Expression of Critical Sulfur- and Iron-Oxidation Genes and the Community Dynamics During Bioleaching of Chalcopyrite Concentrate by Moderate Thermophiles.

Sulfate adenylyltransferase gene and 4Fe-4S ferredoxin gene are the key genes related to sulfur and iron oxidations during bioleaching system, respectively. In order to better understand the bioleaching and microorganism synergistic mechanism in chalcopyrite bioleaching by mixed culture of moderate thermophiles, expressions of the two energy metabolism genes and community dynamics of free and attached microorganisms were investigated. Specific primers were designed for real-time quantitative PCR to study the expression of these genes. Real-time PCR results showed that sulfate adenylyltransferase gene was more highly expressed in Sulfobacillus thermosulfidooxidans than that in Acidithiobacillus caldus, and expression of 4Fe-4S ferredoxin gene was higher in Ferroplasma thermophilum than that in S. thermosulfidooxidans and Leptospirillum ferriphilum. The results indicated that in the bioleaching system of chalcopyrite concentrate, sulfur and iron oxidations were mainly performed by S. thermosulfidooxidans and F. thermophilum, respectively. The community dynamics results revealed that S. thermosulfidooxidans took up the largest proportion during the whole period, followed by F. thermophilum, A. caldus, and L. ferriphilum. The CCA analysis showed that 4Fe-4S ferredoxin gene expression was mainly affected (positively correlated) by high pH and elevated concentration of ferrous ion, while no factor was observed to prominently influence the expression of sulfate adenylyltransferase gene.

Altered responsiveness to TGF-ß results in reduced Papss2 expression and alterations in the biomechanical properties of mouse articular cartilage.

INTRODUCTION: Previous studies have indicated that transforming growth factor ß (TGF-ß) signaling has a critical role in cartilage homeostasis and repair, yet the mechanisms of TGF-ß's chondroprotective effects are not known. Our objective in this study was to identify downstream targets of TGF-ß that could act to maintain biochemical and biomechanical properties of cartilage. METHODS: Tibial joints from 20-week-old mice that express a dominant-negative mutation of the TGF-ß type II receptor (DNIIR) were graded histologically for osteoarthritic changes and tested by indentation to evaluate their mechanical properties. To identify gene targets of TGF-ß, microarray analysis was performed using bovine articular chondrocytes grown in micromass culture that were either treated with TGF-ß or left untreated. Phosphoadenosine phosphosynthetase 2 (PAPSS2) was identified as a TGF-ß-responsive gene. Papss2 expression is crucial for proper sulfation of cartilage matrix, and its deficiency causes skeletal defects in mice and humans that overlap with those seen in mice with mutations in TGF-ß-signaling genes. Regulation of Papss2 was verified by real time RT-PCR and Western blot analyses. Alterations in sulfation of glycosaminoglycans were analyzed by critical electrolyte concentration and Alcian blue staining and immunofluorescence for chondroitin-4-sulfate, unsulfated chondroitin and the aggrecan core protein. RESULTS: DNIIR mutants showed reduced mechanical properties and osteoarthritis-like changes when compared to wild-type control mice. Microarray analysis identified a group of genes encoding matrix-modifying enzymes that were regulated by TGF-ß. Papss2 was upregulated in bovine articular chondrocytes after treatment with TGF-ß and downregulated in cartilage from DNIIR mice. Articular cartilage in DNIIR mice demonstrated reduced Alcian blue staining at critical electrolyte concentrations and reduced chondroitin-4-sulfate staining. Staining for unsulfated chondroitin sulfate was increased, whereas staining for the aggrecan core protein was comparable in DNIIR and wild-type mice. CONCLUSION: TGF-ß maintains biomechanical properties and regulates expression of Papss2 and sulfation of glycosaminoglycans in mouse articular cartilage.

Expression of genes for sulfur oxidation in the intracellular chemoautotrophic symbiont of the deep-sea bivalve Calyptogena okutanii.

