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Anaerobic oxidation of methane (AOM) mediated by anaerobic methanotrophic archaea (ANME) is the primary process that provides energy to cold seep ecosystems by converting methane into inorganic carbon. Notably, cold seep ecosystems are dominated by highly divergent heterotrophic microorganisms. The role of the AOM process in supporting heterotrophic population remains unknown. We investigate the acetogenic capacity of ANME-2a in a simulated cold seep ecosystem using high-pressure biotechnology, where both AOM activity and acetate production are detected. The production of acetate from methane is confirmed by isotope-labeling experiments. A complete archaeal acetogenesis pathway is identified in the ANME-2a genome, and apparent acetogenic activity of the key enzymes ADP-forming acetate-CoA ligase and acetyl-CoA synthetase is demonstrated. Here, we propose a modified model of carbon cycling in cold seeps: during AOM process, methane can be converted into organic carbon, such as acetate, which further fuels the heterotrophic community in the ecosystem., Ocean cold seeps are poorly understood relative to related systems like hydrothermal vents. Here the authors use high pressure bioreactors and microbial communities from a cold seep mud volcano and find a previously missing step of methane conversion to acetate that likely fuels heterotrophic communities.

4-alpha-Glucanotransferase (GTase) enzymes (EC 2.4.1.25) modulate the size of alpha-glucans by cleaving and reforming alpha-1,4 glycosidic bonds in alpha-glucans, an essential process in starch and glycogen metabolism in plants and microorganisms. The glycoside hydrolase family 57 enzyme (GTase57) studied in the current work catalyzes both disproportionation and cyclization reactions. Amylose was converted into cyclic amylose (with a minimum size of 17 glucose monomers) as well as to a spectrum of maltodextrins, but in contrast to glycoside hydrolase family 13 cyclodextrin glucanotransferases (CGTases), no production of cyclodextrins (C6-C8) was observed. GTase57 also effectively produced alkyl-glycosides with long alpha-glucan chains from dodecyl-beta-D-maltoside and starch, demonstrating the potential of the enzyme to produce novel variants of surfactants. Importantly, the GTase57 has excellent thermostability with a maximal activity at 95 A degrees C and an activity half-life of 150 min at 90 A degrees C which is highly advantageous in this manufacturing process suggesting that enzymes from this relatively uncharacterized family, GH57, can be powerful biocatalysts for the production of large head group glucosides from soluble starch.

Laminarinase A (LamA) from Pyrococcus furiosus is a hyperthermostable endo-β-1,3-glucanase (EC 3.2.1.39) belonging to the glycosyl hydrolase family GH16. Here, we report the two-step immobilization of LamA on macroporous acrylic epoxy beads, extra-functionalized with disulfide groups. To facilitate initial immobilization via thiol-disulfide exchange, we introduced, by site-directed mutagenesis, a superficial cysteine residue near the protein C-terminal end. The thus-obtained S296C variant showed similar catalytic properties as native LamA. The activity of immobilized S296C displayed an inverse relationship with particle size. Use of conventional beads (150-300 μm in diameter) obstructed the catalytic efficiency due to pore diffusion limitation of the polysaccharide substrate. Bifunctional attachment to milled beads (20-40 μm) resulted in high enzyme load and outstanding catalytic features. Bifunctional immobilized S296C showed extreme pH stability and could be repeatedly used at 60 °C without significant activity loss.

Indexación: Web of Science; Scopus. Rubrerythrins (RBRs) are non-heme di-iron proteins belonging to the ferritin-like superfamily. They are involved in oxidative stress defense as peroxide scavengers in a wide range of organisms. The vast majority of RBRs, including classical forms of this protein, contain a C-terminal rubredoxin-like domain involved in electron transport that is used during catalysis in anaerobic conditions. Rubredoxin is an ancient and large protein family of short length (

Increased hydrogen (H2) levels are known to inhibit H2 formation in Caldicellulosiruptor saccharolyticus. To investigate this organism's strategy for dealing with elevated H2 levels the effect of the hydrogen partial pressure ( P H 2 ) on fermentation performance was studied by growing cultures under high and low P H 2 in a glucose limited chemostat setup. Transcriptome analysis revealed the upregulation of genes involved in the disposal of reducing equivalents under high P H 2 , like lactate dehydrogenase and alcohol dehydrogenase as well as the NADH-dependent and ferredoxin-dependent hydrogenases. These findings are in line with the observed shift in fermentation profiles from acetate production to the production of acetate, lactate and ethanol under high P H 2 . Moreover, differential transcription was observed for genes involved in carbon metabolism, fatty acid biosynthesis and several transport systems. In addition, presented transcription data provide evidence for the involvement of the redox sensing Rex protein in gene regulation under high P H 2 cultivation conditions.

An intein-driven protein splicing approach allowed for the covalent linkage between the N- and C-termini of a polypeptide chain to create circular variants of the endo-β-1,3-1,4-glucanase, LicA, from Bacillus licheniformis. Two circular variants, LicA-C1 and LicA-C2, which have connecting loops of 20 and 14 amino acids, respectively, showed catalytic activities that are approximately two and three times higher, respectively, compared to that of the linear LicA (LicA-L1). The thermal stability of the circular variants was significantly increased compared to the linear form. Whereas the linear glucanase lost half of its activity after 3 min at 65 °C, the two circular variants have 6-fold (LicA-C1) and 16-fold (LicA-C2) increased half-life time of inactivation. In agreement with this, fluorescence spectroscopy and differential scanning calorimetry studies revealed that circular enzymes undergo structural changes at higher temperatures compared to that of the linear form. The effect of calcium on the conformational stability and function of the circular LicAs was also investigated, and we observed that the presence of calcium ions results in increased thermal stability. The impact of the length of the designed loops on thermal stability of the circular proteins is discussed, and it is suggested that cyclization may be an efficient strategy for the increased stability of proteins.

