Microbially promoted calcite precipitation in the pelagic redoxcline: Elucidating the formation of the turbid layer.

Large bell-shaped calcite formations called "Hells Bells" were discovered underwater in the stratified cenote El Zapote on the Yucatán Peninsula, Mexico. Together with these extraordinary speleothems, divers found a white, cloudy turbid layer into which some Hells Bells partially extend. Here, we address the central question if the formation of the turbid layer could be based on microbial activity, more specifically, on microbially induced calcite precipitation. Metagenomic and metatranscriptomic profiling of the microbial community in the turbid layer, which overlaps with the pelagic redoxcline in the cenote, revealed chemolithoautotrophic Hydrogenophilales and unclassified ß-Proteobacteria as the metabolic key players. Bioinformatic and hydrogeochemical data suggest chemolithoautotrophic oxidation of sulfide to zero-valent sulfur catalyzed by denitrifying organisms due to oxygen deficiency. Incomplete sulfide oxidation via nitrate reduction and chemolithoautotrophy are both proton-consuming processes, which increase the pH in the redoxcline favoring authigenic calcite precipitation and may contribute to Hells Bells growth. The observed mechanism of microbially induced calcite precipitation is potentially applicable to many other stagnant sulfate-rich water bodies.

Persulfide Dioxygenase From Acidithiobacillus caldus: Variable Roles of Cysteine Residues and Hydrogen Bond Networks of the Active Site.

Persulfide dioxygenases (PDOs) are abundant in Bacteria and also crucial for H2S detoxification in mitochondria. One of the two pdo-genes of the acidophilic bacterium Acidithiobacillus caldus was expressed in Escherichia coli. The protein (AcPDO) had 0.77 ± 0.1 Fe/subunit and an average specific sulfite formation activity of 111.5 U/mg protein (Vmax) at 40°C and pH 7.5 with sulfur and GSH following Michaelis-Menten kinetics. KM for GSH and Kcat were 0.5 mM and 181 s-1, respectively. Glutathione persulfide (GSSH) as substrate gave a sigmoidal curve with a Vmax of 122.3 U/mg protein, a Kcat of 198 s-1 and a Hill coefficient of 2.3 ± 0.22 suggesting positive cooperativity. Gel permeation chromatography and non-denaturing gels showed mostly tetramers. The temperature optimum was 40-45°C, the melting point 63 ± 1.3°C in thermal unfolding experiments, whereas low activity was measurable up to 95°C. Site-directed mutagenesis showed that residues located in the predicted GSH/GSSH binding site and in the central hydrogen bond networks including the iron ligands are essential for activity. Among these, the R139A, D141A, and H171A variants were inactive concomitant to a decrease of their melting points by 3-8 K. Other variants were inactivated without significant melting point change. Two out of five cysteines are likewise essential, both of which lie presumably in close proximity at the surface of the protein (C87 and C224). MalPEG labeling experiments suggests that they form a disulfide bridge. The reducing agent Tris(2-carboxyethyl)phosphine was inhibitory besides N-ethylmaleimide and iodoacetamide suggesting an involvement of cysteines and the disulfide in catalysis and/or protein stabilization. Mass spectrometry revealed modification of C87, C137, and C224 by 305 mass units equivalent to GSH after incubation with GSSH and with GSH in case of the C87A and C224A variants. The results of this study suggest that disulfide formation between the two essential surface-exposed cysteines and Cys-S-glutathionylation serve as a protective mechanism against uncontrolled thiol oxidation and the associated loss of enzyme activity.

The Sulfur Oxygenase Reductase Activity Assay: Catalyzing a Reaction with Elemental Sulfur as Substrate at High Temperatures.

The sulfur oxygenase reductase (SOR) reaction is a dioxygen-dependent disproportionation of elemental sulfur (S0), catalyzed at optimal temperatures between 65 °C and 85 °C. Thiosulfate and sulfite are formed as oxidized products as well hydrogen sulfide as reduced product. External co-factors are not required. Usually, the SOR assay is performed in a milliliter scale in S0-containing Tris-buffer at high temperatures followed by colorimetric product quantification. In order to make the SOR assay more sensitive and better reproducible, several modifications were implemented compared to the original SOR assay (Kletzin, 1989). Here we present the modified SOR assay and the following quantification of the reaction products.

