bluuebunny/crossref_metadata_2025 huggingface dataset:
Reverse micelles in organic solvents: a medium for the biotechnological use of extreme halophilic enzymes at low salt concentration
Authors: Marhuenda-Egea Frutos C. , Piera-Velázquez Sonsoles , Cadenas Chiquinquirá , Cadenas Eduardo
Published: September 2009

Alkaline p‐nitrophenylphosphate phosphatase (pNPPase) from the halophilic archaeobacterium Halobacterium salinarum (previously halobium) was solubilized at low salt concentration in reverse micelles of hexadecyltrimethylammoniumbromide in cyclohexane with 1‐butanol as cosurfactant. The enzyme maintained its catalytic properties under these conditions. The thermodynamic “solvation–stabilization hypothesis” has been used to explain the bell‐shaped dependence of pNPPase activity on the water content of reverse micelles, in terms of protein–solvent interactions. According to this model, the stability of the folded protein depends on a network of hydrated ions associated with acidic residues at the protein surface. At low salt concentration and low water content (the ratio of water concentration to surfactant concentration; w0), the network of hydrated ions within the reverse micelles may involve the cationic heads of the surfactant. The bell‐shaped profile of the relationship between enzyme activity and w0 varied depending on the concentrations of NaCl and Mn2+.
Structural analysis by reductive cleavage with LiAlH4 of an allyl ether choline‐phospholipid, archaetidylcholine, from the hyperthermophilic methanoarchaeon Methanopyrus kandleri
Authors: Nishihara Masateru , Morii Hiroyuki , Matsuno Koji , Ohga Mami , Stetter Karl O. , Koga Yosuke
Published: September 2009

A choline‐containing phospholipid (PL‐4) in Methanopyrus kandleri cells was identified as archaetidylcholine, which has been described by Sprott et al. (1997). The PL‐4 consisted of a variety of molecular species differing in hydrocarbon composition. Most of the PL‐4 was acid‐labile because of its allyl ether bond. The identity of PL‐4 was confirmed by thin‐layer chromatography (TLC) followed by positive staining with Dragendorff‐reagent and fast‐atom bombardment–mass spectrometry. A new method of LiAlH4 hydrogenolysis was developed to cleave allyl ether bonds and recover the corresponding hydrocarbons. We confirmed the validity of the LiAlH4 method in a study of the model compound synthetic unsaturated archaetidic acid (2,3‐di‐O‐geranylgeranyl‐sn‐glycerol‐1‐phosphate). Saturated ether bonds were not cleaved by the LiAlH4 method. The hydrocarbons formed following LiAlH4 hydrogenolysis of PL‐4 were identified by gas–liquid chromatography and mass spectrometry. Four kinds of hydrocarbons with one to four double bonds were detected: 47% of the hydrocarbons had four double bonds; 11% had three double bonds; 14% had two double bonds; 7% had one double bond; and 6% were saturated species. The molecular species composition of PL‐4 was also estimated based on acid lability: 77% of the molecular species had two acid‐labile hydrocarbons; 11% had one acid‐labile and one acid‐stable hydrocarbon; and 11% had two acid‐stable hydrocarbons. To our knowledge, this is the first report of a specific chemical degradation method for the structural analysis of allyl ether phospholipid in archaea.
Distribution and phylogenies of enzymes of the Embden‐Meyerhof‐Parnas pathway from archaea and hyperthermophilic bacteria support a gluconeogenic origin of metabolism
Authors: Ronimus Ron S. , Morgan Hugh W.
Published: September 2009

Enzymes of the gluconeogenic/glycolytic pathway (the Embden‐Meyerhof‐Parnas (EMP) pathway), the reductive tricarboxylic acid cycle, the reductive pentose phosphate cycle and the Entner‐Doudoroff pathway are widely distributed and are often considered to be central to the origins of metabolism. In particular, several enzymes of the lower portion of the EMP pathway (the so‐called trunk pathway), including triosephosphate isomerase (TPI; EC 5.3.1.1), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH; EC 1.2.1.12/13), phosphoglycerate kinase (PGK; EC 2.7.2.3) and enolase (EC 4.2.1.11), are extremely well conserved and universally distributed among the three domains of life. In this paper, the distribution of enzymes of gluconeogenesis/glycolysis in hyperthermophiles—microorganisms that many believe represent the least evolved organisms on the planet—is reviewed. In addition, the phylogenies of the trunk pathway enzymes (TPIs, GAPDHs, PGKs and enolases) are examined. The enzymes catalyzing each of the six‐carbon transformations in the upper portion of the EMP pathway, with the possible exception of aldolase, are all derived from multiple gene sequence families. In contrast, single sequence families can account for the archaeal and hyperthermophilic bacterial enzyme activities of the lower portion of the EMP pathway. The universal distribution of the trunk pathway enzymes, in combination with their phylogenies, supports the notion that the EMP pathway evolved in the direction of gluconeogenesis, i.e., from the bottom up.
Novel archaeal macrocyclic diether core membrane lipids in a methane‐derived carbonate crust from a mud volcano in the Sorokin Trough, NE Black Sea
Authors: Stadnitskaia Alina , Baas Marianne , Ivanov Michael K. , Van Weering Tjeerd C. E. , Sinninghe Damsté Jaap S.
Published: September 2009

