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This study was to understand the effect of LED light intensity, color, and illumination pattern on the growth of and bacteriorhodopsin (BR) accumulation in archaeon Halobacterium halobium. Experimental results showed that archaeon growth and BR content increased with increasing white light intensity. Green and white LED lights were found to be the most effective for archaeon growth and BR accumulation; here, effectiveness is defined based on photons shined on the bioreactor. The 12-h light/12-h dark cycle illumination pattern resulted in longer lag phase but achieved higher final cell growth and BR accumulation than continuous white light or an instant flash of light/dark illumination. The fade pattern that had smooth transitioning among green, blue, and red lights was better than the jump pattern without transitioning. The highest cell dry weight and BR content of H. halobium were 1.84 g l-1 and 11.76 mg l-1, respectively, under 29.85 µmol/m2s LED white light illumination. The green LED light of 1.39 µmol/m²s had the highest energy-specific conversion effectiveness and saved energy consumption by 84 and 87% per cell biomass and BR dry weight, respectively, compared to the worst case of LED white light of 29.85 µmol/m2s. [ABSTRACT FROM AUTHOR]

The acidophilic and thermophilic pullulanases have many potential applications in the processes of starch liquefaction and saccharification. In this study, a gene encoding an amylopullulanase from Thermofilum pendens (TPApu) was heterologously expressed in Escherichia coli . Although TPApu possessed the same continuous GH57N_Apu domain and the succeeding α-helical region as other two amylopullulanases from Staphylothermus marinus (SMApu) and Caldivirga maquilingensis (CMApu), it only showed maximal amino acid identities of 25.7–28.7% with CMApu and SMApu. The purified TPApu appeared as a single band of SDS-PAGE with a molecular mass of 65.5 kDa and exhibited the maximal activity at pH 3.5 and 95–100 °C. TPApu had the highest catalytic efficiency towards pullulan (kcat/km, 8.79 s −1 mL mg −1 ) and α-cyclodextrin (kcat/km, 0.36 s −1 mM −1 ). In the initial stages, the ring-opening reactions of γ-cyclodextrin, 6- O -glucosyl-β-cyclodextrin, 6- O -maltosyl-β-cyclodextrin and the debranching reactions of 6- O -maltooctaosyl-β-cyclodextrin were firstly catalyzed. In the subsequent reactions, a serial of maltooligosaccharides were produced. As the most acidophilic amylopullulanase among thermophilic pullulanases reported to date, TPApu preferred to debranch the DP6–12 side chains of amylopectin at pH 4.5 and 100 °C. [ABSTRACT FROM AUTHOR]

Application of halophiles can decrease the cost of polyhydroxyalkanoate (PHA) production or bioplastic which are an alternative to the petroleum-derived plastic. Extremely halophilic archaeon, Natrinema ajinwuensis RM-G10 accumulated 61.02 ± 0.68% PHA of its cell dry mass at 72 h in repeated batch cultures yielding 0.210 ± 0.001 g L −1 h −1 volumetric productivity after selection of the best cultivation conditions. Transmission electron microscopy showed the presence of PHA granules inside the archaeal cells. Characterization by gas chromatographic analysis, gas chromatographic- mass spectrophotometric analysis, thermogravimetric analysis, differential scanning calorimetric analysis, X-ray diffraction analysis, Fourier transform infra red spectroscopy and nuclear magnetic resonance spectroscopy revealed the polymer to be poly(3-hydroxybutyrate- co -3-hydroxyvalerate) with 13.93 mol% 3-hydroxyvalerate content and having 35.45% crystallinity, −12.3 °C glass transition temperature, 143 °C and 157.5 °C melting temperatures and 284 °C degradation temperature. This is the first report on production enhancement (on a small scale) and characterization of the polyhydroxyalkanoate produced by Natrinema ajinwuensis (as synonym) ≡ Natrinema altunense strain RM-G10 and the Natrinema genus in general. [ABSTRACT FROM AUTHOR]

The DNA coding sequence of TaqStoffel polymerase was fused with the DNA-binding domain of Pyrococcus furiosus ligase. The resulting novel recombinant gene was cloned and expressed in E. coli. The recombinant enzyme was purified and its enzymatic features were studied. The fusion protein ( PfuDBDlig- TaqS) was found to have enhanced processivity as a result of the conversion of the TaqDNA polymerase from a relatively low processive to a highly processive enzyme. The abovementioned processivity enhancement was about threefold as compared to the recombinant TaqStoffel DNA polymerase ( TaqS), and the recombinant fusion protein was more thermostable. It had a half-life of 23 min at 99 °C as compared to 10 min for TaqS. The fusion protein also showed a significantly higher resistance to PCR inhibitors such as heparin or lactoferrin and the fusion polymerase-amplified GC-rich templates much more efficiently and was efficient even with 78% GC pairs. [ABSTRACT FROM AUTHOR]