A Sulfur Oxygenase from the Haloalkaliphilic Bacterium Thioalkalivibrio paradoxus with Atypically Low Reductase Activity.

Sequence comparisons showed that the sulfur oxygenase reductase (SOR) of the haloalkaliphilic bacterium Thioalkalivibrio paradoxus Arh 1 (TpSOR) is branching deeply within dendrograms of these proteins (29 to 34% identity). A synthetic gene encoding TpSOR expressed in Escherichia coli resulted in a protein 14.7 ± 0.9 nm in diameter and an apparent molecular mass of 556 kDa. Sulfite and thiosulfate were formed from elemental sulfur in a temperature range of 10 to 98°C (optimum temperature ≈ 80°C) and a pH range of 6 to 11.5 (optimum pH ≈ 9; 308 ± 78 U/mg of protein). Sulfide formation had a maximum specific activity of 0.03 U/mg, or <1% of the corresponding activity of other SORs. Hence, reductase activity seems not to be an integral part of the reaction mechanism. TpSOR was most active at NaCl or glycine betaine concentrations of 0 to 1 M, although 0.2% of the maximal activity was detected even at 5 M NaCl and 4 M betaine. The melting point of TpSOR was close to 80°C, when monitored by circular dichroism spectroscopy or differential scanning fluorimetry; however, the denaturation kinetics were slow: 55% of the residual activity remained after 25 min of incubation at 80°C. Site-directed mutagenesis showed that the active-site residue Cys44 is essential for activity, whereas alanine mutants of the two other conserved cysteines retained about 0.5% residual activity. A model of the sulfur metabolism in T. paradoxus is discussed. IMPORTANCE: Sulfur oxygenase reductases (SORs) are the only enzymes catalyzing an oxygen-dependent disproportionation of elemental sulfur and/or polysulfides to sulfite, thiosulfate, and hydrogen sulfide. SORs are known from mesophilic and extremophilic archaea and bacteria. All SORs seem to form highly thermostable 24-subunit hollow spheres. They carry a low-potential mononuclear nonheme iron in the active site and an indispensable cysteine; however, their exact reaction mechanisms are unknown. Typically, the reductase activity of SORs is in the range of 5 to 50% of the oxygenase activity, but mutagenesis studies had so far failed to identify residues crucial for the reductase reaction. We describe here the first SOR, which is almost devoid of the reductase reaction and which comes from a haloalkaliphilic bacterium.

Cytochromes c in Archaea: distribution, maturation, cell architecture, and the special case of Ignicoccus hospitalis.

Cytochromes c (Cytc) are widespread electron transfer proteins and important enzymes in the global nitrogen and sulfur cycles. The distribution of Cytc in more than 300 archaeal proteomes deduced from sequence was analyzed with computational methods including pattern and similarity searches, secondary and tertiary structure prediction. Two hundred and fifty-eight predicted Cytc (with single, double, or multiple heme c attachment sites) were found in some but not all species of the Desulfurococcales, Thermoproteales, Archaeoglobales, Methanosarcinales, Halobacteriales, and in two single-cell genome sequences of the Thermoplasmatales, all of them Cren- or Euryarchaeota. Other archaeal phyla including the Thaumarchaeota are so far free of these proteins. The archaeal Cytc sequences were bundled into 54 clusters of mutual similarity, some of which were specific for Archaea while others had homologs in the Bacteria. The cytochrome c maturation system I (CCM) was the only one found. The highest number and variability of Cytc were present in those species with known or predicted metal oxidation and/or reduction capabilities. Paradoxical findings were made in the haloarchaea: several Cytc had been purified biochemically but corresponding proteins were not found in the proteomes. The results are discussed with emphasis on cell morphologies and envelopes and especially for double-membraned Archaea-like Ignicoccus hospitalis. A comparison is made with compartmentalized bacteria such as the Planctomycetes of the Anammox group with a focus on the putative localization and roles of the Cytc and other electron transport proteins.

Three multihaem cytochromes c from the hyperthermophilic archaeon Ignicoccus hospitalis: purification, properties and localization.