A methane‐derived carbonate crust was collected from the recently discovered NIOZ mud volcano in the Sorokin Trough, NE Black Sea during the 11th Training‐through‐Research cruise of the R/V Professor Logachev. Among several specific bacterial and archaeal membrane lipids present in this crust, two novel macrocyclic diphytanyl glycerol diethers, containing one or two cyclopentane rings, were detected. Their structures were tentatively identified based on the interpretation of mass spectra, comparison with previously reported mass spectral data, and a hydrogenation experiment. This macrocyclic type of archaeal core membrane diether lipid has so far been identified only in the deep‐sea hydrothermal vent methanogen Methanococcus jannaschii. Here, we provide the first evidence that these macrocyclic diethers can also contain internal cyclopentane rings. The molecular structure of the novel diethers resembles that of dibiphytanyl tetraethers in which biphytane chains, containing one and two pentacyclic rings, also occur. Such tetraethers were abundant in the crust. Compound‐specific isotope measurements revealed δ13C values of –104 to –111‰ for these new archaeal lipids, indicating that they are derived from methanotrophic archaea acting within anaerobic methane‐oxidizing consortia, which subsequently induce authigenic carbonate formation.
Remarkable sequence signatures in archaeal genomes
Authors: Fadiel Ahmed , Lithwick Stuart , Ganji Gopi , Scherer Stephen W.
Published: September 2009

Complete archaeal genomes were probed for the presence of long (≥ 25 bp) oligonucleotide repeats (words). We detected the presence of many words distributed in tandem with narrow ranges of periodicity (i.e., spacer length between repeats). Similar words were not identified in genomes of non‐archaeal species, namely Escherichia coli, Bacillus subtilis, Haemophilus influenzae, Mycoplasma genitalium and Mycoplasma pneumoniae. BLAST similarity searches against the GenBank nucleotide sequence database revealed that these words were archaeal species‐specific, indicating that they are of a signature character. Sequence analysis and genome viewing tools showed these repeats to be restricted to non‐coding regions. Thus, archaea appear to possess a non‐coding genomic signature that is absent in bacterial species. The identification of a species‐specific genomic signature would be of great value to archaeal genome mapping, evolutionary studies and analyses of genome complexity.
Archaeosomes varying in lipid composition differ in receptor‐mediated endocytosis and differentially adjuvant immune responses to entrapped antigen
Authors: Sprott G. Dennis , Sad Subash , Fleming L. Perry , DiCaire Chantal J. , Patel Girishchandra B. , Krishnan Lakshmi
Published: September 2009

Archaeosomes prepared from total polar lipids extracted from six archaeal species with divergent lipid compositions had the capacity to deliver antigen for presentation via both MHC class I and class II pathways. Lipid extracts from Halobacterium halobium and from Halococcus morrhuae strains 14039 and 16008 contained archaetidylglycerol methylphosphate and sulfated glycolipids rich in mannose residues, and lacked archaetidylserine, whereas the opposite was found in Methanobrevibacter smithii, Methanosarcina mazei and Methanococcus jannaschii. Annexin V labeling revealed a surface orientation of phosphoserine head groups in M. smithii, M. mazei and M. jannaschii archaeosomes. Uptake of rhodamine‐labeled M. smithii or M. jannaschii archaeosomes by murine peritoneal macrophages was inhibited by unlabeled liposomes containing phosphatidylserine, by the sulfhydryl inhibitor N‐ethylmaleimide, and by ATP depletion using azide plus fluoride, but not by H. halobium archaeosomes. In contrast, N‐ethylmaleimide failed to inhibit uptake of the four other rhodamine‐labeled archaeosome types, and azide plus fluoride did not inhibit uptake of H. halobium or H. morrhuae archaeosomes. These results suggest endocytosis of archaeosomes rich in surface‐exposed phosphoserine head groups via a phosphatidylserine receptor, and energy‐independent surface adsorption of certain other archaeosome composition classes. Lipid composition affected not only the endocytic mechanism, but also served to differentially modulate the activation of dendritic cells. The induction of IL‐12 secretion from dendritic cells exposed to H. morrhuae 14039 archaeosomes was striking compared with cells exposed to archaeosomes from 16008. Thus, archaeosome types uniquely modulate antigen delivery and dendritic cell activation.
Genomic comparison of archaeal conjugative plasmids from Sulfolobus
Authors: Greve Bo , Jensen Susanne , Brügger Kim , Zillig Wolfram , Garrett Roger A.
Published: September 2009

All of the known self‐transmissable plasmids of the Archaea have been found in the genus Sulfolobus. To gain more insight into archaeal conjugative processes, four newly isolated self‐transmissable plasmids, pKEF9, pHVE14, pARN3 and pARN4, were sequenced and subjected to a comparative sequence analysis with two earlier sequenced plasmids, pNOB8 and pING1. The analyses revealed three conserved and functionally distinct sections in the genomes. Section A is considered to encode the main components of the conjugative apparatus, where two genes show low but significant sequence similarity to sections of genes encoding bacterial conjugative proteins. A putative origin of replication is located in section B, which is highly conserved in sequence and contains several perfect and imperfect direct and inverted repeats. Further downstream, in section C, an operon encoding six to nine smaller proteins is implicated in the initiation and regulation of replication. Each plasmid carries an integrase gene of the type that does not partition on integration, and there is strong evidence for their integration into host chromosomes, where they may facilitate intercellular exchange of chromosomal genes. Two plasmids contain hexameric short regularly spaced repeats (SRSR), which have been implicated in plasmid maintenance, and each plasmid carries multiple recombination motifs, concentrated in the variable regions, which likely provide sites for genomic rearrangements.
Description of Thermococcus kodakaraensis sp. nov., a well studied hyperthermophilic archaeon previously reported as Pyrococcus sp. KOD1
Authors: Atomi Haruyuki , Fukui Toshiaki , Kanai Tamotsu , Morikawa Masaaki , Imanaka Tadayuki
Published: September 2009