Phthalocyanines (Pc) are non-natural organic dyes with wide and deep impact in materials science, based on their intense absorption at the near-infrared (NIR), longlived fluorescence and high singlet oxygen (O-1(2)) quantum yields. However, Pcs tend to stack in buffer solutions, losing their ability to generate singlet oxygen, which limits their scope of application. Furthermore, Pcs are challenging to organize in crystalline structures. Protein cages, on the other hand, are very promising biological building blocks that can be used to organize different materials into crystalline nanostructures. Here, we combine both kinds of components into photoactive biohybrid crystals. Toward this end, a hierarchical organization process has been designed in which (a) a supramolecular complex is formed between octacationic zinc Pc (1) and a tetraanionic pyrene (2) derivatives, driven by electrostatic and pi-pi interactions, and (b) the resulting tetracationic complex acts as a molecular glue that binds to the outer surface anionic patches of the apoferritin (aFt) protein cage, inducing cocrystallization. The obtained ternary face-centered cubic (fcc) packed cocrystals, with diameters up to 100 mu m, retain the optical properties of the pristine dye molecules, such as fluorescence at 695 nm and efficient light-induced O-1(2) production. Considering that O-1(2) is utilized in important technologies such as photodynamic therapy (PDT), water treatments, diagnostic arrays and as an oxidant in organic synthesis, our results demonstrate a powerful methodology to create functional biohybrid systems with unprecedented long-range order. This approach should greatly aid the development of nanotechnology and biomedicine.

The hyperthermophilic archaeon Sulfolobus solfataricus has been shown to exhibit a complex transcriptional response to UV irradiation involving 55 genes. Among the strongest UV-induced genes was a putative pili biogenesis operon encoding a potential secretion ATPase, two pre-pilins, a putative transmembrane protein and a protein of unknown function. Electron microscopy and image reconstruction of UV-treated cells showed straight pili with 10 nm in diameter, variable in length, not bundled or polarized and composed of three evenly spaced helices, thereby clearly being distinguishable from archaeal flagella. A deletion mutant of SSO0120, the central type II/IV secretion ATPase, did not produce pili. It could be complemented by reintroducing the gene on a plasmid vector. We have named the operon ups operon for UV-inducible pili operon of Sulfolobus. Overexpression of the pre-pilins, Ups-A/B (SSO0117/0118) in Sulfolobus resulted in production of extremely long filaments. Pronounced cellular aggregation was observed and quantified upon UV treatment. This aggregation was a UV-dose-dependent, dynamic process, not inducible by other physical stressors (such as pH or temperature shift) but stimulated by chemically induced double-strand breaks in DNA. We hypothesize that pili formation and subsequent cellular aggregation enhance DNA transfer among Sulfolobus cells to provide increased repair of damaged DNA via homologous recombination.

The superoxide radical O 2 ·̅ is a toxic by-product of oxygen metabolism. Two O 2 ·̅ detoxifying enzymes have been described so far, superoxide dismutase and superoxide reductase (SOR), both forming H 2 O 2 as a reaction product. Recently, the SOR active site, a ferrous iron in a [Fe 2+ (N-His) 4 (S-Cys)] pentacoordination, was shown to have the ability to form a complex with the organometallic compound ferrocyanide. Here, we have investigated in detail the reactivity of the SOR–ferrocyanide complex with O 2 ·̅ by pulse and γ-ray radiolysis, infrared, and UV-visible spectroscopies. The complex reacts very efficiently with O 2 ·̅ . However, the presence of the ferrocyanide adduct markedly modifies the reaction mechanism of SOR, with the formation of transient intermediates different from those observed for SOR alone. A one-electron redox chemistry appears to be carried out by the ferrocyanide moiety of the complex, whereas the SOR iron site remains in the reduced state. Surprisingly, the toxic H 2 O 2 species is no longer the reaction product. Accordingly, in vivo experiments showed that formation of the SOR–ferrocyanide complex increased the antioxidant capabilities of SOR expressed in an Escherichia coli sodA sodB recA mutant strain. Altogether, these data describe an unprecedented O 2 ·̅ detoxification activity, catalyzed by the SOR–ferrocyanide complex, which does not conduct to the production of the toxic H 2 O 2 species.

Sugar uptake in Sulfolobus solfataricus, a thermoacidophilic archaeon, occurs through high-affinity binding of protein-dependent ABC transporters. We have investigated the expression patterns of two sugar transport operons, that is, the glucose and arabinose transporters. Analysis of the araS promoter activity, and the mRNA and protein levels in S. solfataricus cells grown on different carbon sources showed that expression of the arabinose transporter gene cluster is highly regulated and dependent on the presence of arabinose in the medium. Glucose in the growth medium repressed the expression of the arabinose transport genes. By means of primer extension, the transcriptional start site for the arabinose operon was mapped. Interestingly, expression of the arabinose transporter is down-regulated by addition of a selective set of amino acids to the medium. Expression of the glucose transporter genes appeared constitutive. These data confirm the earlier observation of a catabolite repression-like system in S. solfataricus.