Three different multihaem cytochromes c were purified from cell extracts of the hyperthermophilic archaeon Ignicoccus hospitalis. One tetrahaem cytochrome, locus tag designation Igni_0530, was purified from membrane fractions together with the iron-sulfur protein Igni_0529. Two octahaem cytochromes, Igni_0955 and Igni_1359, were purified from soluble fractions but were also present in the membrane fraction. N-terminal sequencing showed that three of the four proteins had their signal peptides cleaved off, while results were ambiguous for Igni_0955. In contrast, mass spectrometry of Igni_0955 and Igni_1359 resulted in single mass peaks including the signal sequences and eight haems per subunit and so both forms might be present in the cell. Igni_0955 and Igni_1359 belong to the hydroxylamine dehydrogenase (HAO) family (29 % mutual identity). HAO or reductase activities with inorganic sulfur compounds were not detected. Igni_0955 was reduced by enriched I. hospitalis hydrogenase at a specific activity of 243 nmol min(-1) (mg hydrogenase)(-1) while activity was non-existent for Igni_0530 and low for Igni_1359. Immuno-electron microscopy of ultra-thin sections showed that Igni_0955 and Igni_1359 are located in both I. hospitalis membranes and also in the intermembrane compartment. We concluded that these cytochromes might function as electron shuttles between the hydrogenase in the outer cellular membrane and cellular reductases, whereas Igni_0530 might be part of the sulfur-reducing mechanism.

An efficient strategy for small-scale screening and production of archaeal membrane transport proteins in Escherichia coli.

BACKGROUND: Membrane proteins play a key role in many fundamental cellular processes such as transport of nutrients, sensing of environmental signals and energy transduction, and account for over 50% of all known drug targets. Despite their importance, structural and functional characterisation of membrane proteins still remains a challenge, partially due to the difficulties in recombinant expression and purification. Therefore the need for development of efficient methods for heterologous production is essential. METHODOLOGY/PRINCIPAL FINDINGS: Fifteen integral membrane transport proteins from Archaea were selected as test targets, chosen to represent two superfamilies widespread in all organisms known as the Major Facilitator Superfamily (MFS) and the 5-Helix Inverted Repeat Transporter superfamily (5HIRT). These proteins typically have eleven to twelve predicted transmembrane helices and are putative transporters for sugar, metabolite, nucleobase, vitamin or neurotransmitter. They include a wide range of examples from the following families: Metabolite-H(+)-symporter; Sugar Porter; Nucleobase-Cation-Symporter-1; Nucleobase-Cation-Symporter-2; and neurotransmitter-sodium-symporter. Overproduction of transporters was evaluated with three vectors (pTTQ18, pET52b, pWarf) and two Escherichia coli strains (BL21 Star and C43 (DE3)). Thirteen transporter genes were successfully expressed; only two did not express in any of the tested vector-strain combinations. Initial trials showed that seven transporters could be purified and six of these yielded quantities of ≥ 0.4 mg per litre suitable for functional and structural studies. Size-exclusion chromatography confirmed that two purified transporters were almost homogeneous while four others were shown to be non-aggregating, indicating that they are ready for up-scale production and crystallisation trials. CONCLUSIONS/SIGNIFICANCE: Here, we describe an efficient strategy for heterologous production of membrane transport proteins in E. coli. Small-volume cultures (10 mL) produced sufficient amount of proteins to assess their purity and aggregation state. The methods described in this work are simple to implement and can be easily applied to many more membrane proteins.

SAD phasing towards structure determination of a thermostable Rieske ferredoxin with a novel stabilizing disulfide bridge.

Rieske proteins and Rieske ferredoxins are ubiquitous electron-transfer metalloproteins that are characterized by a [2Fe-2S] cluster coordinated by pairs of cysteine and histidine residues. The thermoacidophilic archaeon Acidianus ambivalens contains a Rieske ferredoxin termed RFd2, which has an hitherto unknown additional region of 40-44 residues at the C-terminus with a Cx3C motif that introduces a novel disulfide bond within the Rieske fold. RFd2 was crystallized with the aim of determining its three-dimensional structure in order to understand the contribution of this as yet unique disulfide bridge to the function and stability of RFd2. RFd2 crystals were successively improved, increasing their diffraction to 1.9 Šresolution. Molecular replacement did not solve the RFd2 structure, but a highly multiple in-house diffraction data set collected at the Cu Kα edge led to solution of the phase problem.