A hyperthermophilic archaeal strain, KOD1, isolated from a solfatara on Kodakara Island, Japan, has previously been reported as Pyrococcus sp. KOD1. However, a detailed phylogenetic tree, made possible by the recent accumulation of 16S rRNA sequences of various species in the order Thermococcales, indicated that strain KOD1 is a member of the genus Thermococcus. We performed DNA–DNA hybridization tests against species that displayed high similarity in terms of 16S ribosomal DNA sequences, including Thermococcus peptonophilus and Thermococcus stetteri. Hybridization results and differences in growth characteristics and substrate utilization differentiated strain KOD1 from T. peptonophilus and T. stetteri at the species level. Our results indicate that strain KOD1 represents a new species of Thermococcus, which we designate as Thermococcus kodakaraensis KOD1 sp. nov.
A proposal to rename the hyperthermophile Pyrococcus woesei as Pyrococcus furiosus subsp. woesei
Authors: Kanoksilapatham Wirojne , González Juan M. , Maeder Dennis L. , DiRuggiero Jocelyne , Robb Frank T.
Published: September 2009

Pyrococcus species are hyperthermophilic members of the order Thermococcales, with optimal growth temperatures approaching 100 °C. All species grow heterotrophically and produce H2 or, in the presence of elemental sulfur (S°), H2S. Pyrococcus woesei and P. furiosus were isolated from marine sediments at the same Vulcano Island beach site and share many morphological and physiological characteristics. We report here that the rDNA operons of these strains have identical sequences, including their intergenic spacer regions and part of the 23S rRNA. Both species grow rapidly and produce H2 in the presence of 0.1% maltose and 10–100 µM sodium tungstate in S°‐free medium. However,P. woesei shows more extensive autolysis than P. furiosus in the stationary phase. Pyrococcus
furiosus and P. woesei share three closely related families of insertion sequences (ISs). A Southern blot performed with IS probes showed extensive colinearity between the genomes of P. woesei and P. furiosus. Cloning and sequencing of ISs that were in different contexts in P. woesei and P. furiosus revealed that the napA gene in P. woesei is disrupted by a type III IS element, whereas in P. furiosus, this gene is intact. A type I IS element, closely linked to the napA gene, was observed in the same context in both P. furiosus and P. woesei genomes. Our results suggest that the IS elements are implicated in genomic rearrangements and reshuffling in these closely related strains. We propose to rename P. woesei a subspecies of P. furiosus based on their identical rDNA operon sequences, many common IS elements that are shared genomic markers, and the observation that all P. woesei nucleotide sequences deposited in GenBank to date are > 99% identical to P. furiosus sequences.
Transaldolase of Methanocaldococcus jannaschii
Authors: Soderberg Tim , Alver Robert C.
Published: September 2009

The Methanocaldococcus jannaschii genome contains putative genes for all four nonoxidative pentose phosphate pathway enzymes. Open reading frame (ORF) MJ0960 is a member of the mipB/talC family of ‘transaldolase‐like’ genes, so named because of their similarity to the well‐characterized transaldolase B gene family. However, recently, it has been reported that both the mipB and the talC genes from Escherichia coli encode novel enzymes with fructose‐6‐phosphate aldolase activity, not transaldolase activity (Schürmann and Sprenger 2001). The same study reports that other members of the mipB/talC family appear to encode transaldolases. To confirm the function of MJ0960 and to clarify the presence of a nonoxidative pentose phosphate pathway in M. jannaschii, we have cloned ORF MJ0960 from M. jannaschii genomic DNA and purified the recombinant protein. MJ0960 encodes a transaldolase and displays no fructose‐6‐phosphate aldolase activity. It retained full activity for 4 h at 80 °C, and for 3 weeks at 25 °C. Methanocaldococcus jannaschii transaldolase has a maximal velocity (V
max) of 1.0 ± 0.2 µmol min–1 mg–1 at 25 °C, whereas V
max = 12.0 ± 0.5 µmol min–1 mg–1 at 50 °C. Apparent Michaelis constants at 50 °C were K
m = 0.65 ± 0.09 mM for fructose‐6‐phosphate and K
m = 27.8 ± 4.3 µM for erythrose‐4‐phosphate. When ribose‐5‐phosphate replaced erythrose‐4‐phosphate as an aldose acceptor, V
max decreased twofold, whereas the K
m was 150‐fold higher. The molecular mass of the active enzyme is 271 ± 27 kDa as estimated by gel filtration, whereas the predicted monomer size is 23.96 kDa, suggesting that the native form of the protein is probably a decamer. A readily available source of thermophilic pentose phosphate pathway enzymes including transaldolase may have direct application in enzymatic biohydrogen production.
Interactions between RNase P protein subunits in Archaea
Authors: Hall Thomas A. , Brown James W.
Published: September 2009

A yeast two‐hybrid system was used to identify protein–protein interactions between the ribonuclease P (RNase P) protein subunits Mth11p, Mth687p, Mth688p and Mth1618p from the archaeon Methanothermobacter thermoautotrophicus. Clear interactions between Mth688p and Mth687p, and between Mth1618p and Mth11p, were confirmed by HIS3 and LacZ reporter expression. Weaker interactions of Mth687p and Mth688p with Mth11p, and Mth11p with itself, are also suggested. These interactions resemble, and confirm, those previously seen among the homologs of these proteins in the more complex yeast RNase P holoenzyme.
In vitro selection of an archaeal RNase P RNA mimics natural variation
Authors: Williams Daniel , Brown James W.
Published: September 2009