Many ammonia-rich biomass sources, such as manures and protein-rich substrates, are potential inhibitors of the anaerobic digestion (AD) process. It was previously demonstrated that bioaugmentation of Methanoculleus bourgensis MS2 T in an ammonia inhibited process in a continuous stirred tank reactor (CSTR), resulted in up to 90% recovery of the methane production compared to the uninhibited production. However, cultivation of pure strains has practical difficulties due to the need of special growth media and sterile conditions. In contrast, acclimatized enriched cultures have minor sterility requirements. In the current study, an enriched ammonia-tolerant methanogenic culture was bioaugmented in a CSTR reactor operating under ammonia-induced, inhibited-steady-state. The results demonstrated that bioaugmentation, completely counteracted the ammonia toxicity effect. This indicates that a commercial application of bioaugmentation could improve up to 36% the methane production, the greenhouse gas reduction efficiency and the gross revenue of ammonia inhibited full scale biogas reactors. 16S rRNA gene sequencing showed that bioaugmentation changed the microbial composition of the reactors resulting in higher bacterial and lower archaeal community diversity. The bioaugmented reactor showed a fourfold increase of the abundance of the bioaugmented methanogens compared to the control reactor. This indicates that ammonia-tolerant methanogens established well in the ammonia-inhibited reactor and dominated over the domestic methanogenic population. Finally, this study showed that the enriched culture alleviated ammonia toxicity 25% more efficiently than the previously used pure culture. [ABSTRACT FROM AUTHOR]

Nitriles are important chemical building blocks for the synthesis of intermediates in fine chemical and pharmaceutical industries. Here, we report a new highly thermostable nitrilase from an Antarctic Pyrococcus sp. MC-FB, a hyperthermophilic archaeon. A gene that encoded a nitrilase was identified and subsequently cloned and overexpressed in Escherichia coli. The recombinant nitrilase, named NitMC-FB, is active as a homodimer (60 kDa) with an optimal temperature and pH of 90 °C and 7.0, respectively. NitMC-FB hydrolyzes preferentially aromatic nitriles, being the first aromatic nitrilase from an archaeon described so far. The K and V parameters were determined to be 13.9 mM and 3.7 μmol/min*mg, respectively, with 2-cyanopyridine as the substrate. Additionally, the recombinant nitrilase is highly thermostable with a half-life of 8 h at 90 °C. [ABSTRACT FROM AUTHOR]

Copyright of Egyptian Journal of Experimental Biology (Botany) is the property of Egyptian Society of Experimental Biology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Egyptian Journal of Experimental Biology (Zoology) is the property of Egyptian Society of Experimental Biology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The crystal structure of the Ta1207 protein from Thermoplasma acidophilum is reported. Ta1207 was identified in a screen for high-molecular-weight protein complexes in T. acidophilum. In solution, Ta1207 forms homopentamers of 188 kDa. The crystal structure of recombinant Ta1207 solved by Se-MAD at 2.4 Å resolution revealed a complex with fivefold symmetry. In the crystal lattice, calcium ions induce the formation of a nanocage from two pentamers. The Ta1207 protomers comprise two domains with the same novel α/β topology. A deep pocket with a binding site for a negatively charged group suggests that Ta1207 functions as an intracellular receptor for an unknown ligand. Homologues of Ta1207 occur only in Thermoplasmatales and its function might be related to the extreme lifestyle of these archaea. The thermostable Ta1207 complex might provide a useful fivefold-symmetric scaffold for future nanotechnological applications. [ABSTRACT FROM AUTHOR]

In recent years, there has been a large increase in the amount of experimental evidence for diverse archaeal organisms, and these findings allow for a comprehensive analysis of archaeal genetic organization. However, studies about regulatory mechanisms in this cellular domain are still limited. In this context, we identified a repertoire of 86 DNA-binding transcription factors (TFs) in the archaeon Pyrococcus furiosus DSM 3638, that are clustered into 32 evolutionary families. In structural terms, 45% of these proteins are composed of one structural domain, 41% have two domains, and 14% have three structural domains. The most abundant DNA-binding domain corresponds to the winged helix-turn-helix domain; with few alternative DNA-binding domains. We also identified seven regulons, which represent 13.5% (279 genes) of the total genes in this archaeon. These analyses increase our knowledge about gene regulation in P. furiosus DSM 3638 and provide additional clues for comprehensive modeling of transcriptional regulatory networks in the Archaea cellular domain.

Archaea, like bacteria, use type IV pili to facilitate surface adhesion. Moreover, archaeal flagella--structures required for motility--share a common ancestry with type IV pili. While the characterization of archaeal homologs of bacterial type IV pilus biosynthesis components has revealed important aspects of flagellum and pilus biosynthesis and the mechanisms regulating motility and adhesion in archaea, many questions remain. Therefore, we screened a Haloferax volcanii transposon insertion library for motility mutants using motility plates and adhesion mutants, using an adapted air--liquid interface assay. Here, we identify 20 genes, previously unknown to affect motility or adhesion. These genes include potential novel regulatory genes that will help to unravel the mechanisms underpinning these processes. Both screens also identified distinct insertions within the genomic region lying between two chemotaxis genes, suggesting that chemotaxis not only plays a role in archaeal motility, but also in adhesion. Studying these genes, as well as hypothetical genes hvo_2512 and hvo_2876--also critical for both motility and adhesion--will likely elucidate how these two systems interact. Furthermore, this study underscores the usefulness of the transposon library to screen other archaeal cellular processes for specific phenotypic defects. [ABSTRACT FROM AUTHOR]