The structural features of the hyperthermophilic endo-beta-1,3-glucanase from Pyrococcus furiosus were studied using circular dichroism, steady-state and time-resolved fluorescence spectroscopy and anisotropy. Upon heat and chemical treatment the folded and denatured states of the protein were characterized by distinguishable spectral profiles that identified a number of conformational states. The fluorescence methods showed that the spectral differences arose from changes in the local environment around specific tryptophan residues in the native, partially folded, partially unfolded and completely unfolded state. A structural resemblance was observed between the native protein and the structurally perturbed state which resulted after heat treatment at 110 degrees C. The enzyme underwent disruption of the native secondary and tertiary structure only after incubation at biologically extremely high temperatures (i.e. 150 degrees C), whilst in the presence of 8 M of guanidine hydrochloride the protein was partially unfolded.

Amylomaltases are 4-α-glucanotransferases (EC 2.4.1.25) of glycoside hydrolase family 77 that transfer α-1,4-linked glucans to another acceptor, which can be the 4-OH group of an α-1,4-linked glucan or glucose. The amylomaltase-encoding gene (PAE1209) from the hyperthermophilic archaeon Pyrobaculum aerophilum IM2 was cloned and expressed in Escherichia coli , and the gene product (PyAMase) was characterized. PyAMase displays optimal activity at pH 6.7 and 95°C and is the most thermostable amylomaltase described to date. The thermostability of PyAMase was reduced in the presence of 2 mM dithiothreitol, which agreed with the identification of two possible cysteine disulfide bridges in a three-dimensional model of PyAMase. The kinetics for the disproportionation of malto-oligosaccharides, inhibition by acarbose, and binding mode of the substrates in the active site were determined. Acting on gelatinized food-grade potato starch, PyAMase produced a thermoreversible starch product with gelatin-like properties. This thermoreversible gel has potential applications in the food industry. This is the first report on an archaeal amylomaltase.

The thermostable Pyrococcus furiosus beta-glycosidase was used for oligosaccharide production from lactose in a kinetically controlled reaction. Our experiments showed that higher temperatures are beneficial for the absolute as well as relative oligosaccharide yield. However, at reaction temperatures of 80degreesC and higher, the inactivation rate of the enzyme in the presence of sugars was increased by a factor of 2 compared to the inactivation rate in the absence of sugars. This increased enzyme inactivation was caused by the occurrence of Maillard reactions between the sugar and the enzyme. The browning of our reaction mixture due to Maillard reactions was modeled by a cascade of a zeroth- and first-order reaction and related to enzyme inactivation. From these results we conclude that modification of only a small number of amino groups already gives complete inactivation of the enzyme. (C) 2003 Wiley Periodicals.

The cytoplasmic membrane of bacteria and archaea determine to a large extent the composition of the cytoplasm. Since the ion and in particular the proton and/or the sodium ion electrochemical gradients across the membranes are crucial for the bioenergetic conditions of these microorganisms, strategies are needed to restrict the permeation of these ions across their cytoplasmic membrane. The proton and sodium permeabilities of all biological membranes increase with the temperature. Psychrophilic and mesophilic bacteria, and mesophilic, (hyper)thermophilic and halophilic archaea are capable of adjusting the lipid composition of their membranes in such a way that the proton permeability at the respective growth temperature remains low and constant (homeo-proton permeability). Thermophilic bacteria, however, have more difficulties to restrict the proton permeation across their membrane at high temperatures and these organisms have to rely on the less permeable sodium ions for maintaining a high sodium-motive force for driving their energy requiring membrane-bound processes. Transport of solutes across the bacterial and archaeal membrane is mainly catalyzed by primary ATP driven transport systems or by proton or sodium motive force driven secondary transport systems. Unlike most bacteria, hyperthermophilic bacteria and archaea prefer primary ATP-driven uptake systems for their carbon and energy sources. Several high-affinity ABC transporters for sugars from hyperthermophiles have been identified and characterized. The activities of these ABC transporters allow these organisms to thrive in their nutrient-poor environments.

Desulfovibrio gigas NCIMB 9332 cells grown in ethanol-containing medium with 0.1 mu M tungstate contained a benzylviologen-linked aldehyde oxidoreductase, The enzyme was purified to electrophoretic homogeneity and found to be a homodimer with a subunit M(r) of 62,000, It contained 0.68 a 0.08 W, 4.8 Fe, and 3.2 +/- 0.2 labile S per subunit, After acid iodine oxidation of the purified enzyme, a fluorescence spectrum typical for form A of molybdopterin was obtained, Acetahdehyde, propionaldehyde, and benzaldehyde were excellent substrates, with apparent K-m values of 12.5, 10.8, and 20 mu M, respectively, The natural electron acceptor is not yet known; benzylviologen was used as an artificial electron acceptor (apparent K-m, 0.55 mM), The enzyme was activated by potassium ions and strongly inhibited by cyanide, arsenite, and iodoacetate, In the as-isolated enzyme, electron paramagnetic resonance studies readily detected W(V) as a complex signal with g values in the range of 1.84 to 1.97, The dithionite-reduced enzyme exhibited a broad signal at low temperature,vith g = 2.04 and 1.92; this is indicative of a [4Fe-4S](1+) cluster interacting with a second paramagnet, possibly the S = 1 system of W(TV), Until now W-containing aldehyde oxidoreductases had only been found in two Clostridium strains and two hyperthermophilic archaea, The D. gigas enzyme is the first example of such an enzyme in a gram-negative bacterium.

