically placed) archaebacteria. These confirm the general rule that methanogens and extreme halophiles have spacers that contain a single tRNAala gene, while tRNA genes are not found in the spacer region of the true extreme thermophiles. The present study also shows that the spacer regions from the sulfate reducing Archaeglobus and the extreme thermophile Thermococcus (both of which cluster phylogenetically with the methanogens and extreme halophiles) contain each a tRNAala gene. Thus, not only all methanogens and extreme halophiles show this characteristic, but all organisms on the "methanogen branch" of the archaebacterial tree appear to do so. The finding of a tRNA gene in the spacer region of the extreme thermophile Thermococcus celer is the first known phenotypic property that links this organism with its phylogenetic counterparts, the methanogens, rather than with its phenotypic counterparts, the sulfur-dependent extreme thermophiles.

A deoxyinosine specific endonuclease from hyperthermophile, Archaeoglobus fulgidus: a homolog of Escherichia coli endonuclease V.

Deoxyadenosine undergoes spontaneous deamination to deoxyinosine in DNA. Based on amino acids sequence homology, putative homologs of endonuclease V were identified in several organisms including archaebacteria, eubacteria as well as eukaryotes. The translated amino acid sequence of the Archaeoglobus fulgidus nfi gene shows 39% identity and 55% similarity to the E. coli nfi gene. A. fulgidus endonuclease V was cloned and expressed in E. coli as a C-terminal hexa-histidine fusion protein. The C-terminal fusion protein was purified to apparent homogeneity by a combination of Ni(++) affinity and MonoS cation exchange liquid chromatography. The purified C-terminal fusion protein has a molecular weight of about 25kDa and showed endonuclease activity towards DNA containing deoxyinosine. A. fulgidus endonuclease V has an absolute requirement for Mg(2+) and an optimum reaction temperature at 85 degrees C. However, in contrast to E. coli endonuclease V, which has a wide substrate spectrum, endonuclease V from A. fulgidus recognized only deoxyinosine. These data suggest that the deoxyinosine cleavage activity is a primordial activity of endonuclease V and that multiple enzymatic activities of E. coli endonuclease V were acquired later during evolution.

Measurement of word frequencies in genomic DNA sequences based on partial alignment and fuzzy set.

Accompanied with the rapid increase of the amount of data registered in the databases of biological sequences, the need for a fast method of sequence comparison applicable to sequences of large size is also increasing. In general, alignment is used for sequence comparison. However, the alignment may not be appropriate for comparison of sequences of large size such as whole genome sequences due to its large time complexity. In this article, we propose a semi alignment-free method of sequence comparison based on word frequency distributions, in which we partially use the alignment to measure word frequencies along with the idea of fuzzy set theory. Experiments with ten bacterial genome sequences demonstrated that the fuzzy measurements has the effect that facilitates discrimination between close relatives and distant relatives.

Carbon dioxide fixation in 'Archaeoglobus lithotrophicus': are there multiple autotrophic pathways?

Several representatives of the euryarchaeal class Archaeoglobi are able to grow facultative autotrophically using the reductive acetyl-CoA pathway, with 'Archaeoglobus lithotrophicus' being an obligate autotroph. However, genome sequencing revealed that some species harbor genes for key enzymes of other autotrophic pathways, i.e. 4-hydroxybutyryl-CoA dehydratase of the dicarboxylate/hydroxybutyrate cycle and the hydroxypropionate/hydroxybutyrate cycle and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the Calvin-Benson cycle. This raised the question of whether only one or multiple autotrophic pathways are operating in these species. We searched for the presence of enzyme activities specific for the dicarboxylate/hydroxybutyrate or the hydroxypropionate/hydroxybutyrate cycles in 'A. lithotrophicus', but such enzymes could not be detected. Low Rubisco activity was detected that could not account for the carbon dioxide (CO(2)) fixation rate; in addition, phosphoribulokinase activity was not found. The generation of ribulose 1,5-bisphosphate from 5-phospho-D-ribose 1-pyrophosphate was observed, but not from AMP; these sources for ribulose 1,5-bisphosphate have been proposed before. Our data indicate that the reductive acetyl-CoA pathway is the only functioning CO(2) fixation pathway in 'A. lithotrophicus'.

Archaeoglobus infectus sp. nov., a novel thermophilic, chemolithoheterotrophic archaeon isolated from a deep-sea rock collected at Suiyo Seamount, Izu-Bonin Arc, western Pacific Ocean.

A novel thermophilic, strictly anaerobic archaeon, designated strain Arc51T, was isolated from a rock sample collected from a deep-sea hydrothermal field in Suiyo Seamount, Izu-Bonin Arc, western Pacific Ocean. Cells of the isolate were irregular cocci with single flagella and exhibited blue-green fluorescence at 436 nm. The optimum temperature, pH and NaCl concentration for growth were 70 degrees C, pH 6.5 and 3 % (w/v), respectively. Strain Arc51T could grow on thiosulfate or sulfite as an electron acceptor in the presence of hydrogen. This strain required acetate as a carbon source for its growth, suggesting that the reductive acetyl CoA pathway for CO2 fixation was incomplete. In addition, coenzyme M (2-mercaptoethanesulfonic acid), which is a known methyl carrier in methanogenesis, was also a requirement for growth of the strain. Analysis of the 16S rRNA gene sequence revealed that the isolate was similar to members of the genus Archaeoglobus, with sequence similarities of 93.6-97.2 %; the closest relative was Archaeoglobus veneficus. Phylogenetic analyses of the dsrAB and apsA genes, encoding the alpha and beta subunits of dissimilatory sulfite reductase and the alpha subunit of adenosine-5'-phosphosulfate reductase, respectively, produced results similar to those inferred from comparisons based on the 16S rRNA gene sequence. On the basis of phenotypic and phylogenetic data, strain Arc51T represents a novel species of the genus Archaeoglobus, for which the name Archaeoglobus infectus sp. nov. is proposed. The type strain is Arc51T (=NBRC 100649T=DSM 18877T).