An extracellular halophilic alpha-amylase (AmyA) was produced by the haloarchaeon Haloterrigena turkmenica grown in medium enriched with 0.2% (w/v) starch. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) analyses showed a major band at 66.0 kDa and a peak of 54.0 kDa, respectively. Analysis of tryptic fragments of the protein present in the major SDS-PAGE band by nano-LC-ESI–MS/MS led to identification of the alpha-amylase catalytic region, encoded by the htur2110 gene, as the protein possessing the described activity. Optimal values for activity were 55 °C, pH 8.5 and 2 M NaCl, and high thermostability was showed at 55 °C and 3 M NaCl. AmyA activity was enhanced by Triton X-100 and was not influenced by n -hexane and chloroform. Starch hydrolysis produced different oligomers with maltose as the smallest end-product. The efficiency of AmyA in degrading starch contained in agronomic residues was tested in grape cane chosen as model substrate. Preliminary results showed that starch was degraded making the enzyme a potential candidate for utilization of agro-industrial waste in fuel and chemicals production. AmyA is one of the few investigated amylases produced by haloarchaea, and the first alpha-amylase described among microorganisms belonging to the genus Haloterrigena . [ABSTRACT FROM AUTHOR]

Bioleaching is an alternative technology for recovering metals from mineral ores; thermophilic microorganisms instead of mesophiles can greatly improve metal solubilization, particularly from refractory mineral species. Acidianus copahuensis is a new species of thermophilic archaea, recently isolated in our laboratory. The effect of culture growth history on the attachment of A. copahuensis to a chalcopyrite concentrate was investigated in shake flasks at 65 °C. Cells adapted to growth with chalcopyrite as energy source showed higher attachment to the mineral concentrate. A. copahuensis cells reached 100% of copper extraction in the bioleaching of chalcopyrite concentrate carried out in shake flasks cultures incubated at 65 °C and pH 2.0. This high bioleaching yield was achieved even at that not so low initial pH value probably because this archaeal species is able to form sufficient amounts of ferric iron but keeping low redox potential (Eh) values. This work also describes the effect of the addition of other energy sources on the bioleaching activity. A. copahuensis achieved the best copper extraction without any addition but copper extraction decreased when alternative substrates were added. [ABSTRACT FROM AUTHOR]

The maltooligosyl trehalose synthase gene from the hyperthermophilic archaeon Sulfolobus tokodaii strain 7 was cloned and the recombinant peotein was expressed in E. coli. The protein was purified to homogeneity by nickel column chromatography. The archaeal enzyme could catalyze an intramolecular transglycosylation reaction and convert the glycosidic bond at the reducing end of dextrins from α-1, 4 (reducing end) into α-1, 1 (non-reducing end). The most suitable temperature was 75°C and the optimal pH was 5. Substrate specificity investigation revealed that maltodextrin and maltooligosaccharide were used as substrates by the enzyme but maltose, chitooligosaccharide, sucrose and β- cyclodextrin weren't used. [ABSTRACT FROM AUTHOR]

Background Haloferax mediterranei is a denitrifying haloarchaeon using nitrate as a respiratory electron acceptor under anaerobic conditions in a reaction catalysed by pNarGH. Other ions such as bromate, perchlorate and chlorate can also be reduced. Methods Hfx. mediterranei cells were grown anaerobically with nitrate as electron acceptor and chlorate reductase activity measured in whole cells and purified nitrate reductase. Results No genes encoding (per)chlorate reductases have been detected either in the Hfx. mediterranei genome or in other haloarchaea. However, a gene encoding a chlorite dismutase that is predicted to be exported across the cytoplasmic membrane has been identified in Hfx. mediterranei genome. Cells did not grow anaerobically in presence of chlorate as the unique electron acceptor. However, cells anaerobically grown with nitrate and then transferred to chlorate-containing growth medium can grow a few generations. Chlorate reduction by the whole cells, as well as by pure pNarGH, has been characterised. No clear chlorite dismutase activity could be detected. Conclusions Hfx. mediterranei pNarGH has its active site on the outer-face of the cytoplasmic membrane and reacts with chlorate and perchlorate. Biochemical characterisation of this enzymatic activity suggests that Hfx. mediterranei or its pure pNarGH could be of great interest for waste water treatments or to better understand biological chlorate reduction in early Earth or Martian environments. General significance Some archaea species reduce (per)chlorate. However, results here presented as well as those recently reported by Liebensteiner and co-workers [1] suggest that complete perchlorate reduction in archaea follows different rules in terms of biological reactions. [ABSTRACT FROM AUTHOR]