Background Superoxide reductases (SOR) catalyse the reduction of superoxide anions to hydrogen peroxide and are involved in the oxidative stress defences of anaerobic and facultative anaerobic organisms. Genes encoding SOR were discovered recently and suffer from annotation problems. These genes, named sor, are short and the transfer of annotations from previously characterized neelaredoxin, desulfoferrodoxin, superoxide reductase and rubredoxin oxidase has been heterogeneous. Consequently, many sor remain anonymous or mis-annotated. Description SORGOdb is an exhaustive database of SOR that proposes a new classification based on domain architecture. SORGOdb supplies a simple user-friendly web-based database for retrieving and exploring relevant information about the proposed SOR families. The database can be queried using an organism name, a locus tag or phylogenetic criteria, and also offers sequence similarity searches using BlastP. Genes encoding SOR have been re-annotated in all available genome sequences (prokaryotic and eukaryotic (complete and in draft) genomes, updated in May 2010). Conclusions SORGOdb contains 325 non-redundant and curated SOR, from 274 organisms. It proposes a new classification of SOR into seven different classes and allows biologists to explore and analyze sor in order to establish correlations between the class of SOR and organism phenotypes. SORGOdb is freely available at http://sorgo.genouest.org/index.php.

Soft x-ray magnetic circular dichroism was used to study a paramagnetic bioinorganic system. We measured the Fe L edges of Pyrococcus furiosus rubredoxin, using circularly polarized synchrotron radiation, a split-coil superconducting magnet, low sample temperatures, and fluorescence detection. The observed dichroism effect is strong (30%) and in general agreement with the calculation. The method is element- and oxidation state-specific, and the data can be interpreted by established theoretical procedures. Soft x-ray magnetic circular dichroism demonstrates enormous potential as a probe for studying paramagnetic systems in biology, chemistry, and material science.

A key element during the flow of genetic information in living systems is fidelity. The accuracy of DNA replication influences the genome size as well as the rate of genome evolution. The large amount of energy invested in gene expression implies that fidelity plays a major role in fitness. On the other hand, an increase in fidelity generally coincides with a decrease in velocity. Hence, an important determinant of the evolution of life has been the establishment of a delicate balance between fidelity and variability. This paper reviews the current knowledge on quality control in archaeal information processing. While the majority of these processes are homologous in Archaea, Bacteria, and Eukaryotes, examples are provided of nonorthologous factors and processes operating in the archaeal domain. In some instances, evidence for the existence of certain fidelity mechanisms has been provided, but the factors involved still remain to be identified.

In eukarya and bacteria, lysine methylation is relatively rare and is catalysed by sequence-specific lysine methyltransferases that typically have only a single-protein target. Using RNA polymerase purified from the thermophilic crenarchaeumSulfolobus solfataricus, we identified 21 methyllysines distributed across 9 subunits of the enzyme. The modified lysines were predominantly inα-helices and showed no conserved sequence context. A limited survey of theThermoproteus tenaxproteome revealed widespread modification with 52 methyllysines in 30 different proteins. These observations suggest the presence of an unusual lysine methyltransferase with relaxed specificity in the crenarchaea. Since lysine methylation is known to enhance protein thermostability, this may be an adaptation to a thermophilic lifestyle. The implications of this modification for studies and applications of recombinant crenarchaeal enzymes are discussed.

Item does not contain fulltext Insight into the hyperthermostable endo-beta-1,3-glucanase pfLamA from Pyrococcus furiosus is obtained by using NMR spectroscopy. pfLamA functions optimally at 104 degrees C and recently the X-ray structure of pfLamA has been obtained at 20 degrees C, a temperature at which the enzyme is inactive. In this study, near-complete (>99%) NMR assignments are presented of chemical shifts of pfLamA in presence and absence of calcium at 62 degrees C, a temperature at which the enzyme is biologically active. The protein contains calcium and the effects of calcium on the protein are assessed. Calcium binding results in relatively small chemical shift changes in a region distant from the active site of pfLamA and thus causes only minor conformational modifications. Removal of calcium does not significantly alter the denaturation temperature of pfLamA, implying that calcium does not stabilize the enzyme against global unfolding. The data obtained form the basis for elucidation of the molecular origins involved in conformational stability and biological activity of hyperthermophilic endo-beta-1,3-glucanases at extreme temperatures.

Hydrogen produced from biomass by bacteria and archaea is an attractive renewable energy source. However, to make its application more feasible, microorganisms are needed with high hydrogen productivities. For several reasons, hyperthermophilic and extremely thermophilic bacteria and archaea are promising is this respect. In addition to the high polysaccharide-hydrolysing capacities of many of these organisms, an important advantage is their ability to use most of the reducing equivalents (e.g. NADH, reduced ferredoxin) formed during glycolysis for the production of hydrogen, enabling H2/hexose ratios of between 3.0 and 4.0. So, despite the fact that the hydrogen-yielding reactions, especially the one from NADH, are thermodynamically unfavourable, high hydrogen yields are obtained. In this review we focus on three different mechanisms that are employed by a few model organisms, viz. Caldicellulosiruptor saccharolyticus and Thermoanaerobacter tengcongensis, Thermotoga maritima, and Pyrococcus furiosus, to efficiently produce hydrogen. In addition, recent developments to improve hydrogen production by hyperthermophilic and extremely thermophilic bacteria and archaea are discussed.