To understand sulfur oxidation in thioauto-trophic deep-sea clam symbionts, we analyzed the recently reported genomes of two chemoautotrophic symbionts of Calyptogena okutanii (Candidatus Vesicomyosocius okutanii strain HA: Vok) and C. magnifica (Candidatus Ruthia magnifica strain Cm: Rma), and examined the sulfur oxidation gene expressions in the Vok by RT-PCR. Both symbionts have genes for sulfide-quinone oxidoreductase (sqr), dissimilatory sulfite reductase (dsr), reversible dissimilatory sulfite reductase (rdsr), sulfur-oxidizing multienzyme system (sox)(soxXYZA and soxB but lacking soxCD), adenosine phosphosulfate reductase (apr), and ATP sulfurylase (sat). While these genomes share 29 orthologous genes for sulfur oxidation implying that both symbionts possess the same sulfur oxidation pathway, Rma has a rhodanese-related sulfurtransferase putative gene (Rmag0316) that has no corresponding ortholog in Vok, and Vok has one unique dsrR (COSY0782). We propose that Calyptogena symbionts oxidize sulfide and thiosulfate, and that sulfur oxidation proceeds as follows. Sulfide is oxidized to sulfite by rdsr. Sulfite is oxidized to sulfate by apr and sat. Thiosulfate is oxidized to zero-valence sulfur by sox, which is then reduced to sulfide by dsr. In addition, thiosulfate may also be oxidized into sulfate by another component of sox. The result of the RT-PCR showed that genes (dsrA, dsrB, dsrC, aprA, aprB, sat, soxB, and sqr) encoding key enzymes catalyzing sulfur oxidation were all equally expressed in the Vok under three different environmental conditions (aerobic, semioxic, and aerobic under high pressure at 9 MPa), indicating that all sulfur oxidation pathways function simultaneously to support intracellular symbiotic life.

[Expression and purification of ATP sulfurylase from Saccharomyces cerevisias in Escherichia coli and its application in pyrosequencing].

ATP sulfurylase (ATPS,EC 2.7.7.4) reversibly catalyzes the reaction between ATP and sulfate to produce APS and pyrophosphate (PPi), and has been used in pyrosequencing. The gene coding ATP sulfurylase was amplified from the genomic DNA of Saccharomyces cerevisias (CICC 1202), and cloned into prokaryotic expression plasmid pET28a( + ) to provide a recombinant expression plasmid pET28a( + )-ATPS. Upon IPTG induction, ATP sulfurylase was produced by E. coli BL21 (DE3) harboring the recombinant expression plasmid pET28a( + )-ATPS. The relative molecular weight of recombinant ATP sulfurylase with His tag was about 60 kD. The recombinant ATP sulfurylase with electrophoretic pure grade was obtained only by two purification steps: His * Bind Resin affinity chromatography and ultrafiltration. The specific activity of the purified recombinant ATP sulfurylase was as high as 5.1 x 10(4) u/mg. The successful application of the enzyme in pyrosequencing was also demostrated.

Soybean ATP sulfurylase, a homodimeric enzyme involved in sulfur assimilation, is abundantly expressed in roots and induced by cold treatment.

