Identification and sequence determination of the capsid protein gene of human astrovirus serotype 1

Abstract Pulse labelling experiments and enzyme studies show that Thermoproteus tenax is able to degrade glucose via the Embden-Meyerhof-Parnas (EMP) pathway. T. tenax is the first archeum for which the glycolytic EMP pathway could be established. One of the key enzymes, 6-phosphofructokinase of T. tenax depends on (pyrophosphate-fructose-6-phosphate-1-phosphotransferase) instead of ATP as found in some bacterial and eucaryal species. In addition to the intermediates of the EMP pathway the intermediary products of the non-phosphorylated Entner-Doudoroff pathway were also detected by pulse labelling experiments indicating that under chemoorganotrophic conditions at least 2 glycolytic pathways are operative in T. tenax .     

Quinones from Archaebacteria, I. New types of menaquinones from the thermophilic archaebacterium Thermoproteus tenax

From the archaebacterium Thermoproteus tenax, strain Kra-1 a mixture of 6 quinones of menaquinone and phylloquinone type with isopentyl side chains, MK-6(12H), MK-6-(10H), MK-5(10H), MK-5(8H), MK-4(8H), MK-4(6H) and two analogous quinones, containing in addition a methyl group in the naphthoquinone system, were isolated and characterized.     

CC1, a novel crenarchaeal DNA binding protein

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

Phosphorylation of serine-rich protein encoded by open reading frame 3 of the TT virus genome

TT virus (TTV) is a newly discovered human virus with a single-stranded, circular DNA genome. The TTV DNA sequence includes two major open reading frames (ORFs), ORF1 and ORF2. Recently, spliced TTV mRNAs were detected and revealed two additional coding regions, ORF3 and ORF4. We found sequence similarity between the TTV ORF3 protein and hepatitis C virus (HCV) nonstructural 5A (NS5A) protein, which is a phosphoprotein and is thought to associate with various cellular proteins. To test whether the TTV ORF3 protein is phosphorylated, the state of phosphorylation was analyzed with a transient protein production system. The TTV ORF3 protein was phosphorylated at the serine residues in its C-terminal portion. Furthermore, the TTV ORF3 gene generated two forms of proteins with a different phosphorylation state, similar to the HCV NS5A region, suggesting that TTV ORF3 protein has function(s) similar to phosphorylated viral proteins such as the HCV NS5A protein.     

Enzymological characteristics of a novel archaeal dye-linked d-lactate dehydrogenase showing loose binding of FAD

Extremophiles - A gene-encoding a dye-linked d-lactate dehydrogenase (Dye-DLDH) homolog was identified in the genome of the hyperthermophilic archaeon Thermoproteus tenax. The gene was expressed in...     

Reconstruction of the central carbohydrate metabolism of Thermoproteus tenax by use of genomic and biochemical data

The hyperthermophilic, facultatively heterotrophic crenarchaeum Thermoproteus tenax was analyzed using a low-coverage shotgun-sequencing approach. A total of 1.81 Mbp (representing 98.5% of the total genome), with an average gap size of 100 bp and 5.3-fold coverage, are reported, giving insights into the genome of T. tenax. Genome analysis and biochemical studies enabled us to reconstruct its central carbohydrate metabolism. T. tenax uses a variant of the reversible Embden-Meyerhof-Parnas (EMP) pathway and two different variants of the Entner-Doudoroff (ED) pathway (a nonphosphorylative variant and a semiphosphorylative variant) for carbohydrate catabolism. For the EMP pathway some new, unexpected enzymes were identified. The semiphosphorylative ED pathway, hitherto supposed to be active only in halophiles, is found in T. tenax. No evidence for a functional pentose phosphate pathway, which is essential for the generation of pentoses and NADPH for anabolic purposes in bacteria and eucarya, is found in T. tenax. Most genes involved in the reversible citric acid cycle were identified, suggesting the presence of a functional oxidative cycle under heterotrophic growth conditions and a reductive cycle for CO2 fixation under autotrophic growth conditions. Almost all genes necessary for glycogen and trehalose metabolism were identified in the T. tenax genome.     

Ultrastructure of the cell envelope of the archaebacteria Thermoproteus tenax and Thermoproteus neutrophilus.

The ultrastructures of the regular surface layers (S-layers) of the extremely thermophilic archaebacteria Thermoproteus tenax and Thermoproteus neutrophilus were examined by freeze-etching, freeze-drying, and negative staining methods combined with optical and digital image enhancement. In both strains, a monolayer of macromolecules arranged in hexagonal arrays with center-to-center spacings of approximately 30 nm was the only component of the cell wall. The gross morphologies of the S-layer lattices of the two organisms were similar and showed the same handedness in the arrangement of the protomers of the morphological units. Striking differences were found in the anionic charge distributions on the surfaces of the two S-layer proteins as determined by labeling with polycationic ferritin. Analysis of the lattice orientation, together with the number and distribution of lattice faults on intact cells, provided a strong indication that the S-layers of both organisms have a shape-determining function.     