DNA microarray technology allows for a quick and easy comparison of complete transcriptomes, resulting in improved molecular insight in fluctuations of gene expression. After emergence of the microarray technology about a decade ago, the technique has now matured and has become routine in many molecular biology laboratories. Numerous studies have been performed that have provided global transcription patterns of many organisms under a wide range of conditions. Initially, implementation of this high-throughput technology has lead to high expectations for ground breaking discoveries. Here an evaluation is performed of the insight that transcriptome analysis has brought about in the field of hyperthermophilic archaea. The examples that will be discussed have been selected on the basis of their impact, in terms of either biological insight or technological progress.

Attachment of microorganisms to surfaces is a prerequisite for colonization and biofilm formation. The hyperthermophilic crenarchaeote Sulfolobus solfataricus was able to attach to a variety of surfaces, such as glass, mica, pyrite, and carbon-coated gold grids. Deletion mutant analysis showed that for initial attachment the presence of flagella and pili is essential. Attached cells produced extracellular polysaccharides containing mannose, galactose, and N -acetylglucosamine. Genes possibly involved in the production of the extracellular polysaccharides were identified.

Ss-LrpB is an Lrp-like transcriptional regulator from Sulfolobus solfataricus. Previously, in vitro binding of Ss-LrpB to the control region of its own gene has been extensively studied. However, nothing was known about the physiological role of this regulator yet. Here, using the knowledge of the DNA-binding sequence specificity of Ss-LrpB, several potential binding sites were predicted in silico in promoter regions of genes located adjacently to the Ss-lrpB gene. These genes include an operon encoding a pyruvate ferredoxin oxidoreductase (porDAB) and two genes encoding putative permeases. In vitro protein-DNA interaction studies allowed the identification of the Ss-LrpB binding sites in the cognate control regions. Intriguingly, the binding site organization in the por operator is identical to that in the Ss-lrpB control region. An Ss-lrpB gene disruption mutant was constructed and the gene expression of the above-mentioned targets in this mutant was analysed by qRT-PCR and compared with isogenic wild type. Our data demonstrate that in vivo Ss-LrpB acts as an activator at the promoters of the three predicted targets. Based on these results, it appears that not all regulators belonging to the archaeal Lrp family perform a function related to the amino acid metabolism, unlike the bacterial Lrp-like regulators.

Carboxylic ester hydrolyzing enzymes constitute a large group of enzymes that are able to catalyze the hydrolysis, synthesis or transesterification of an ester bond. They can be found in all three domains of life, including the group of hyperthermophilic bacteria and archaea. Esterases from the latter group often exhibit a high intrinsic stability, which makes them of interest them for various biotechnological applications. In this review, we aim to give an overview of all characterized carboxylic ester hydrolases from hyperthermophilic microorganisms and provide details on their substrate specificity, kinetics, optimal catalytic conditions, and stability. Approaches for the discovery of new carboxylic ester hydrolases are described. Special attention is given to the currently characterized hyperthermophilic enzymes with respect to their biochemical properties, 3D structure, and classification.

The hyperthermophilic archaeon Sulfolobus solfataricus has been shown to exhibit a complex transcriptional response to UV irradiation involving 55 genes. Among the strongest UV-induced genes was a putative pili biogenesis operon encoding a potential secretion ATPase, two pre-pilins, a putative transmembrane protein and a protein of unknown function. Electron microscopy and image reconstruction of UV-treated cells showed straight pili with 10 nm in diameter, variable in length, not bundled or polarized and composed of three evenly spaced helices, thereby clearly being distinguishable from archaeal flagella. A deletion mutant of SSO0120, the central type II/IV secretion ATPase, did not produce pili. It could be complemented by reintroducing the gene on a plasmid vector. We have named the operon ups operon for UV-inducible pili operon of Sulfolobus. Overexpression of the pre-pilins, Ups-A/B (SSO0117/0118) in Sulfolobus resulted in production of extremely long filaments. Pronounced cellular aggregation was observed and quantified upon UV treatment. This aggregation was a UV-dose-dependent, dynamic process, not inducible by other physical stressors (such as pH or temperature shift) but stimulated by chemically induced double-strand breaks in DNA. We hypothesize that pili formation and subsequent cellular aggregation enhance DNA transfer among Sulfolobus cells to provide increased repair of damaged DNA via homologous recombination.

Background The ubiquitous Rad50 and Mre11 proteins play a key role in many processes involved in the maintenance of genome integrity in Bacteria and Eucarya, but their function in the Archaea is presently unknown. We showed previously that in most hyperthermophilic archaea, rad50-mre11 genes are linked to nurA encoding both a single-strand endonuclease and a 5' to 3' exonuclease, and herA, encoding a bipolar DNA helicase which suggests the involvement of the four proteins in common molecular pathway(s). Since genetic tools for hyperthermophilic archaea are just emerging, we utilized immuno-detection approaches to get the first in vivo data on the role(s) of these proteins in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius. Results We first showed that S. acidocaldarius can repair DNA damage induced by high doses of gamma rays, and we performed a time course analysis of the total levels and sub-cellular partitioning of Rad50, Mre11, HerA and NurA along with the RadA recombinase in both control and irradiated cells. We found that during the exponential phase, all proteins are synthesized and display constant levels, but that all of them exhibit a different sub-cellular partitioning. Following gamma irradiation, both Mre11 and RadA are immediately recruited to DNA and remain DNA-bound in the course of DNA repair. Furthermore, we show by immuno-precipitation assays that Rad50, Mre11 and the HerA helicase interact altogether. Conclusion Our analyses strongly support that in Sulfolobus acidocaldarius, the Mre11 protein and the RadA recombinase might play an active role in the repair of DNA damage introduced by gamma rays and/or may act as DNA damage sensors. Moreover, our results demonstrate the functional interaction between Mre11, Rad50 and the HerA helicase and suggest that each protein play different roles when acting on its own or in association with its partners. This report provides the first in vivo evidence supporting the implication of the Mre11 protein in DNA repair processes in the Archaea and showing its interaction with both Rad50 and the HerA bipolar helicase. Further studies on the functional interactions between these proteins, the NurA nuclease and the RadA recombinase, will allow us to define their roles and mechanism of action.