Effect of nitrate and nitrite on sulfide production by two thermophilic, sulfate-reducing enrichments from an oil field in the North Sea.

Thermophilic sulfate-reducing bacteria (tSRB) can be major contributors to the production of H(2)S (souring) in oil reservoirs. Two tSRB enrichments from a North Sea oil field, NS-tSRB1 and NS-tSRB2, were obtained at 58 degrees C with acetate-propionate-butyrate and with lactate as the electron donor, respectively. Analysis by rDNA sequencing indicated the presence of Thermodesulforhabdus norvegicus in NS-tSRB1 and of Archaeoglobus fulgidus in NS-tSRB2. Nitrate (10 mM) had no effect on H(2)S production by mid-log phase cultures of NS-tSRB1 and NS-tSRB2, whereas nitrite (0.25 mM or higher) inhibited sulfate reduction. NS-tSRB1 did not recover from inhibition, whereas sulfate reduction activity of NS-tSRB2 recovered after 500 h. Nitrite was also effective in souring inhibition and H(2)S removal in upflow bioreactors, whereas nitrate was similarly ineffective. Hence, nitrite may be preferable for souring prevention in some high-temperature oil fields because it reacts directly with sulfide and provides long-lasting inhibition of sulfate reduction.

Enzyme-driven speciation: crystallizing Archaea via lipid capture.

As the origin(s) of life on Earth remains an open question, detailed characteristics about the "last universal ancestor" (LUA) continue to be obscured. Here we provide arguments that strengthen the bacterial-like nature of the LUA. Our view attempts to recreate the evolution of archaeal lipids, the major components of the distinctive membrane that encapsulates these ancient prokaryotes. We show that (S)- 3-O-geranylgeranylglyceryl phosphate synthase (GGGPS), a TIM-barrel protein that performs the committed step in archaeal lipid synthesis, likely evolved from the duplication and fusion of a (betaalpha)4 half-barrel ancestor. By comparison to the well-characterized HisA and HisF TIM-barrel proteins, we propose a time line for the invention of this diagnostic archaeal biosynthetic pathway. After excluding the possibility of horizontal gene transfer, we conclude that the evolutionary history of GGGPS mirrors the emergence of Archaea from the LUA. We illustrate aspects of this "lipid capture" model that support its likelihood in recreating key evolutionary events and, as our hypothesis is built on a single initiating event, we suggest that the appearance of GGGPS represents an example of enzyme-driven speciation.

Characterization of AMA, a new AAA protein from Archaeoglobus and methanogenic archaea.

We have previously reported a new group of AAA proteins, which is only found in Archaeoglobus and methanogenic archaea (AMA). The proteins are phylogenetically basal to the metalloprotease clade and their N-terminal domain is homologous to the beta-clam part of the N-domain of CDC48-like proteins. Here we report the biochemical and biophysical characterization of Archaeoglobus fulgidus AMA, and of its isolated N-terminal (AMA-N) and ATPase (AMA-DeltaN) domains. AfAMA forms hexameric complexes, as does AMA-N, while AMA-DeltaN only forms dimers. The ability to hexamerize is dependent on the integrity of a GYPL motif in AMA-N, which resembles the pore motif of FtsH and HslU. While the physiological function of AMA is unknown, we show that it has ATP-dependent chaperone activity and can prevent the thermal aggregation of proteins in vitro. The ability to interact with non-native proteins resides in the N-domain and is energy-independent.

Effects of dissolved sulfide, pH, and temperature on growth and survival of marine hyperthermophilic Archaea.

The ability of metabolically diverse hyperthermophilic archaea to withstand high temperatures, low pHs, high sulfide concentrations, and the absence of carbon and energy sources was investigated. Close relatives of our study organisms, Methanocaldococcus jannaschii, Archaeoglobus profundus, Thermococcus fumicolans, and Pyrococcus sp. strain GB-D, are commonly found in hydrothermal vent chimney walls and hot sediments and possibly deeper in the subsurface, where highly dynamic hydrothermal flow patterns and steep chemical and temperature gradients provide an ever-changing mosaic of microhabitats. These organisms (with the possible exception of Pyrococcus strain GB-D) tolerated greater extremes of low pH, high sulfide concentration, and high temperature when actively growing and metabolizing than when starved of carbon sources and electron donors/acceptors. Therefore these organisms must be actively metabolizing in the hydrothermal vent chimneys, sediments, and subsurface in order to withstand at least 24 h of exposure to extremes of pH, sulfide, and temperature that occur in these environments.