Archaeal and bacterial RNase P RNAs are similar in sequence and secondary structure, but in the absence of protein, the archaeal RNAs are much less active and require extreme ionic conditions for activity. To assess how readily the activity of the archaeal RNA alone could be improved by small changes in sequence, in vitro selection was used to generate variants of a Methanobacterium formicicum RNase P RNA: Bacillus subtilus pre‐tRNAAsp self‐cleaving conjugate RNA. Functional variants were generated with a spectrum of mutations that were predominately consistent with natural variation in this RNA. Variants generated from the selection had cleavage rates comparable to that of wild type; variants with improved cleavage rates or lower ionic requirements were not obtained. This suggests that the RNase P RNAs of Bacteria and Archaea are globally optimized and the basis for the large biochemical differences between these two types of RNase P RNA is distributed in the molecule.
Novel RepA‐MCM proteins encoded in plasmids pTAU4, pORA1 and pTIK4 from Sulfolobus neozealandicus
Authors: Greve Bo , Jensen Susanne , Phan Hoa , Brügger Kim , Zillig Wolfram , She Qunxin , Garrett Roger A.
Published: September 2009

Three plasmids isolated from the crenarchaeal thermoacidophile Sulfolobus neozealandicus were characterized. Plasmids pTAU4 (7,192 bp), pORA1 (9,689 bp) and pTIK4 (13,638 bp) show unusual properties that distinguish them from previously characterized cryptic plasmids of the genus Sulfolobus. Plasmids pORA1 and pTIK4 encode RepA proteins, only the former of which carries the novel polymerase–primase domain of other known Sulfolobus plasmids. Plasmid pTAU4 encodes a mini‐chromosome maintenance protein homolog and no RepA protein; the implications for DNA replication are considered. Plasmid pORA1 is the first Sulfolobus plasmid to be characterized that does not encode the otherwise highly conserved DNA‐binding PlrA protein. Another encoded protein appears to be specific for the New Zealand plasmids. The three plasmids should provide useful model systems for functional studies of these important crenarchaeal proteins.
Characterization of an archaeal malic enzyme from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1
Authors: Fukuda Wakao , Sari Ismail Yulia , Fukui Toshiaki , Atomi Haruyuki , Imanaka Tadayuki
Published: September 2009

Although the interconversion between C4 and C3 compounds has an important role in overall metabolism, limited information is available on the properties and regulation of enzymes acting on these metabolites in hyperthermophilic archaea. Malic enzyme is one of the enzymes involved in this interconversion, catalyzing the oxidative decarboxylation of malate to pyruvate as well as the reductive carboxylation coupled with NAD(P)H. This study focused on the enzymatic properties and expression profile of an uncharacterized homolog of malic enzyme identified in the genome of a heterotrophic, hyperthermophilic archaeon T hermococcus kodakaraensis KOD1 (Tk‐Mae). The amino acid sequence of Tk‐Mae was 52–58% identical to those of malic enzymes from bacteria, whereas the similarities to the eukaryotic homologs were lower. Several catalytically important regions and residues were conserved in the primary structure of Tk‐Mae. The recombinant protein, which formed a homodimer, exhibited thermostable malic enzyme activity with strict divalent cation dependency. The enzyme preferred NADP+ rather than NAD+, but did not catalyze the decarboxylation of oxaloacetate, unlike the usual NADP‐dependent malic enzymes. The apparent Michaelis constant (Km) of Tk‐Mae for malate (16.9 mM) was much larger than those of known enzymes, leading to no strong preference for the reaction direction. Transcription of the gene encoding Tk‐Mae and intracellular malic enzyme activity in T. kodakaraensis were constitutively weak, regardless of the growth substrates. Possible roles of Tk‐Mae are discussed based on these results and the metabolic pathways of T. kodakaraensis deduced from the genome sequence.
Biosynthesis of ribose‐5‐phosphate and erythrose‐4‐phosphate in archaea: a phylogenetic analysis of archaeal genomes
Authors: Soderberg Tim
Published: September 2009

A phylogenetic analysis of the genes encoding enzymes in the pentose phosphate pathway (PPP), the ribulose monophosphate (RuMP) pathway, and the chorismate pathway of aromatic amino acid biosynthesis, employing data from 13 complete archaeal genomes, provides a potential explanation for the enigmatic phylogenetic patterns of the PPP genes in archaea. Genomic and biochemical evidence suggests that three archaeal species (Methanocaldococcus jannaschii, Thermoplasma acidophilum and Thermoplasma volcanium) produce ribose‐5‐phosphate via the nonoxidative PPP (NOPPP), whereas nine species apparently lack an NOPPP but may employ a reverse RuMP pathway for pentose synthesis. One species (Halobacterium sp. NRC‐1) lacks both the NOPPP and the RuMP pathway but may possess a modified oxidative PPP (OPPP), the details of which are not yet known. The presence of transketolase in several archaeal species that are missing the other two NOPPP genes can be explained by the existence of differing requirements for erythrose‐4‐phosphate (E4P) among archaea: six species use transketolase to make E4P as a precursor to aromatic amino acids, six species apparently have an alternate biosynthetic pathway and may not require the ability to make E4P, and one species (Pyrococcus horikoshii) probably does not synthesize aromatic amino acids at all.
Recombinant production of Zymomonas mobilis pyruvate decarboxylase in the haloarchaeon Haloferax volcanii
Authors: Kaczowka Steven J. , Reuter Christopher J. , Talarico Lee A. , Maupin-Furlow Julie A.
Published: September 2009