Nanomaterials as solid supports can improve the efficiency of immobilized enzymes by reducing diffusional limitation as well as by increasing the surface area per mass unit and therefore improving enzyme loading. In this work, beta-glucosidase from almonds was immobilized on two smectite nanoclays. The resulting hybrid biocatalysts were characterized by a combination of powder X-ray diffraction, X-ray photoelectron spectroscopy, thermogravimetric. analysis, differential thermal analysis, and infrared spectroscopy. Biochemical studies showed an improved thermostability of the immobilized enzyme as well as enhanced performance at higher temperatures and in a wider pH range. Chemistry of Materials

Iron-peroxide intermediates are central in the reaction cycle of many iron-containing biomolecules. We trapped iron(III)-(hydro) peroxo species in crystals of superoxide reductase (SOR), a nonheme mononuclear iron enzyme that scavenges superoxide radicals. X-ray diffraction data at 1.95 angstrom resolution and Raman spectra recorded in crystallo revealed iron-(hydro) peroxo intermediates with the ( hydro) peroxo group bound end-on. The dynamic SOR active site promotes the formation of transient hydrogen bond networks, which presumably assist the cleavage of the iron- oxygen bond in order to release the reaction product, hydrogen peroxide. ispartof: Science vol:316 issue:5823 pages:449-453 status: published

Prolidase deficiency (PD) is a rare autosomal recessive connective tissue disorder caused by mutations in the prolidase gene. The PD patients show a wide range of clinical outcomes characterised mainly by intractable skin ulcers, mental retardation and recurrent respiratory infections. Here we describe five different PEPD mutations in six European patients. We identified two new PEPD mutant alleles: a 13 bp duplication in exon 8, which is the first reported duplication in the prolidase gene and a point mutation resulting in a change in amino acid E412, a highly conserved residue among different species. The E412K substitution is responsible for the first reported phenotypic variability within a family with severe and asymptomatic outcomes.

The alpha-galactosidase (AGA) from Bifidobacterium adolescentis DSM 20083 has a high transglycosylation activity. The optimal conditions for this activity are pH 8, and 37 degrees C. At high melibiose concentration (600 mM), approximately 64% of the enzyme-substrate encounters resulted in transglycosylation. Examination of the acceptor specificity showed that AGA required a hydroxyl group at C-6 for transglycosylation. Pentoses, hexuronic acids, deoxyhexoses, and alditols did not serve as acceptor molecules. Disaccharides were found to be good acceptors. A putative 3D-structure of the catalytic site of AGA was obtained by homology modeling. Based on this structure and amino acid sequence alignments, site-directed mutagenesis was performed to increase the transglycosylation efficiency of the enzyme, which resulted in four positive mutants. The positive single mutations were combined, resulting in six double mutants. The mutant H497M had an increase in transglycosylation of 16%, whereas most of the single mutations showed an increase of 2%-5% compared to the wild-type AGA. The double mutants G382C-Y500L, and H497M-Y500L had an increase in transglycosylation activity of 10%-16%, compared to the wild-type enzyme, whereas the increase for the other double mutants was low (4%-7%). The results show that with a single mutation (H497M) the transglycosylation efficiency can be increased from 64% to 75% of all enzyme-substrate encounters. Combining successful single mutants in double mutations did not necessarily result in an extra increase in transglycosylation efficiency. The donor and acceptor specificity did not change in the mutants, whereas the thermostability of the mutants with G382C decreased drastically.

Archaea and bacteria that optimally grow at temperatures above 60C and 80C are referred to as thermophiles and hyperthermophiles, respectively (Stetter, 1996). Since their discovery in the late 1960s (Brock and Freeze, 1969), attempts were made to reveal the secrets of the thermal resistance of these microorganisms, initially by physiological, biochemical and genetic analysis (Allers and Mevarech, 2005). In addition, the sequencing of the genomes of many thermophiles during the last decade has allowed for a series of genome-based research lines. Comparative genomics is the in silico analysis of genome data that aims to predict the metabolic potential of an organism, including the interconversions of metabolites and the regulation thereof (Ettema et al., 2005; Makarova and Koonin, 2003). Functional genomics is the experimental analysis at the level of RNA (transcriptomics), protein (proteomics), as well as metabolites (metabolomics). In general, such holistic studies aim at addressing the phenotypic response of an organism either to different cultivation conditions, or to genotypic variations. Here we review recent developments of functional genomics of thermophiles in general, and of the thermo-acidophilic archaeon Sulfolobus solfataricus in particular.