A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus DSM3638 exhibited an extended exponential phase and atypical cell aggregation behavior. Genomic DNA from the mutant culture was sequenced and compared to wild-type (WT) DSM3638, revealing 145 genes with one or more insertions, deletions, or substitutions (12 silent, 33 amino acid substitutions, and 100 frame shifts). Approximately, half of the mutated genes were transposases or hypothetical proteins. The WT transcriptome revealed numerous changes in amino acid and pyrimidine biosynthesis pathways coincidental with growth phase transitions, unlike the mutant whose transcriptome reflected the observed prolonged exponential phase. Targeted gene deletions, based on frame-shifted ORFs in the mutant genome, in a genetically tractable strain of P. furiosus (COM1) could not generate the extended exponential phase behavior observed for the mutant. For example, a putative radical SAM family protein (PF2064) was the most highly up-regulated ORF (>25-fold) in the WT between exponential and stationary phase, although this ORF was unresponsive in the mutant; deletion of this gene in P. furiosus COM1 resulted in no apparent phenotype. On the other hand, frame-shifting mutations in the mutant genome negatively impacted transcription of a flagellar biosynthesis operon (PF0329-PF0338).Consequently, cells in the mutant culture lacked flagella and, unlike the WT, showed minimal evidence of exopolysaccharide-based cell aggregation in post-exponential phase. Electron microscopy of PF0331-PF0337 deletions in P. furiosus COM1 showed that absence of flagella impacted normal cell aggregation behavior and, furthermore, indicated that flagella play a key role, beyond motility, in the growth physiology of P. furiosus. [ABSTRACT FROM AUTHOR]

Archaea, like bacteria, use type IV pili to facilitate surface adhesion. Moreover, archaeal flagella—structures required for motility—share a common ancestry with type IV pili. While the characterization of archaeal homologs of bacterial type IV pilus biosynthesis components has revealed important aspects of flagellum and pilus biosynthesis and the mechanisms regulating motility and adhesion in archaea, many questions remain. Therefore, we screened a Haloferax volcanii transposon insertion library for motility mutants using motility plates and adhesion mutants, using an adapted air–liquid interface assay. Here, we identify 20 genes, previously unknown to affect motility or adhesion. These genes include potential novel regulatory genes that will help to unravel the mechanisms underpinning these processes. Both screens also identified distinct insertions within the genomic region lying between two chemotaxis genes, suggesting that chemotaxis not only plays a role in archaeal motility, but also in adhesion. Studying these genes, as well as hypothetical genes hvo_2512 and hvo_2876—also critical for both motility and adhesion—will likely elucidate how these two systems interact. Furthermore, this study underscores the usefulness of the transposon library to screen other archaeal cellular processes for specific phenotypic defects.

A gene for membrane-integral inorganic pyrophosphatase (miPPase) was found in the composite genome of the extremophile archaeon Candidatus Korarchaeum cryptofilum (CKc). This korarchaeal genome shows unusual partial similarity to both major archaeal phyla Crenarchaeota and Euryarchaeota. Thus this Korarchaeote might have retained features that represent an ancestral archaeal form, existing before the occurrence of the evolutionary bifurcation into Crenarchaeota and Euryarchaeota. In addition, CKc lacks five genes that are common to early genomes at the LUCA border. These two properties independently suggest a pre-LUCA evolutionary position of this extremophile. Our finding of the miPPase gene in the CKc genome points to a role for the enzyme in the energy conversion of this very early archaeon. The structural features of its miPPase indicate that it can pump protons through membranes. An miPPase from the extremophile bacterium Caldicellulosiruptor saccharolyticus also has a sequence indicating a proton pump. Recent analysis of the three-dimensional structure of the miPPase from Vigna radiata has resulted in the recognition of a strongly acidic substrate (orthophosphate: Pi, pyrophosphate: PPi) binding pocket, containing 11 Asp and one Glu residues. Asp (aspartic acid) is an evolutionarily very early proteinaceous amino acid as compared to the later appearing Glu (glutamic acid). All the Asp residues are conserved in the miPPase of CKc, V. radiata and other miPPases. The high proportion of Asp, as compared to Glu, seems to strengthen our argument that biological energy conversion with binding and activities of orthophosphate (Pi) and energy-rich pyrophosphate (PPi) in connection with the origin and early evolution of life may have started with similar or even more primitive acidic peptide funnels and/or pockets. [ABSTRACT FROM AUTHOR]

Nicotinamidase is involved in the maintenance of NAD homeostasis and in the NAD salvage pathway of most prokaryotes, and it is considered as a possible drug target. The gene ( ASAC_0847) encoding a hypothetical nicotinamidase has been found in the genome of the thermophilic archaeon Acidilobus saccharovorans. The product of this gene, NA_As0847, has been expressed in Escherichia coli, isolated, and characterized as a Fe-containing nicotinamidase ( k/ K = 427 mM·sec)/pyrazinamidase ( k/ K = 331 mM·sec). NA_As0847 is a homodimer with molecular mass 46.4 kDa. The enzyme has high thermostability (T (60°C) = 180 min, T (80°C) = 35 min) and thermophilicity (T = 90°C, E = 30.2 ± 1.0 kJ/mol) and broad pH interval of activity, with the optimum at pH 7.5. Special features of NA_As084 are the presence of Fe instead of Zn in the active site of the enzyme and inhibition of the enzyme activity by Zn at micromolar concentrations. Analysis of the amino acid sequence revealed a new motif of the metal-binding site ( DX HXXX DXX EXXX WXX H) for homological archaeal nicotinamidases. [ABSTRACT FROM AUTHOR]