Soybeans are a rich source of protein and a key feed ingredient in livestock production, but lack sufficient levels of cysteine and methionine to meet the nutritional demands of swine or poultry as feed components. Although engineering the sulfur assimilatory pathway could lead to increased sulfur-containing amino acid content, little is known about this pathway in legumes. Here, we describe the cloning and characterization of soybean ATP sulfurylase (ATPS), which acts as the metabolic entry point into the sulfur assimilation pathway. Analysis of the ATPS clone isolated from a soybean seedling cDNA library revealed an open-reading frame, encoding a 52 kDa polypeptide with an N-terminal chloroplast/plastid transit peptide, which was related to the enzymes from Arabidopsis, potato, human, and yeast. Soybean ATP sulfurylase was expressed in Escherichia coli and purified to apparent homogeneity. Based on gel-filtration chromatography, the enzyme functions as a 100 kDa homodimer. Analysis of genomic DNA by Southern blotting revealed that multiple genes encode ATP sulfurylase in soybean. Analysis of the transcript profiles retrieved from a soybean EST database indicated that ATP sulfurylase mRNA was most abundant in root tissue. Cold treatment induced mRNA accumulation and enhanced the specific activity of ATP sulfurylase in root tissue. Northern blot analysis indicated a decline in the ATP sulfurylase transcript levels during seed development. Likewise, ATP sulfurylase specific activity also declined in the later stages of seed development. Increasing the expression levels of this key enzyme during soybean seed development could lead to an increase in the availability of sulfur amino acids, thereby enhancing the nutritional value of the crop.

Suppression of DHEA sulfotransferase (Sult2A1) during the acute-phase response.

The acute-phase response (APR) induces alterations in lipid metabolism, and our data suggest that this is associated with suppression of type II nuclear hormone receptors that are key regulators of fatty acid, cholesterol, and bile acid metabolism. Recently, the farnesoid X receptor (FXR), constitutive androstane receptor (CAR), and pregnane X receptor (PXR) were found to regulate DHEA sulfotransferase (Sult2A1), which plays an important role in DHEA sulfation and detoxification of bile acids. Because FXR, PXR, and CAR are suppressed during the APR, we hypothesized that Sult2A1 is downregulated during the APR. To induce the APR, mice were treated with LPS, which will then trigger the release of various cytokines, and the mRNA levels of Sult2A1 and the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (PAPSS2), as well as the enzyme activity of Sult2A1, were determined in the liver. We found that mRNA levels of Sult2A1 decrease in a time- and dose-dependent manner during the LPS-induced APR. Similar changes were observed in the mRNA levels of PAPSS2, the major synthase of PAPS in the liver. Moreover, hepatic Sult2A1 activity and serum levels of DHEA-sulfate (DHEA-S) were significantly decreased in LPS-treated animals. These results suggest that decreased levels or activities of FXR, PXR, and CAR during the APR could contribute to decreases in Sult2A1, resulting in decreased sulfation of DHEA and lower circulating level of DHEA-S. Finally, we found that both TNF and IL-1 caused a significant decrease in the mRNA level of Sult2A1 in Hep3B human hepatoma cells, suggesting that the proinflammatory cytokines TNF and IL-1 mediate the inhibitory effect of LPS on Sult2A1 mRNA level. Our study provides a possible mechanism by which infection and inflammation are associated with altered steroid metabolism and cholestasis.

Increasing sulphite formation in Saccharomyces cerevisiae by overexpression of MET14 and SSU1.

Saccharomyces cerevisiae produces sulphite as an intermediate product during the assimilatory reduction of sulphate to sulphide. Three genes, MET3, MET14 and MET16, are essential for this reduction. We investigated the level of transcription of these genes in strains of S. cerevisiae with high, medium and low sulphite formation. The level of MET14- and MET16-mRNA varied with sulphite production, whereas the level of MET3-mRNA was very weak in almost all strains. We also analysed the effect of overexpression of MET14 and MET16 on sulphite formation. Two strains with low sulphite production were transformed with high-copy plasmids containing either or both MET14 and MET16. The overexpression of these two genes leads to a two- to three-fold sulphite formation. In addition, inactivation of MET10, encoding a subunit of the sulphite reductase, also leads to a distinct increase in sulphite formation; however, the cells became methionine auxotroph. The overexpression of SSU1, a gene encoding a putative sulphite pump, yields a slight increase in sulphite accumulation, whereas overexpression of SSU1, together with MET14, increases sulphite formation up to 10-fold. Furthermore, sulphite formation strongly depends on growth conditions, e.g. yeast transformants growing in wort produce much higher amounts of sulphite when compared to growth in minimal media. The addition of glucose can also increase the sulphite formation in strains overexpressing MET14 and/or SSU1 under oxygen-limiting conditions, while the addition of glucose has no significant effect under aerobic conditions.