Archaea and bacteria that optimally grow at temperatures above 60C and 80C are referred to as thermophiles and hyperthermophiles, respectively (Stetter, 1996). Since their discovery in the late 1960s (Brock and Freeze, 1969), attempts were made to reveal the secrets of the thermal resistance of these microorganisms, initially by physiological, biochemical and genetic analysis (Allers and Mevarech, 2005). In addition, the sequencing of the genomes of many thermophiles during the last decade has allowed for a series of genome-based research lines. Comparative genomics is the in silico analysis of genome data that aims to predict the metabolic potential of an organism, including the interconversions of metabolites and the regulation thereof (Ettema et al., 2005; Makarova and Koonin, 2003). Functional genomics is the experimental analysis at the level of RNA (transcriptomics), protein (proteomics), as well as metabolites (metabolomics). In general, such holistic studies aim at addressing the phenotypic response of an organism either to different cultivation conditions, or to genotypic variations. Here we review recent developments of functional genomics of thermophiles in general, and of the thermo-acidophilic archaeon Sulfolobus solfataricus in particular.

The structural features of the hyperthermophilic endo-beta-1,3-glucanase from Pyrococcus furiosus were studied using circular dichroism, steady-state and time-resolved fluorescence spectroscopy and anisotropy. Upon heat and chemical treatment the folded and denatured states of the protein were characterized by distinguishable spectral profiles that identified a number of conformational states. The fluorescence methods showed that the spectral differences arose from changes in the local environment around specific tryptophan residues in the native, partially folded, partially unfolded and completely unfolded state. A structural resemblance was observed between the native protein and the structurally perturbed state which resulted after heat treatment at 110 degrees C. The enzyme underwent disruption of the native secondary and tertiary structure only after incubation at biologically extremely high temperatures (i.e. 150 degrees C), whilst in the presence of 8 m of guanidine hydrochloride the protein was partially unfolded.

Some sulfate-reducing and microaerophilic bacteria rely on the enzyme superoxide reductase (SOR) to eliminate the toxic superoxide anion radical (O-2.(-)). SOR catalyses the one-electron reduction Of O-2.(-) to hydrogen peroxide at a nonheme ferrous iron center. The structures of Desulfoarculus baarsii SOR (mutant E47A) alone and in complex with ferrocyanide were solved to 1.15 and 1.7 Angstrom resolution, respectively. The latter structure, the first ever reported of a complex between ferrocyanide and a protein, reveals that this organo-metallic compound entirely plugs the SOR active site, coordinating the active iron through a bent cyano bridge. The subtle structural differences between the mixed-valence and the fully reduced SOR-ferrocyanide adducts were investigated by taking advantage of the photoelectrons induced by X-rays. The results reveal that photo-reduction from Fe(III) to Fe(II) of the iron center, a very rapid process under a powerful synchrotron beam, induces an expansion of the SOR active site. ispartof: Structure vol:12 issue:9 pages:1729-1740 status: published

Three NADH oxidase encoding genes noxA-1 , noxB-1 and noxC were cloned from the genome of Archaeoglobus fulgidus , expressed in Escherichia coli , and the gene products were purified and characterized. Expression of noxA-1 and noxB-1 resulted in active gene products of the expected size. The noxC gene was expressed as well but the protein produced showed no activity in the standard Nox assay. NoxA-1 and NoxB-1 are both FAD-containing enzymes with subunit molecular masses of 48 and 69 kDa, respectively. NoxA-1 exists predominantly as homodimer, NoxB-1 as monomer. NoxA-1 and NoxB-1 showed pH optimum of 8.0 and 6.5, with specific NADH oxidase activities of 5.8 U.mg(-1) and 4.1 U.mg(-1) , respectively. Both enzymes were specific for NADH as electron donor, but with different apparent K (m) values (NoxA-1, 0.13 mm; NoxB-1, 0.011 mm). The apparent K (m) values for oxygen differed significantly (NoxA-1, 0.06 mm; NoxB-1, 2.9 mm). In contrast with all mesophilic homologues, both enzymes were found to produce predominantly H-2 O-2 instead of H-2 O. Despite apparent similarities, NoxB-1 is essentially different from NoxA-1. Whereas NoxA-1 resembles typical H-2 O-producing Nox enzymes that are expected to have a role in oxidative stress defence, NoxB-1 belongs to a small group of enzymes that is involved in catalysing the reduction of unsaturated acids and aldehydes, suggesting a role in fatty acid oxidation. Moreover, NoxB-1 contains a ferredoxin-like motif, which is absent in NoxA-1.

Substrate and product inhibition of hydrogen production during sucrose fermentation by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was studied. The inhibition kinetics were analyzed with a noncompetitive, nonlinear inhibition model. Hydrogen was the most severe inhibitor when allowed to accumulate in the culture. Concentrations of 5-10 mM H2 in the gas phase ( partial hydrogen pressure (pH2) of (1-2) · 104 Pa) initiated a metabolic shift to lactate formation. The extent of inhibition by hydrogen was dependent on the density of the culture. The highest tolerance for hydrogen was found at low volumetric hydrogen production rates, as occurred in cultures with low cell densities. Under those conditions the critical hydrogen concentration in the gas phase was 27.7 mM H2 ( pH2 of 5.6 · 104 Pa); above this value hydrogen production ceased completely. With an efficient removal of hydrogen sucrose fermentation was mainly inhibited by sodium acetate. The critical concentrations of sucrose and acetate, at which growth and hydrogen production was completely inhibited (at neutral pH and 70°C), were 292 and 365 mM, respectively. Inorganic salts, such as sodium chloride, mimicked the effect of sodium acetate, implying that ionic strength was responsible for inhibition. Undissociated acetate did not contribute to inhibition of cultures at neutral or slightly acidic pH. Exposure of exponentially growing cultures to concentrations of sodium acetate or sodium chloride higher than ca. 175 mM caused cell lysis, probably due to activation of autolysins. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 255-262, 2003. (Less)