Sequencing, cloning, and high-level expression of the pfp gene, encoding a PP(i)-dependent phosphofructokinase from the extremely thermophilic eubacterium Dictyoglomus thermophilum

The sequencing, cloning, and expression of the pfp gene from Dictyoglomus thermophilum, which consists of 1,041 bp and encodes a pyrophosphate-dependent phosphofructokinase, are described. A phylogenetic analysis indicates that the enzyme is closely related to the pyrophosphate-dependent enzyme from Thermoproteus tenax. The recombinant and native enzymes share a high degree of similarity for most properties examined.     

The PrP-like protein Doppel binds copper

Doppel (Dpl) is a glycosylphosphatidylinositol-anchored protein expressed in the testis. It exhibits 26% sequence identity with the prion protein (PrP) but lacks the octarepeat region implicated as the major copper-binding domain. Contrary to expectations, Cu(II) induced a 26% reduction in the intrinsic fluorescence of Dpl(27-154) and a calculated K(d) for a single-site model of 0.16 +/- 0.08 microm. Other metals had minimal effects on fluorescence quenching. Matrix-assisted laser desorption ionization mass spectrometry of a Dpl peptide revealed binding of copper (but not other metals) to the helical alphaB/B'-loop-alphaC subregion of Dpl. Fluorescence quenching and equilibrium dialysis analyses of this Dpl(101-145) peptide were compatible with a binding site of K(d) = 0.4 microm. Diethylpyrocarbonate footprinting (Qin, K., Yang, Y., Mastrangelo, P., and Westaway, D. (2002) J. Biol. Chem. 277, 1981-1990) of Dpl(27-154) defined one residue/molecule was protected by copper from diethylpyrocarbonate adduct formation, and reiteration of this analysis with Dpl(101-145) suggested that His(131) may contribute to Cu(II) binding. Taken together, our data indicate that the alpha-helical region of mouse Dpl possesses a selective copper-binding site with a submicromolar K(d) and perhaps one or more lower affinity sites. Although metallated forms of Dpl might exist in vivo, analyses of Tg(Dpl)10329 mice were inconsistent with reports that Dpl expression is associated with increased carbonylation and nitrosylation of brain proteins. Thus, rather than comprising an important source of free radical damage, copper binding may serve to modulate the activity, stability, or localization of the Dpl protein.     

Characterization of glycerate kinase (2-phosphoglycerate forming), a key enzyme of the nonphosphorylative Entner-Doudoroff pathway, from the thermoacidophilic euryarchaeon Picrophilus torridus

Picrophilus torridus has been shown to degrade glucose via a nonphosphorylative Entner-Doudoroff (ED) pathway. Here we report the characterization of a key enzyme of this pathway, glycerate kinase (2-phosphoglycerate forming). The enzyme was purified 5,100-fold to homogeneity. The 95 kDa homodimeric protein catalyzed the ATP-dependent phosphorylation of glycerate specifically to 2-phosphoglycerate. The enzyme showed highest activity at 60 degrees C and pH 7.3, with ATP as phosphoryl donor and Mg(2+) as divalent cation. By MALDI-TOF analysis, ORF Pto1442 was identified in the genome of P. torridus as the encoding gene, designated gck. Homologs with high sequence identity were identified in the genomes of the archaea Thermoplasma and Sulfolobus spp. and Thermoproteus tenax, for which the operation of nonphosphorylative ED pathways, involving 2-phosphoglycerate forming glycerate kinases, has been proposed.     

A novel trehalose synthesizing pathway in the hyperthermophilic Crenarchaeon <Emphasis Type="Italic">Thermoproteus tenax</Emphasis>: the unidirectional TreT pathway

In the genome of the hyperthermophilic archaeon Thermoproteus tenax a gene (treS/P) encoding a protein with similarity to annotated trehalose phosphorylase (TreP), trehalose synthase (TreS) and more recently characterized trehalose glycosyltransferring synthase (TreT) was identified. The treS/P gene as well as an upstream located ORF of unknown function (orfY) were cloned, heterologously expressed in E. coli and purified. The enzymatic characterization of the putative TreS/P revealed TreT activity. However, contrary to the previously characterized reversible TreT from Thermococcus litoralis and Pyrococcus horikoshii, the T. tenax enzyme is unidirectional and catalyzes only the formation of trehalose from UDP (ADP)-glucose and glucose. The T. tenax enzyme differs from the reversible TreT of T. litoralis by its preference for UDP-glucose as co-substrate. Phylogenetic and comparative gene context analyses reveal a conserved organization of the unidirectional TreT and OrfY gene cluster that is present in many Archaea and a few Bacteria. In contrast, the reversible TreT pathway seems to be restricted to only a few archaeal (e.g. Thermococcales) and bacterial (Thermotogales) members. Here we present a new pathway exclusively involved in trehalose synthesis--the unidirectional TreT pathway--and discuss its physiological role as well as its phylogenetic distribution.