Aim: To characterize, TK1111, a cupin-type phosphoglucose isomerase from a hyperthermophilic archaeon Thermococcus kodakarensis. Methods: The genome of T. kodakarensis was searched in order to find an open reading frame annotated as glucose-6-phosphate isomerase. The structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. Effect of metal ions on the restoration of enzyme activity was examined. Results: Genome search of T. kodakarensis revealed the presence of an open reading frame, TK1111, annotated as glucose-6-phosphate isomerase. TK1111 exhibited high homology with archaeal PGIs related to the cupin-superfamily instead of well-known family of PGI enzymes. The gene encoding TK1111 consisted of 567 nucleotides corresponding to a polypeptide of 189 amino acids. The structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. Recombinant TK1111 displayed highest activity (43 U/mg) at 90 °C and pH 6.0. To our knowledge this is the only PGI from cupin-superfamily exhibiting highest activity in the acidic pH. TK1111 catalyzed the conversion of fructose 6-phosphate to glucose 6-phosphate following Michaelis-Menten kinetics with a Km and Vmax values of 2.2 mM and 12.5 µmol min-1 mg-1, respectively at 37 °C. Addition of Zn2+ ions in the assay mixture remarkably enhanced the enzyme activity in contrast to the PGI from Thermococcus litoralis, another member of the same genus exhibiting 75% identity to TK1111. The enzyme activity was completely diminished when TK1111 was heated at 70 °C in the presence of EDTA. Complete restoration of the enzyme activity was observed when TK1111 was incubated at room temperature in the presence of Zn2+. Conclusion: TK1111 from T. kodakaraensis is a novel phosphoglucose isomerase that belongs to cupintype isomerases. To our knowledge this is the only PGI from cupin-family exhibiting highest activity in the acidic pH. Enzyme activity was remarkably enhanced in the presence of Zn2+ in contrast to other members of the family. [ABSTRACT FROM AUTHOR]

The gene encoding a β-glucosidase from the archaeon Thermofilum pendens (Tpbgl) was cloned and expressed in Escherichia coli. The purified recombinant enzyme had a molecular mass of 77.8 kDa and released glucose or mannose from p-nitrophenyl-β-d-glucopyranoside (pNPG), cellobiose, mannobiose, and genistin. Peak Tpbgl activity was detected at 90°C, and 50% activity remained after incubation for 60 min at 95°C. The optimal pH for pNPG hydrolysis was 3.5. When the enzyme was incubated with pNPG in the presence of ethanol and propanol, the glucose moiety was transferred to acceptor alcohols. Tpbgl is the archaeal β-glucosidase from glucoside hydrolase family 3 and found to be most heat stable under extremely acidic conditions (pH 3.5). The kinetic parameters revealed that Tpbgl had the highest catalytic efficiency toward pNPG (k cat /K m = 3.05) with strong substrate affinity for such natural substrates as cellobiose (K m = 0.149) and mannobiose (K m = 0.147). Genistin solubilized in 10–40% DMSO was hydrolyzed to genistein with nearly 99% conversion, indicating that high concentrations of the water-insoluble isoflavone glycoside can be treated by the enzyme. Our results indicate that Tpbgl has great potential in cellulose saccharification and the glucoside hydrolysis of natural compounds. [Copyright &y& Elsevier]

The extreme acid conditions required for scorodite (FeAsO·2HO) biomineralization (pH below 1.3) are suboptimal for growth of most thermoacidophilic Archaea. With the objective to develop a continuous process suitable for biomineral production, this research focuses on growth kinetics of thermoacidophilic Archaea at low pH conditions. Ferrous iron oxidation rates were determined in batch-cultures at pH 1.3 and a temperature of 75°C for Acidianus sulfidivorans, Metallosphaera prunea and a mixed Sulfolobus culture. Ferrous iron and CO in air were added as sole energy and carbon source. The highest growth rate (0.066 h) was found with the mixed Sulfolobus culture. Therefore, this culture was selected for further experiments. Growth was not stimulated by increase of the CO concentration or by addition of sulphur as an additional energy source. In a CSTR operated at the suboptimal pH of 1.1, the maximum specific growth rate of the mixed culture was 0.022 h, with ferrous iron oxidation rates of 1.5 g L d. Compared to pH 1.3, growth rates were strongly reduced but the ferrous iron oxidation rate remained unaffected. Influent ferrous iron concentrations above 6 g L caused instability of Fe oxidation, probably due to product (Fe) inhibition. Ferric-containing, nano-sized precipitates of K-jarosite were found on the cell surface. Continuous cultivation stimulated the formation of an exopolysaccharide-like substance. This indicates that biofilm formation may provide a means of biomass retention. Our findings showed that stable continuous cultivation of a mixed iron-oxidizing culture is feasible at the extreme conditions required for continuous biomineral formation. [ABSTRACT FROM AUTHOR]

Using citrate synthase from the hyperthermophile Pyrococcus furiosus ( PfCS) as our test molecule, we show through guanidine hydrochloride-induced unfolding that the dimer separates into folded, but inactive, monomers before individual subunit unfolding takes place. Given that forces across the dimer interface are vital for thermostability, a robust computational method was derived that uses the University of Houston Brownian Dynamics (UHBD) program to calculate both the hydrophobic and electrostatic contribution to the dimerisation energy at 100°C. The results from computational and experimental determination of the lowered stability of interface mutants were correlated, being both of the same order of magnitude and placing the mutant proteins in the same order of stability. This computational method, optimised for hyperthermophilic molecules and tested in the laboratory, after further testing on other examples, could be of widespread use in the prediction of thermostabilising mutations in other oligomeric proteins for which dissociation is the first step in unfolding. [ABSTRACT FROM AUTHOR]