The cytoplasmic membrane of bacteria and archaea determine to a large extent the composition of the cytoplasm. Since the ion and in particular the proton and/or the sodium ion electrochemical gradients across the membranes are crucial for the bioenergetic conditions of these microorganisms, strategies are needed to restrict the permeation of these ions across their cytoplasmic membrane. The proton and sodium permeabilities of all biological membranes increase with the temperature. Psychrophilic and mesophilic bacteria, and mesophilic, (hyper)thermophilic and halophilic archaea are capable of adjusting the lipid composition of their membranes in such a way that the proton permeability at the respective growth temperature remains low and constant (homeo-proton permeability). Thermophilic bacteria, however, have more difficulties to restrict the proton permeation across their membrane at high temperatures and these organisms have to rely on the less permeable sodium ions for maintaining a high sodium-motive force for driving their energy requiring membrane-bound processes. Transport of solutes across the bacterial and archaeal membrane is mainly catalyzed by primary ATP driven transport systems or by proton or sodium motive force driven secondary transport systems. Unlike most bacteria, hyperthermophilic bacteria and archaea prefer primary ATP-driven uptake systems for their carbon and energy sources. Several high-affinity ABC transporters for sugars from hyperthermophiles have been identified and characterized. The activities of these ABC transporters allow these organisms to thrive in their nutrient-poor environments.

14 pages, 10 figures, 6 tables.-- PMID: 10924516 [PubMed]., The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AF218569., A new class of glutamate dehydrogenase (GDH) is reported. The GDH of Streptomyces clavuligerus was purified to homogeneity and characterized. It has a native molecular mass of 1,100 kDa and exists as an alpha(6) oligomeric structure composed of 183-kDa subunits. GDH, which requires AMP as an essential activator, shows a maximal rate of catalysis in 100 mm phosphate buffer, pH 7.0, at 30 degrees C. Under these conditions, GDH displayed hyperbolic behavior toward ammonia (K(m), 33 mm) and sigmoidal responses to changes in alpha-ketoglutarate (S(0.5) 1.3 mm; n(H) 1.50) and NADH (S(0.5) 20 microm; n(H) 1.52) concentrations. Aspartate and asparagine were found to be allosteric activators. This enzyme is inhibited by an excess of NADH or NH(4)(+), by some tricarboxylic acid cycle intermediates and by ATP. This GDH seems to be a catabolic enzyme as indicated by the following: (i) it is NAD-specific; (ii) it shows a high value of K(m) for ammonia; and (iii) when S. clavuligerus was cultured in minimal medium containing glutamate as the sole source of carbon and nitrogen, a 5-fold increase in specific activity of GDH was detected compared with cultures provided with glycerol and ammonia. GDH has 1,651 amino acids, and it is encoded by a DNA fragment of 4,953 base pairs (gdh gene). It shows strong sequence similarity to proteins encoded by unidentified open reading frames present in the genomes of species belonging to the genera Mycobacterium, Rickettsia, Pseudomonas, Vibrio, Shewanella, and Caulobacter, suggesting that it has a broad distribution. The GDH of S. clavuligerus is the first member of a class of GDHs included in a subfamily of GDHs (large GDHs) whose catalytic requirements and evolutionary implications are described and discussed., This investigation was supported by Comisión Interministerial de Ciencia y Tecnología, Madrid, Spain, Grant AMB97-0603-C02-01, Fondo Europeo de Desarrollo Regional Grant 1FD97-0245, and Junta de Castilla y León Grant LE 42/96.

Desulfovibrio gigas NCIMB 9332 cells grown in ethanol-containing medium with 0.1 mu M tungstate contained a benzylviologen-linked aldehyde oxidoreductase, The enzyme was purified to electrophoretic homogeneity and found to be a homodimer with a subunit M(r) of 62,000, It contained 0.68 a 0.08 W, 4.8 Fe, and 3.2 +/- 0.2 labile S per subunit, After acid iodine oxidation of the purified enzyme, a fluorescence spectrum typical for form A of molybdopterin was obtained, Acetahdehyde, propionaldehyde, and benzaldehyde were excellent substrates, with apparent K-m values of 12.5, 10.8, and 20 mu M, respectively, The natural electron acceptor is not yet known; benzylviologen was used as an artificial electron acceptor (apparent K-m, 0.55 mM), The enzyme was activated by potassium ions and strongly inhibited by cyanide, arsenite, and iodoacetate, In the as-isolated enzyme, electron paramagnetic resonance studies readily detected W(V) as a complex signal with g values in the range of 1.84 to 1.97, The dithionite-reduced enzyme exhibited a broad signal at low temperature,vith g = 2.04 and 1.92; this is indicative of a [4Fe-4S](1+) cluster interacting with a second paramagnet, possibly the S = 1 system of W(TV), Until now W-containing aldehyde oxidoreductases had only been found in two Clostridium strains and two hyperthermophilic archaea, The D. gigas enzyme is the first example of such an enzyme in a gram-negative bacterium.