The sulfur oxygenase reductase from the mesophilic bacterium Halothiobacillus neapolitanus is a highly active thermozyme.

A biochemical, biophysical, and phylogenetic study of the sulfur oxygenase reductase (SOR) from the mesophilic gammaproteobacterium Halothiobacillus neapolitanus (HnSOR) was performed in order to determine the structural and biochemical properties of the enzyme. SOR proteins from 14 predominantly chemolithoautotrophic bacterial and archaeal species are currently available in public databases. Sequence alignment and phylogenetic analysis showed that they form a coherent protein family. The HnSOR purified from Escherichia coli after heterologous gene expression had a temperature range of activity of 10 to 99°C with an optimum at 80°C (42 U/mg protein). Sulfite, thiosulfate, and hydrogen sulfide were formed at various stoichiometries in a range between pH 5.4 and 11 (optimum pH 8.4). Circular dichroism (CD) spectroscopy and dynamic light scattering showed that the HnSOR adopts secondary and quaternary structures similar to those of the 24-subunit enzyme from the hyperthermophile Acidianus ambivalens (AaSOR). The melting point of the HnSOR was ≈20°C lower than that of the AaSOR, when analyzed with CD-monitored thermal unfolding. Homology modeling showed that the secondary structure elements of single subunits are conserved. Subtle changes in the pores of the outer shell and increased flexibility might contribute to activity at low temperature. We concluded that the thermostability was the result of a rigid protein core together with the stabilizing effect of the 24-subunit hollow sphere.

The complete genome sequence of Thermoproteus tenax: a physiologically versatile member of the Crenarchaeota.

Here, we report on the complete genome sequence of the hyperthermophilic Crenarchaeum Thermoproteus tenax (strain Kra1, DSM 2078(T)) a type strain of the crenarchaeotal order Thermoproteales. Its circular 1.84-megabase genome harbors no extrachromosomal elements and 2,051 open reading frames are identified, covering 90.6% of the complete sequence, which represents a high coding density. Derived from the gene content, T. tenax is a representative member of the Crenarchaeota. The organism is strictly anaerobic and sulfur-dependent with optimal growth at 86°C and pH 5.6. One particular feature is the great metabolic versatility, which is not accompanied by a distinct increase of genome size or information density as compared to other Crenarchaeota. T. tenax is able to grow chemolithoautotrophically (CO2/H2) as well as chemoorganoheterotrophically in presence of various organic substrates. All pathways for synthesizing the 20 proteinogenic amino acids are present. In addition, two presumably complete gene sets for NADH:quinone oxidoreductase (complex I) were identified in the genome and there is evidence that either NADH or reduced ferredoxin might serve as electron donor. Beside the typical archaeal A0A1-ATP synthase, a membrane-bound pyrophosphatase is found, which might contribute to energy conservation. Surprisingly, all genes required for dissimilatory sulfate reduction are present, which is confirmed by growth experiments. Mentionable is furthermore, the presence of two proteins (ParA family ATPase, actin-like protein) that might be involved in cell division in Thermoproteales, where the ESCRT system is absent, and of genes involved in genetic competence (DprA, ComF) that is so far unique within Archaea.

Substrate pathways and mechanisms of inhibition in the sulfur oxygenase reductase of acidianus ambivalens.

BACKGROUND: The sulfur oxygenase reductase (SOR) is the initial enzyme of the sulfur oxidation pathway in the thermoacidophilic Archaeon Acidianus ambivalens. The SOR catalyzes an oxygen-dependent sulfur disproportionation to H(2)S, sulfite and thiosulfate. The spherical, hollow, cytoplasmic enzyme is composed of 24 identical subunits with an active site pocket each comprising a mononuclear non-heme iron site and a cysteine persulfide. Substrate access and product exit occur via apolar chimney-like protrusions at the fourfold symmetry axes, via narrow polar pores at the threefold symmetry axes and via narrow apolar pores within in each subunit. In order to investigate the function of the pores we performed site-directed mutagenesis and inhibitor studies. RESULTS: Truncation of the chimney-like protrusions resulted in an up to sevenfold increase in specific enzyme activity compared to the wild type. Replacement of the salt bridge-forming Arg(99) residue by Ala at the threefold symmetry axes doubled the activity and introduced a bias toward reduced reaction products. Replacement of Met(296) and Met(297), which form the active site pore, lowered the specific activities by 25-55% with the exception of an M(296)V mutant. X-ray crystallography of SOR wild type crystals soaked with inhibitors showed that Hg(2+) and iodoacetamide (IAA) bind to cysteines within the active site, whereas Zn(2+) binds to a histidine in a side channel of the enzyme. The Zn(2+) inhibition was partially alleviated by mutation of the His residue. CONCLUSIONS: The expansion of the pores in the outer shell led to an increased enzyme activity while the integrity of the active site pore seems to be important. Hg(2+) and IAA block cysteines in the active site pocket, while Zn(2+) interferes over a distance, possibly by restriction of protein flexibility or substrate access or product exit.