Pyrobaculum islandicum is a hyperthermophilic archaeon. P. islandicum cells have been suggested to multiply by constriction, budding and branching, as no septa were observed in cells by phase-contrast light microscopy. In this study, we observed the cells using transmission electron microscopy, scanning electron microscopy, and light microscopy with dark-field image analyses, and we report binary fission via septum formation to be the main mode of P. islandicum’s proliferation. “Long cells” reported previously were found to comprise several cylindrical cells that align in tandem. [ABSTRACT FROM AUTHOR]

The haloarchaeon Haloferax mediterranei is able to assimilate nitrate or nitrite using the assimilatory nitrate pathway. An assimilatory nitrate reductase (Nas) and an assimilatory nitrite reductase (NiR) catalyze the first and second reactions, respectively. The genes involved in this process are transcribed as two messengers, one polycistronic ( nasABC; nasA encodes Nas) and one monocistronic ( nasD; codes for NiR). Here we report the Hfx mediterranei growth as well as the Nas and NiR activities in presence of high nitrate, nitrite and salt concentrations, using different approaches such as physiological experiments and enzymatic activities assays. The nasA and nasD expression profiles are also analysed by real-time quantitative PCR. The results presented reveal that the assimilatory nitrate/nitrite pathway in Hfx mediterranei takes place even if the salt concentration is higher than those usually present in the environments where this microorganism inhabits. This haloarchaeon grows in presence of 2 M nitrate or 50 mM nitrite, which are the highest nitrate and nitrite concentrations described from a prokaryotic microorganism. Therefore, it could be attractive for bioremediation applications in sewage plants where high salt, nitrate and nitrite concentrations are detected in wastewaters and brines. [ABSTRACT FROM AUTHOR]

Strain B31T is a Gram-staining-negative, motile, and extremely halophilic archaeon that was isolated from salt-fermented seafood. Its morphology, physiology, biochemical features, and 16S rRNA gene sequence were determined. Phylogenetic analysis of its 16S rRNA gene sequence and composition of its major polar lipids placed this archaeon in the genus Halorubrum of the family Halobacteriaceae. Strain B31T showed 97.3, 97.2, and 96.9 % 16S rRNA similarity to the type strains of Halorubrum alkaliphilum, Hrr. tibetense, and Hrr. vacuolatum, respectively. Its major polar lipids were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and sulfated diglycosyl diether (S-DGD). Genomic DNA from strain B31T has a 61.7 mol% G+C content. Analysis of 16S rRNA gene sequences, as well as physiological and biochemical tests, identified genotypic and phenotypic differences between strain B31T and other Halorubrum species. The type strain of the novel species is B31T (=JCM 15757T =DSM 19504T). [ABSTRACT FROM AUTHOR]

The article presents a study which identifies glutamate decarboxylase (GAD) from the hyperthermophilic archaea, Pyrococcus horikoshii, which aims to develop a hyperthermostable GAD for industrial use. The study sought to express and purified the GAD homologs from the archaea through the expression system of Escherichia coli. The identified archaeal GAD enzyme has lower GAD activity that other GAD and has potential use in the industrial production of ᵧ-amino-butyric acid (GABA).

The partial amino acid sequence of the sulfolobal thermoprotein biochemically characterized as poly(ADP-ribose)polymerase-like enzyme overlaps those of DING proteins. This group of proteins, widely occurring in animals, plants and eubacteria, shows a characteristic and highly conserved N-terminus, DINGGGATL. The sequence of the N-terminal region and of the analyzed tryptic peptides of the sulfolobal thermozyme shows a high similarity with most of the DING proteins from databases. This is the first example of a DING protein from a sulfolobal source. [ABSTRACT FROM AUTHOR]

The extremely thermophilic anaerobic archaeon strain, HJ21, was isolated from a deep-sea hydrothermal vent, could produce hyperthermophilic α-amylase, and later was identified as Thermococcus from morphological, biochemical, and physiological characteristics and the 16S ribosomal RNA gene sequence. The extracellular thermostable α-amylase produced by strain HJ21 exhibited maximal activity at pH 5.0. The enzyme was stable in a broad pH range from pH 5.0 to 9.0. The optimal temperature of α-amylase was observed at 95°C. The half-life of the enzyme was 5 h at 90°C. Over 40% and 30% of the enzyme activity remained after incubation at 100°C for 2 and 3 h, respectively. The enzyme did not require Ca2+ for thermostability. This α-amylase gene was cloned, and its nucleotide sequence displayed an open reading frame of 1,374 bp, which encodes a protein of 457 amino acids. Analysis of the deduced amino acid sequence revealed that four homologous regions common in amylases were conserved in the HJ21 α-amylase. The molecular weight of the mature enzyme was calculated to be 51.4 kDa, which correlated well with the size of the purified enzyme as shown by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [ABSTRACT FROM AUTHOR]