The unusual physiological properties of archaea (e.g., growth in extreme salt concentration, temperature and pH) make them ideal platforms for metabolic engineering. Towards the ultimate goal of modifying an archaeon to produce bioethanol or other useful products, the pyruvate decarboxylase gene of Zymomonas mobilis (Zm pdc) was expressed in Haloferax volcanii. This gene has been used successfully to channel pyruvate to ethanol in various Gram‐negative bacteria, including Escherichia coli. Although the ionic strength of the H. volcanii cytosol differs over 15‐fold from that of E. coli, gel filtration and circular dichroism revealed no difference in secondary structure between the ZmPDC protein isolated from either of these hosts. Like the E. coli purified enzyme, ZmPDC from H. volcanii catalyzed the nonoxidative decarboxylation of pyruvate. A decrease in the amount of soluble ZmPDC protein was detected as H. volcanii transitioned from log phase to late stationary phase that was inversely proportional to the amount of pdc‐specific mRNA. Based on these results, proteins from non‐halophilic organisms can be actively synthesized in haloarchaea; however, post‐transcriptional mechanisms present in stationary phase appear to limit the amount of recombinant protein expressed.
A study of archaeal enzymes involved in polar lipid synthesis linking amino acid sequence information, genomic contexts and lipid composition
Authors: Daiyasu Hiromi , Kuma Kei-Ichi , Yokoi Toshiro , Morii Hiroyuki , Koga Yosuke , Toh Hiroyuki
Published: September 2009

Cellular membrane lipids, of which phospholipids are the major constituents, form one of the characteristic features that distinguish Archaea from other organisms. In this study, we focused on the steps in archaeal phospholipid synthetic pathways that generate polar lipids such as archaetidylserine, archaetidylglycerol, and archaetidylinositol. Only archaetidylserine synthase (ASS), from Methanothermobacter thermautotrophicus, has been experimentally identified. Other enzymes have not been fully examined. Through database searching, we detected many archaeal hypothetical proteins that show sequence similarity to members of the CDP alcohol phosphatidyltransferase family, such as phosphatidylserine synthase (PSS), phosphatidylglycerol synthase (PGS) and phosphatidylinositol synthase (PIS) derived from Bacteria and Eukarya. The archaeal hypothetical proteins were classified into two groups, based on the sequence similarity. Members of the first group, including ASS from M. thermautotrophicus, were closely related to PSS. The rough agreement between PSS homologue distribution within Archaea and the experimentally identified distribution of archaetidylserine suggested that the hypothetical proteins are ASSs. We found that an open reading frame (ORF) tends to be adjacent to that of ASS in the genome, and that the order of the two ORFs is conserved. The sequence similarity of phosphatidylserine decarboxylase to the product of the ORF next to the ASS gene, together with the genomic context conservation, suggests that the ORF encodes archaetidylserine decarboxylase, which may transform archaetidylserine to archaetidylethanolamine. The second group of archaeal hypothetical proteins was related to PGS and PIS. The members of this group were subjected to molecular phylogenetic analysis, together with PGSs and PISs and it was found that they formed two distinct clusters in the molecular phylogenetic tree. The distribution of members of each cluster within Archaea roughly corresponded to the experimentally identified distribution of archaetidylglycerol or archaetidylinositol. The molecular phylogenetic tree patterns and the correspondence to the membrane compositions suggest that the two clusters in this group correspond to archaetidylglycerol synthases and archaetidylinositol synthases. No archaeal hypothetical protein with sequence similarity to known phosphatidylcholine synthases was detected in this study.
Oligomerization of Sulfolobus solfataricus signature amidase is promoted by acidic pH and high temperature
Authors: D’Abusco Anna Scotto , Casadio Rita , Tasco Gianluca , Giangiacomo Laura , Giartosio Anna , Calamia Valentina , Di Marco Stefania , Chiaraluce Roberta , Consalvi Valerio , Scandurra Roberto , Politi Laura
Published: September 2009

The recombinant amidase from the hyperthermophylic archaeon Sulfolobus solfataricus (SSAM) a signature amidase, was cloned, purified and characterized. The enzyme is active on a large number of aliphatic and aromatic amides over the temperature range 60–95 °C and at pH values between 4.0 and 9.5, with an optimum at pH 5.0. The recombinant enzyme is in the form of a dimer of about 110 kD that reversibly associates into an octamer in a pH‐dependent reaction. The pH dependence of the state of association was studied using gel permeation chromatography, analytical ultracentrifugation and dynamic light scattering techniques.pH 7.0 all three techniques show the presence of two species, in about equal amounts, which is compatible with the existence of a dimeric and an octameric form. In decreasing pH, the dimers formed the octameric species and in increasing pH, the octameric species was converted to dimers. Above pH 8.0, only dimers were present, below pH 3.0 only octamers were present. The association of dimers into octamers decreased in non‐polar solvents and increased with temperature. A mutant (Y41C) was obtained that did not show this behavior.
Succession of methanogenic archaea in rice straw incorporated into a Japanese rice field: estimation by PCR‐DGGE and sequence analyses
Authors: Sugano Atsuo , Tsuchimoto Hidetaka , Cho Tun Cho , Kimura Makoto , Asakawa Susumu
Published: September 2009