An Extracellular Tetrathionate Hydrolase from the Thermoacidophilic Archaeon Acidianus Ambivalens with an Activity Optimum at pH 1.

BACKGROUND: The thermoacidophilic and chemolithotrophic archaeon Acidianus ambivalens is routinely grown with sulfur and CO(2)-enriched air. We had described a membrane-bound, tetrathionate (TT) forming thiosulfate:quinone oxidoreductase. Here we describe the first TT hydrolase (TTH) from Archaea. RESULTS: A. ambivalens cells grown aerobically with TT as sole sulfur source showed doubling times of 9 h and final cell densities of up to 8 × 10(8)/ml. TTH activity (≈0.28 U/mg protein) was found in cell-free extracts of TT-grown but not of sulfur-grown cells. Differential fractionation of freshly harvested cells involving a pH shock showed that about 92% of the TTH activity was located in the pseudo-periplasmic fraction associated with the surface layer, while 7.3% and 0.3% were present in the soluble and membrane fractions, respectively. The enzyme was enriched 54-fold from the cytoplasmic fraction and 2.1-fold from the pseudo-periplasmic fraction. The molecular mass of the single subunit was 54 kDa. The optimal activity was at or above 95°C at pH 1. Neither PQQ nor divalent cations had a significant effect on activity. The gene (tth1) was identified following N-terminal sequencing of the protein. Northern hybridization showed that tth1 was transcribed in TT-grown cells in contrast to a second paralogous tth2 gene. The deduced amino acid sequences showed similarity to the TTH from Acidithiobacillus and other proteins from the PQQ dehydrogenase superfamily. It displayed a ß-propeller structure when being modeled, however, important residues from the PQQ-binding site were absent. CONCLUSION: The soluble, extracellular, and acidophilic TTH identified in TT-grown A. ambivalens cells is essential for TT metabolism during growth but not for the downstream processing of the TQO reaction products in S°-grown cells. The liberation of TTH by pH shock from otherwise intact cells strongly supports the pseudo-periplasm hypothesis of the S-layer of Archaea.

Role of a novel disulfide bridge within the all-beta fold of soluble Rieske proteins.

Rieske proteins and Rieske ferredoxins are present in the three domains of life and are involved in a variety of cellular processes. Despite their functional diversity, these small Fe-S proteins contain a highly conserved all-beta fold, which harbors a [2Fe-2S] Rieske center. We have identified a novel subtype of Rieske ferredoxins present in hyperthermophilic archaea, in which a two-cysteine conserved SKTPCX((2-3))C motif is found at the C-terminus. We establish that in the Acidianus ambivalens representative, Rieske ferredoxin 2 (RFd2), these cysteines form a novel disulfide bond within the Rieske fold, which can be selectively broken under mild reducing conditions insufficient to reduce the [2Fe-2S] cluster or affect the secondary structure of the protein, as shown by visible circular dichroism, absorption, and attenuated total reflection Fourier transform IR spectroscopies. RFd2 presents all the EPR, visible absorption, and visible circular dichroism spectroscopic features of the [2Fe-2S] Rieske center. The cluster has a redox potential of +48 mV (25 degrees C and pH 7) and a pK (a) of 10.1 +/- 0.2. These shift to +77 mV and 8.9 +/- 0.3, respectively, upon reduction of the disulfide. RFd2 has a melting temperature near the boiling point of water (T(m) = 99 degrees C, pH 7.0), but it becomes destabilized upon disulfide reduction (DeltaT(m) = -9 degrees C, DeltaC(m) = -0.7 M guanidinium hydrochloride). This example illustrates how the incorporation of an additional structural element such as a disulfide bond in a highly conserved fold such as that of the Rieske domain may fine-tune the protein for a particular function or for increased stability.