Aims: To isolate Ferroplasma thermophilum L1T from a low pH environment and to understand its role in bioleaching of chalcopyrite. Methods and Results: Using serial dilution method, a moderately thermophilic and acidophilic ferrous iron-oxidizing archaeon, named L1T, was isolated from a chalcopyrite-leaching bioreactor. The morphological, biochemical and physiological characteristics of strain L1T and its role in bioleaching of chalcopyrite were studied. Strain L1T was a nonmotile coccus that lacked cell wall. Strain L1T had a temperature optimum of 45°C and the optimum pH for growth was 1·0. Strain L1T was capable of chemomixotrophic growth on ferrous iron and yeast extract. Results of fatty acid analysis, DNA–DNA hybridization, G+C content, and analysis based on 16S rRNA gene sequence indicated that strain L1T should be grouped in the genus Ferroplasma, and represented a new species, Ferroplasma thermophilum. Ferroplasma thermophilum in combination with Acidithiobacillus caldus and Leptospirillum ferriphilum could improve the copper dissolution in bioleaching of chalcopyrite. Conclusions: A novel extremely acidophilic, moderately thermophilic archaeon isolated from a bioleaching reactor has been identified as F. thermophilum that played an important role in bioleaching of chalcopyrite at low pH. Significance and Impact of the Study: This study contributes to understand the characteristics of F. thermophilum L1T and its role in bioleaching of sulfide ores. [ABSTRACT FROM AUTHOR]

Abstract: Osmotically inducible protein C (OsmC) is involved in the cellular defense mechanism against oxidative stress caused by exposure to hyperoxides or elevated osmolarity. OsmC was identified by two-dimensional electrophoresis (2DE) analysis as a protein that is overexpressed in response to osmotic stress, but not under heat and oxidative stress. Here, an OsmC gene from T. kodakaraensis KOD1 was cloned and expressed in Escherichia coli. TkOsmC showed a homotetrameric structure based on gel filtration and electron microscopic analyses. TkOsmC has a significant peroxidase activity toward both organic and inorganic peroxides in high, but not in low temperature. [Copyright &y& Elsevier]

A gene encoding an intracellular glucoamylase was identified in the genome of the extreme thermoacidophilic Archaeon Thermoplasma acidophilum. The gene taGA, consisting of 1,911 bp, was cloned and successfully expressed in Escherichia coli. The recombinant protein was purified 22-fold to homogeneity using heat treatment, anion-exchange chromatography, and gel filtration. Detailed analysis shows that the glucoamylase, with a molecular weight of 66 kDa per subunit, is a homodimer in its active state. Amylolytic activity was measured over a wide range of temperature (40–90°C) and pH (pH 3.5–7) and was maximal at 75°C and at acidic condition (pH 5). The recombinant archaeal glucoamylase uses a variety of polysaccharides as substrate, including glycogen and amylose. Maximal activity was measured towards amylopectin with a specific activity of 4.2 U/mg and increased almost threefold in the presence of manganese. Calcium ions have a pronounced effect on enzyme stability; in the presence of 5 mM CaCl2, the half-life increased from 15 min to 2 h at 80°C. [ABSTRACT FROM AUTHOR]

The D-trehalose/D-maltose-binding protein (TMBP), a monomeric protein of 48 kDa, is one component of the trehalose and maltose (Mal) uptake system. In the hyperthermophilic archaeon Thermococcus litoralis, this is mediated by a protein-dependent ATP-binding cassette system transporter. The gene coding for a thermostable TMBP from the archaeon T. litoralis has been cloned, and the recombinant protein has been expressed in E. coli. The recombinant TMBP has been purified to homogeneity and characterized. It exhibits the same functional and structural properties as the native one. In fact, it is highly thermostable and binds sugars, such as maltose, trehalose and glucose, with high affinity. In this work, we have immobilized TMBP on a porous silicon wafer. The immobilization of TMBP to the chip was monitored by reflectivity and Fourier Transformed Infrared spectroscopy. Furthermore, we have tested the optical response of the protein-Chip complex to glucose binding. The obtained data suggest the use of this protein for the design of advanced optical non-consuming analyte biosensors for glucose detection. [ABSTRACT FROM AUTHOR]

Physiology, regulation and biochemical aspects of the nitrogen assimilation are well known in Prokarya or Eukarya but they are poorly described in Archaea domain. The haloarchaeon Haloferax mediterranei can use different nitrogen inorganic sources (NO, NO or NH) for growth. Different approaches were considered to study the effect of NH on nitrogen assimilation in Hfx. mediterranei cells grown in KNO3 medium. The NH addition to KNO3 medium caused a decrease of assimilatory nitrate (Nas) and nitrite reductases (NiR) activities. Similar effects were observed when nitrate-growing cells were transferred to NH media. Both activities increased when NH was removed from culture, showing that the negative effect of NH on this pathway is reversible. These results suggest that ammonium causes the inhibition of the assimilatory nitrate pathway, while nitrate exerts a positive effect. This pattern has been confirmed by RT-PCR. In the presence of both NO and NH, NH was preferentially consumed, but NO uptake was not completely inhibited by NH at prolonged time scale. The addition of MSX to NH or NO cultures results in an increase of Nas and NiR activities, suggesting that NH assimilation, rather than NHper se, has a negative effect on assimilatory nitrate reduction in Hfx. mediterranei. [ABSTRACT FROM AUTHOR]