The succession and phylogenetic profiles of methanogenic archaeal communities associated with rice straw decomposition in rice‐field soil were studied by polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) analysis followed by 16S rDNA sequencing. Nylon bags containing either leaf sheaths or blades were buried in the plowed layer of a Japanese rice field under drained conditions during the off‐crop season and under flooded conditions after transplanting. In addition, rice straw samples that had been buried in the rice field under drained conditions during the off‐crop season were temporarily removed during spring plowing and then re‐buried in the same rice field under flooded conditions at transplanting. Populations of methanogenic archaea were examined by amplification of the 16S rRNA genes in the DNA extracted from the rice straw samples. No PCR product was produced for samples of leaf sheath or blade prior to burial or after burial under drained conditions, indicating that the methanogen population was very small during decomposition of rice straw under oxic conditions. Many common bands were observed in rice straw samples of leaf sheath and blade during decomposition of rice straw under flooded conditions. Cluster analysis based on DGGE patterns divided methanogenic archaeal communities into two groups before and after the mid‐season drainage. Sequence analysis of DGGE bands that were commonly present were closely related to Methanomicrobiales and Rice cluster I. Methanomicrobiales, Rice cluster I and Methanosarcinales were major members before the mid‐season drainage, whereas the DGGE bands that characterized methanogenic archaeal communities after the mid‐season drainage were closely related to Methanomicrobiales. These results indicate that mid‐season drainage affected the methanogenic archaeal communities irrespective of their location on rice straw (sheath and blade) and the previous history of decomposition during the off‐crop season.
Characterization of the Family I inorganic pyrophosphatase from Pyrococcus horikoshii OT3
Authors: Jeon Sung-Jong , Ishikawa Kazuhiko
Published: September 2009

A gene encoding for a putative Family inorganic pyrophosphatase (PPase, EC 3.6.1.1) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (Accession No. 1907) from P. horikoshii showed some identity with other Family I inorganic pyrophosphatases from archaea. The recombinant PPase from P. horikoshii (PhPPase) has a molecular mass of 24.5 kDa, determined by SDS‐PAGE. This enzyme specifically catalyzed the hydrolysis of pyrophosphate and was sensitive to NaF. The optimum temperature and pH for PPase activity were 70 °C and 7.5, respectively. The half‐life of heat inactivation was about 50 min at 105 °C. The heat stability of PhPPase was enhanced in the presence of Mg2+. A divalent cation was absolutely required for enzyme activity, Mg2+ being most effective; Zn2+, Co2+ and Mn2+ efficiently supported hydrolytic activity in a narrow range of concentrations (0.05– 0.5 mM). The Km for pyrophosphate and Mg2+ were 113 and 303 µM, respectively; and maximum velocity, V
max, was estimated at 930 U mg–1.
Trace methane oxidation studied in several Euryarchaeota under diverse conditions
Authors: Moran James J. , House Christopher H. , Freeman Katherine H. , Ferry James G.
Published: September 2009

We used 13C‐labeled methane to document the extent of trace methane oxidation by Archaeoglobus fulgidus, Archaeoglobus lithotrophicus, Archaeoglobus profundus, Methanobacterium thermoautotrophicum, Methanosarcina barkeri and Methanosarcina acetivorans. The results indicate trace methane oxidation during growth varied among different species and among methanogen cultures grown on different substrates. The extent of trace methane oxidation by Mb. thermoautotrophicum (0.05 ± 0.04%, ± 2 standard deviations of the methane produced during growth) was less than that by M. barkeri (0.15 ± 0.04%), grown under similar conditions with H2 and CO2. Methanosarcina acetivorans oxidized more methane during growth on trimethylamine (0.36 ± 0.05%) than during growth on methanol (0.07 ± 0.03%). This may indicate that, in M. acetivorans, either a methyltransferase related to growth on trimethylamine plays a role in methane oxidation, or that methanol is an intermediate of methane oxidation. Addition of possible electron acceptors (O2, NO3–, SO22–, SO32–) or H2 to the headspace did not substantially enhance or diminish methane oxidation in M. acetivorans cultures. Separate growth experiments with FAD and NAD+ showed that inclusion of these electron carriers also did not enhance methane oxidation. Our results suggest trace methane oxidized during methanogenesis cannot be coupled to the reduction of these electron acceptors in pure cultures, and that the mechanism by which methane is oxidized in methanogens is independent of H2 concentration. In contrast to the methanogens, species of the sulfate‐reducing genus Archaeoglobus did not significantly oxidize methane during growth (oxidizing 0.003 ± 0.01% of the methane provided to A. fulgidus, 0.002 ± 0.009% to A. lithotrophicus and 0.003 ± 0.02% to A. profundus). Lack of observable methane oxidation in the three Archaeoglobus species examined may indicate that methyl‐coenzyme M reductase, which is not present in this genus, is required for the anaerobic oxidation of methane, consistent with the “reverse methanogenesis” hypothesis.
Higher‐level classification of the Archaea: evolution of methanogenesis and methanogens
Authors: Bapteste Éric , Brochier Céline , Boucher Yan
Published: September 2009

We used a phylogenetic approach to analyze the evolution of methanogenesis and methanogens. We show that 23 vertically transmitted ribosomal proteins do not support the monophyly of methanogens, and propose instead that there are two distantly related groups of extant archaea that produce methane, which we have named Class I and Class II. Based on this finding, we subsequently investigated the uniqueness of the origin of methanogenesis by studying both the enzymes of methanogenesis and the proteins that synthesize its specific coenzymes. We conclude that hydrogenotrophic methanogenesis appeared only once during evolution. Genes involved in the seven central steps of the methanogenic reduction of carbon dioxide (CO2) are ubiquitous in methanogens and share a common history. This suggests that, although extant methanogens produce methane from various substrates (CO2, formate, acetate, methylated C‐1 compounds), these archaea have a core of conserved enzymes that have undergone little evolutionary change. Furthermore, this core of methanogenesis enzymes seems to originate (as a whole) from the last ancestor of all methanogens and does not appear to have been horizontally transmitted to other organisms or between members of Class I and Class II. The observation of a unique and ancestral form of methanogenesis suggests that it was preserved in two independent lineages, with some instances of specialization or added metabolic flexibility. It was likely lost in the Halobacteriales, Thermoplasmatales and Archaeoglobales. Given that fossil evidence for methanogenesis dates back 2.8 billion years, a unique origin of this process makes the methanogenic archaea a very ancient taxon.
New experimental approaches for investigating interactions between Pyrococcus furiosus carbamate kinase and carbamoyltransferases, enzymes involved in the channeling of thermolabile carbamoyl phosphate
Authors: Massant Jan , Glansdorff Nicolas
Published: September 2009