Acidianus, Sulfolobus and Metallosphaera surface layers: structure, composition and gene expression.

The cell walls of Sulfolobales species consist of proteinaceous S-layers assembled from two polypeptides, SlaA and SlaB. We isolated the large S-layer protein of Acidianus ambivalens and both S-layer subunits of Sulfolobus solfataricus and Metallosphaera sedula, respectively. The slaAB genes, lying adjacently in the chromosomes, are constitutively transcribed as bicistronic operons in A. ambivalens and S. solfataricus. A smaller slaA transcript appeared in Northern hybridizations of A. ambivalens RNA. PCRs experiments showed that 80-85% of the transcripts stop at an oligo-T terminator downstream of slaA while 15-20% are read through to a second terminator downstream of slaB. The bicistronic operons including promoter and terminator regions are conserved in the Sulfolobales. While no SlaA homologue is found outside the Sulfolobales, SlaB is distantly similar to S-layer proteins of other Crenarchaeota, e.g. the Staphylothermus marinus tetrabrachion. Molecular modelling suggests SlaBs to be composed of 2-3 consecutive beta sandwich domains, a coiled-coil domain of 15-17 nm in length and a C-terminal transmembrane helix. Electron microscopy shows crystalline protein arrays with triangular and hexagonal pores. We propose that the mushroom-shaped 'unit cells' of the Sulfolobales' S-layers consist of three SlaBs anchoring the complex in the membrane and six SlaAs forming the detergent-resistant outer sacculus.

The cytochrome ba complex from the thermoacidophilic crenarchaeote Acidianus ambivalens is an analog of bc(1) complexes.

A novel cytochrome ba complex was isolated from aerobically grown cells of the thermoacidophilic archaeon Acidianus ambivalens. The complex was purified with two subunits, which are encoded by the cbsA and soxN genes. These genes are part of the pentacistronic cbsAB-soxLN-odsN locus. The spectroscopic characterization revealed the presence of three low-spin hemes, two of the b and one of the a(s)-type with reduction potentials of +200, +400 and +160 mV, respectively. The SoxN protein is proposed to harbor the heme b of lower reduction potential and the heme a(s), and CbsA the other heme b. The soxL gene encodes a Rieske protein, which was expressed in E. coli; its reduction potential was determined to be +320 mV. Topology predictions showed that SoxN, CbsB and CbsA should contain 12, 9 and one transmembrane alpha-helices, respectively, with SoxN having a predicted fold very similar to those of the cytochromes b in bc(1) complexes. The presence of two quinol binding motifs was also predicted in SoxN. Based on these findings, we propose that the A. ambivalens cytochrome ba complex is analogous to the bc(1) complexes of bacteria and mitochondria, however with distinct subunits and heme types.

The dihydrolipoamide dehydrogenase from the crenarchaeon Acidianus ambivalens.

A dihydrolipoamide dehydrogenase (DLDH) was purified and characterized for the first time from a crenarchaeon, Acidianus ambivalens. The holoenzyme consists of two identical subunits with a molecular mass of 45.4 kDa per monomer. It contains FAD as a prosthetic group and uses NAD+ as the preferential substrate, but can also reduce NADP+. The Michaelis-Menten constants of the forward (NAD+ reduction) and reverse (NADH oxidation) reactions were KM (dihydrolipoamide)=0.70 mM, KM (NAD+)=0.71 mM, KM (lipoamide)=1.26 mM and KM (NADH)=3.15 microM. A comparative study of NADH:lipoamide oxidoreductase and NADH:K3[Fe(CN)6] oxidoreductase activities was performed, the optimal temperature and pH being different for each: 55 degrees C, pH 7 and 89 degrees C, pH 5.5, respectively. Although DLDH is generally part of the alpha-ketoacid dehydrogenase complexes in Bacteria and Eukarya, none of these complexes has yet been isolated from Sulfolobales. The metabolic role of DLDH in these organisms is discussed.

A proteomic approach toward the selection of proteins with enhanced intrinsic conformational stability.