Haloferax mediterranei is a halophilic archaeon that can grow using nitrate as the sole nitrogen source. A ferredoxin that serves as the physiological electron donor to the nitrate and nitrite reductases in this assimilatory process has been characterized. The ferredoxin was found to contain approximately two atoms of iron and two atoms of sulphur, indicative of the binding of a [2Fe–2S] cluster. The electron paramagnetic resonance spectrum of the reduced form of the protein displayed a rhombic signal, with g x=1.91, g y=1.98, g z=2.07, that shows considerable similarity to plant and algal [2Fe–2S] ferredoxins. UV-visible spectropotentiometric analysis determined a midpoint redox potential for the [2Fe–2S]2+/1+ transition of around −285 mV vs. SHE that was independent of salt concentration. UV-visible spectroscopy was also used to establish that the [2Fe–2S] cluster integrity of this protein was maintained over the pH range 5–11. Significantly, the Haloferax mediterranei ferredoxin was shown to be a highly thermostable protein. It was stable up to 60 °C in a low-salt (0.2 M) medium and this increased to 80 °C in a high-salt (4 M) medium. This thermostability at high salt concentration is an essential physiological characteristic because haloarchaea are mainly found in environments where high temperatures and concentrated salt water occur. [ABSTRACT FROM AUTHOR]

Topoisomerase V is a type I topoisomerase without structural or sequence similarities to other topoisomerases. Although it belongs to the type I subfamily of topoisomerases, it is unrelated to either type IA or lB enzymes. We used real-time single-molecule micro- mechanical experiments to show that topoisomerase V relaxes DNA via events that release multiple DNA turns, employing a constrained swiveling mechanism similar to that for type lB enzymes. Relaxation is powered by the torque in the supercoiled DNA and is constrained by friction between the protein and the DNA. Although all type lB enzymes share a common structure and mechanism and type IA and type II enzymes show marked structural and functional similarities, topoisomerase V represents a different type of topoisomerase that relaxes DNA in a similar overall manner as type lB molecules but by using a completely different structural and mechanistic framework. [ABSTRACT FROM AUTHOR]

The crystal structure of the ferritin from the archaeon, hyperthermophile and anaerobe Pyrococcus furiosus (PfFtn) is presented. While many ferritin structures from bacteria to mammals have been reported, until now only one was available from archaea, the ferritin from Archaeoglobus fulgidus (AfFtn). The PfFtn 24-mer exhibits the 432 point-group symmetry that is characteristic of most ferritins, which suggests that the 23 symmetry found in the previously reported AfFtn is not a common feature of archaeal ferritins. Consequently, the four large pores that were found in AfFtn are not present in PfFtn. The structure has been solved by molecular replacement and refined at 2.75-Å resolution to R = 0.195 and R free = 0.247. The ferroxidase center of the aerobically crystallized ferritin contains one iron at site A and shows sites B and C only upon iron or zinc soaking. Electron paramagnetic resonance studies suggest this iron depletion of the native ferroxidase center to be a result of a complexation of iron by the crystallization salt. The extreme thermostability of PfFtn is compared with that of eight structurally similar ferritins and is proposed to originate mostly from the observed high number of intrasubunit hydrogen bonds. A preservation of the monomer fold, rather than the 24-mer assembly, appears to be the most important factor that protects the ferritin from inactivation by heat. [ABSTRACT FROM AUTHOR]

Abstract: 2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the ATP-dependent phosphorylation of 2-keto-3-deoxygluconate, a key intermediate in the modified (semi-phosphorylative) Entner–Doudoroff (ED) glucose metabolic pathway. We identified the gene (ORF ID: ST2478) encoding KDGK in the hyperthermophilic archaeon Sulfolobus tokodaii based on the structure of a gene cluster in a genomic database and functionally expressed it in Escherichia coli. The expressed protein was purified from crude extract by heat treatment and two conventional column chromatography steps, and the partial amino acid sequence in the N-terminal region of the purified enzyme (MAKLIT) was identical to that obtained from the gene sequence. The purified enzyme was extremely thermostable and retained full activity after heating at 80°C for 1h. The enzyme utilized ATP or GTP, but not ADP or AMP, as a phosphoryl donor and 2-keto-3-deoxy-d-gluconate or 2-keto-d-gluconate as a phosphoryl acceptor. Divalent cations including Mg2+, Co2+, Ni2+, Zn2+ or Mn2+ were required for activity, and the apparent Km values for KDG and ATP at 50°C were 0.027mM and 0.057mM, respectively. The presence of KDGK means that the hyperthermophilic archaeon S. tokodaii metabolizes glucose via both modified (semi-phosphorylative) and non-phosphorylative ED pathways. [Copyright &y& Elsevier]

Haloferax mediterranei is a denitrifying halophilic archaeon, able to assimilate nitrate or nitrite in the presence of oxygen by the assimilatory nitrate pathway. It can also grow in the presence of high nitrate or nitrite concentrations under anoxic conditions, using both nitrogen species as electron acceptors. In this study, the ability of H. mediterranei to remove high nitrate and nitrite concentrations from culture media has been demonstrated. This suggests that this haloarchaeon could be applied in water bioremediation processes to repair damage caused by anthropogenic activities. This could be beneficial in regions such as Comunidad Valenciana or Murcia (Spain), where the water tables contain high nitrate and nitrite concentrations due to fertiliser addition, and high salt concentrations due to marine intrusions. [ABSTRACT FROM AUTHOR]