A somewhat neglected but essential aspect of the molecular physiology of hyperthermophiles is the protection of thermolabile metabolites and coenzymes. An example is carbamoyl phosphate (CP), a precursor of pyrimidines and arginine, which is an extremely labile and potentially toxic intermediate. The first evidence for a biologically significant interaction between carbamate kinase (CK) and ornithine carbamoyltransferase (OTC) from Pyrococcus furiosus was provided by affinity electrophoresis and co‐immunoprecipitation in combination with cross‐linking (Massant et al. 2002). Using the yeast two‐hybrid system, Hummel‐Dreyer chromatography and isothermal titration calorimetry, we obtained additional concrete evidence for an interaction between CK and OTC, the first evidence for an interaction between CK and aspartate carbamoyltransferase (ATC) and an estimate of the binding constant between CK and ATC. The physical interaction between CK and OTC or ATC may prevent thermodenaturation of CP in the aqueous cytoplasmic environment. Here we emphasize the importance of developing experimental approaches to investigate the mechanism of thermal protection of metabolic intermediates by metabolic channeling and the molecular basis of transient protein–protein interactions in the physiology of hyperthermophiles.
Role of the precorrin 6‐X reductase gene in cobamide biosynthesis in Methanococcus maripaludis
Authors: Kim Wonduck , Major Tiffany A. , Whitman William B.
Published: September 2009

In Methanococcus maripaludis strain JJ, deletion of the homolog to cbiJ, which encodes the corrin biosynthetic enzyme precorrin 6‐X reductase, yielded an auxotroph that required either cobamide or acetate for good growth. This phenotype closely resembled that of JJ117, a mutant in which tandem repeats were introduced into the region immediately downstream of the homolog of cbiJ. Mutant JJ117 also produced low quantities of cobamides, about 15 nmol g–1 protein or 1–2% of the amount found in wild‐type cells. These results confirm the role of the cbiJ homolog in cobamide biosynthesis in the Archaea and suggest the presence of low amounts of a bypass activity in these organisms.
Transparent Yttrium Aluminium Garnet Obtained by Spark Plasma Sintering of Lyophilized Gels
Authors: Suárez M. , Fernández A. , Menéndez J. L. , Torrecillas R.
Published: September 2009

Lyophilized YAG gel, synthesized by the coprecipitation technique, has been sintered to transparency by spark plasma sintering method at 1500∘C. Whereas conventionally dried gels show large agglomerates, over 1 μm, powders from lyophilized gels show no agglomeration with an average particle size below 100 nm. The absence of agglomerates affects on the optical properties of the sintered materials: conventionally dried powders are opaque after sintering, whereas 0.8 mm thick transparent YAG materials with in‐line transmittances close to 60% at 680 nm and over 80% in the infrared range have been obtained for the lyophilized gels.
Existence and Uniqueness of Periodic Solutions of Mixed Monotone Functional Differential Equations
Authors: Kang Shugui , Cheng Sui Sun
Published: September 2009

This paper deals with the existence and uniqueness of periodic solutions for the first‐order
functional differential equation
y′(t) = −a(t)y(t) + f1(t, y(t − τ(t))) + f2(t, y(t − τ(t)))
with periodic coefficients and delays. We choose the mixed monotone operator theory to approach
our problem because such methods, besides providing the usual existence results,
may also sometimes provide uniqueness as well as additional numerical schemes for the computation
of solutions.
Software Toolchain for Large‐Scale RE‐NFA Construction on FPGA
Authors: Yang Yi-Hua E. , Prasanna Viktor K.
Published: September 2009

We present a software toolchain for constructing large‐scaleregular expression matching(REM) on FPGA. The software automates the conversion of regular expressions into compact and high‐performance nondeterministic finite automata (RE‐NFA). Each RE‐NFA is described as an RTL regular expression matching engine (REME) in VHDL for FPGA implementation. Assuming a fixed number of fan‐out transitions per state, ann‐statem‐bytes‐per‐cycle RE‐NFA can be constructed inO(n×m) time andO(n×m) memory by our software. A large number of RE‐NFAs are placed onto a two‐dimensionalstaged pipeline, allowing scalability to thousands of RE‐NFAs with linear area increase and little clock rate penalty due to scaling. On a PC with a 2 GHz Athlon64 processor and 2 GB memory, our prototype software constructs hundreds of RE‐NFAs used by Snort in less than 10 seconds. We also designed a benchmark generator which can produce RE‐NFAs with configurable pattern complexity parameters, including state count, state fan‐in, loop‐back and feed‐forward distances. Several regular expressions with various complexities are used to test the performance of our RE‐NFA construction software.
Radiation Recall Reaction Induced by Adjuvant Trastuzumab (Herceptin)
Authors: Chung Caroline , Stuart David , Keyes Mira
Published: September 2009

Although concerns of radiation sensitization have been raised with concurrent trastuzumab (Herceptin) administration, there has been no published case of radiation recall reaction associated with trastuzumab. This case describes a clinical presentation consistent with a radiation recall reaction following administration of adjuvant trastuzumab after neoadjuvant FEC-D chemotherapy and locoregional radiotherapy for HER2-positive, locally advanced breast cancer in a premenopausal woman. Although the mechanism and etiology of radiation recall dermatitis remain unclear, this case raises further hypotheses regarding a possible drug dose-dependence and possible predisposing risk factor for the development of radiation recall reactions.
The Prognostic Significance of the Preoperative Full Blood Count after Resection of Colorectal Liver Metastases
Authors: Dajani K. , O'Reilly D. A. , De Liguori Carino N. , Ghaneh P. , Poston G. , Wu A.
Published: September 2009