A detailed understanding of the molecular basis of protein folding and stability determinants partly relies on the study of proteins with enhanced conformational stability properties, such as those from thermophilic organisms. In this study, we set up a methodology aiming at identifying the subset of cytosolic hyperstable proteins using Sulfurispharea sp., a hyperthermophilic archaeon, able to grow between 70 and 97 degrees C, as a model organism. We have thermally and chemically perturbed the cytosolic proteome as a function of time (up to 96 h incubation at 90 degrees C), and proceeded with analysis of the remaining proteins by combining one- and two-dimensional gel electrophoresis, liquid chromatography fractionation, and protein identification by N-terminal sequencing and mass spectrometry methods. In total, 14 proteins with enhanced stabilities which are involved in key cellular processes such as detoxification, nucleic acid processing, and energy metabolism were identified including a superoxide dismutase, a peroxiredoxin, and a ferredoxin. We demonstrate that these proteins are biologically active after extensive thermal treatment of the proteome. The relevance of these and other targets is discussed in terms of the organism's ecology. This work thus illustrates an experimental approach aimed at mining a proteome for hyperstable proteins, a valuable tool for target selection in protein stability and structural studies.

X-ray Structure of a self-compartmentalizing sulfur cycle metalloenzyme.

Numerous microorganisms oxidize sulfur for energy conservation and contribute to the global biogeochemical sulfur cycle. We have determined the 1.7 angstrom-resolution structure of the sulfur oxygenase reductase from the thermoacidophilic archaeon Acidianus ambivalens, which catalyzes an oxygen-dependent disproportionation of elemental sulfur. Twenty-four monomers form a large hollow sphere enclosing a positively charged nanocompartment. Apolar channels provide access for linear sulfur species. A cysteine persulfide and a low-potential mononuclear non-heme iron site ligated by a 2-His-1-carboxylate facial triad in a pocket of each subunit constitute the active sites, accessible from the inside of the sphere. The iron is likely the site of both sulfur oxidation and sulfur reduction.

Novel genes for nitrite reductase and Amo-related proteins indicate a role of uncultivated mesophilic crenarchaeota in nitrogen cycling.

Mesophilic crenarchaeota are frequently found in terrestrial and marine habitats worldwide, but despite their considerable abundance the physiology of these as yet uncultivated archaea has remained unknown. From a 1.2 Gb large-insert environmental fosmid library of a calcareous grassland soil, a 43 kb genomic fragment was isolated with a ribosomal RNA that shows its affiliation to group 1.1b of crenarchaeota repeatedly found in soils. The insert encoded a homologue of a copper-containing nitrite reductase with an unusual C-terminus that encoded a potential amicyanin-like electron transfer domain as well as two proteins related to subunits of ammonia monooxygenases or particulate methane monooxygenases (AmoAB/PmoAB) respectively. Expression of nirK and the amoA-like gene was shown by reverse transcription polymerase chain reaction (PCR) analyses in soil samples, the latter being found at higher levels when the soil was incubated with ammonia (measured by quantitative PCR). Further variants of both genes were amplified from soil samples and were found in the environmental database from the Sargasso Sea plankton. Taken together, our findings suggest that mesophilic terrestrial and marine crenarchaeota might be capable of ammonia oxidation under aerobic and potentially also under anaerobic conditions.

Identification of core active site residues of the sulfur oxygenase reductase from Acidianus ambivalens by site-directed mutagenesis.

The sulfur oxygenase reductase (SOR) is the initial enzyme in the sulfur oxidation pathway of Acidianus ambivalens. The SOR is composed of 308 aa residues, three of which are cysteines, and contains a mononuclear non-heme iron site. Mutations of the suspected iron-binding residues H86, H90 and E114 to alanine resulted in inactive enzyme with no iron incorporated, whereas an E114D mutant showed 1% of wild type activity. The mutation of C31 to alanine and serine caused inactivity of the enzyme, however, the iron content was the same as in the wild type. C101A, C104S/A, and C101/104S/A double mutants caused a decrease in specific activity to 10-43% of the wild type while the C101S mutant showed only 1% activity of the wild type. The drop in activity of the C101S and E114D mutants was accompanied with a proportional decrease in iron content. In all cases the oxygenase and reductase partial reactions were equally affected. It was concluded that the Fe site with H86, H90 and E114 as ligands and C31 constitute the core active site whereas C101 and C104 optimize reaction conditions.