Introduction. Increased preoperative platelet and neutrophil counts are risk factors for decreased survival in several different malignancies. Our aim was to investigate the relationship between overall or disease-free survival after resection of CRLM and the preoperative haematological parameters.
Methods. We reviewed a cohort of 140 patients who underwent resection of CRLM with curative intent, utilising prospectively maintained databases. Patient demographics, operative details, FBC, CRP, INR, histopathology results, and survival data were examined. Kaplan-Meier survival and Cox regression analyses were used to determine the impact of all variables on survival.
Results. 140 patients (96 males) with a median age of 67 years (range 33–82 years) underwent resection of CRLM. A significant correlation was exhibited between preoperative platelet count and neutrophil count (rho = 0.186, ). When modelled as continuous covariates in a Cox regression hazards, an increased preoperative platelet () and neutrophil counts () were significantly associated with overall survival. Of the haematological parameters assessed only preoperative platelet count showed a strong trend of association with disease free survival; however this failed to reach statistical significance (). Conclusions. Increased preoperative platelet and neutrophil counts are independent risk factors for decreased survival in patients undergoing resection of CRLM in our series of patients. These findings require validation in larger studies to determine their relationship with survival. Further research into the role of these cell types in tumour progression, particularly in the development and inhibition of angiogenesis, is warranted.
Analysis and Enhancement of Random Number Generator in FPGA Based on Oscillator Rings
Authors: Wold Knut , Tan Chik How
Published: September 2009

A true random number generator (TRNG) is an important
component in cryptographic systems. Designing a fast
and secure TRNG in an FPGA is a challenging task. In this
paper, we analyze the TRNG designed by Sunar et al. (2007)
based on XOR of the outputs of several oscillator rings. We
propose an enhanced TRNG with better randomness characteristics
that does not require postprocessing and passes
the statistical tests. We have shown by experiment that the
frequencies of the equal length oscillator rings in the TRNG
are not identical. The difference is due to the placement of
the inverters in the FPGA and the resulting routing between
the inverters. We have implemented our proposed TRNG in
an Altera Cyclone II FPGA. Our implementation has passed
the NIST and DIEHARD statistical tests with a throughput
of 100 Mbps and with a usage of less than 100 logic elements
in the FPGA. The restart experiments have shown
that the output from our TRNG behaves truly random and
not pseudorandom.
Distributed Approach for Solving Time-Dependent Problems in Multimodal Transport Networks
Authors: Galvez-Fernandez Carlos , Khadraoui Djamel , Ayed Hedi , Habbas Zineb , Alba Enrique
Published: September 2009

This paper presents an alternative approach for time-dependent multimodal transport problem. We describe a new graph structure to abstract multimodal networks, calledtransfer graph, which adapts to the distributed nature of real information sources of transportation networks. A decomposition of the Shortest Path Problem intransfer graphis proposed to optimize the computation time. This approach was computationally tested in several experimental multimodal networks having different size and complexity. The approach was integrated in the multimodal transport service of the European Carlink platform, where it has been validated in real scenarios. Comparision with other related works is provided.
The Elliptic GL(n) Dynamical Quantum Group as an 𝔥‐Hopf Algebroid
Authors: Hartwig Jonas T.
Published: September 2009

Using the language of 𝔥‐Hopf algebroids which was
introduced by Etingof and Varchenko,
we construct a dynamical quantum group, ℱell(GL(n)),
from the elliptic solution of the quantum dynamical Yang‐Baxter
equation with spectral parameter associated to the
Lie algebra 𝔰𝔩n. We apply the generalized FRST construction
and obtain an 𝔥‐bialgebroid ℱell(M(n)). Natural analogs of
the exterior algebra and their matrix elements, elliptic minors,
are defined and studied. We show how to use the cobraiding to prove that the elliptic determinant
is central. Localizing at this determinant and constructing an antipode we obtain the 𝔥‐Hopf algebroid ℱell(GL(n)).
Wide and Fast Wavelength‐Swept Fiber Laser Based on Dispersion Tuning for Dynamic Sensing
Authors: Yamashita Shinji , Nakazaki Yuichi , Konishi Ryosei , Kusakari Osamu
Published: September 2009

We have developed a unique wide and fast wavelength‐swept fiber laser for dynamic and accurate fiber sensing. The wavelength tuning is based on the dispersion tuning technique, which simply modulates the loss/gain in the dispersive laser cavity. By using wideband semiconductor optical amplifiers (SOAs), the sweep range could be as wide as ∼180 nm. Since the cavity contains no mechanical components, such as tunable filters, we could achieve very high sweep rate, as high as ∼200 kHz. We have realized the swept lasers at three wavelength bands, 1550 nm, 1300 nm, and 800 nm, using SOAs along with erbium‐doped fiber amplifiers (EDFAs), and in two laser configurations, ring and linear ones. We also succeeded in applying the swept laser for a dynamic fiber‐Bragg grating (FBG) sensor system. In this paper, we review our researches on the wide and fast wavelength‐swept fiber lasers.
On Some Properties of Focal Points
Authors: Ferchichi M. R. , Djellit I.
Published: September 2009

We consider some dynamical properties of two‐dimensional maps having an inverse with vanishing denominator. We put in evidence a link between a fixed point of a map with fractional inverse and a focal point of this inverse.