Transcriptional regulation of human 3'-phosphoadenosine 5'-phosphosulfate synthase 1.

Sulfonation, which is essential for normal growth, development and maintenance of the internal milieu, requires the universal sulfonate donor molecule 3'-phosphoadenosine 5'-phosphosulfate (PAPS) produced from ATP and inorganic sulfate by two bifunctional PAPS synthase isozymes. The gene for PAPS synthase 1 containing neither a TATA nor a CCAAT box was found to be under the influence of the Sp1 family of transcription factors. Multiple GC/GT boxes are present in the proximal promoter region and deletion analysis implicated their involvement in transcription, a finding supported by mutational analysis of specific GC/GT boxes. Nuclear extract of SW13 cells, which highly express PAPS synthase 1, contains proteins that bind to probes possessing specific GC/GT boxes; furthermore, the presence of Sp1, Sp2, and Sp3 proteins in nuclear extracts was confirmed by supershift analysis. Cotransfection experiments using SL2 cells yielded additional support for the involvement of Sp1 in transcriptional regulation of the PAPS synthase 1 gene; the involvement of Sp2 and/or Sp3 is presently unclear.

Differential accumulation of transcripts encoding sulfur assimilation enzymes upon sulfur and/or nitrogen deprivation in Arabidopsis thaliana.

Expression of nine genes encoding enzymes involved in the sulfur assimilation pathway was examined by RNA blot hybridization. Significantly increased levels of transcripts encoding ATP sulfurylase and APS reductase were apparent under sulfur deprivation. However, in the absence of nitrogen, their responsiveness to sulfur deprivation was markedly reduced. Results suggest that the sulfur assimilation pathway is regulated at the transcriptional level by both nitrogen and sulfur sources.

Production, purification, and luminometric analysis of recombinant Saccharomyces cerevisiae MET3 adenosine triphosphate sulfurylase expressed in Escherichia coli.

ATP sulfurylase cDNA from MET3 on chromosome X of Saccharomyces cerevisiae was amplified and cloned, and recombinant ATP sulfurylase was expressed in Escherichia coli. The synthesis of ATP sulfurylase was directed by an expression system that employs the regulatory genes of the luminous bacterium Vibrio fischeri. A soluble, biologically active form was purified to electrophoretic homogeneity from lysates of recombinant E. coli by ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration. The specific activity of the purified enzyme was estimated to 140 U/mg. The apparent molecular mass of the recombinant enzyme was determined by gel filtration to be 470 kDa, which indicates that the active enzyme is an octamer of identical subunits (the molecular mass of a single subunit is 59.3 kDa). The ATP sulfurylase activity was monitored in real time by a very sensitive bioluminometric method.

cDNA cloning, expression, and characterization of the human bifunctional ATP sulfurylase/adenosine 5'-phosphosulfate kinase enzyme.

A cDNA encoding the human bifunctional ATP sulfurylase/adenosine 5'-phosphosulfate (APS) kinase was cloned and sequenced. The enzyme contains an APS kinase domain in its N-terminal portion and an ATP sulfurylase domain in its C-terminal portion. Recombinant full-length enzyme and its constituent APS kinase and ATP sulfurylase domains were individually expressed, purified, and shown to have their respective enzymatic activities.

Sulfation in high endothelial venules: cloning and expression of the human PAPS synthetase.

High endothelial venules (HEVs) are specialized postcapillary venules found in lymphoid organs and chronically inflamed tissues that support high levels of lymphocyte extravasation from the blood. Studies with chlorate, a metabolic inhibitor of sulfation, had previously revealed that production of PAPS (3'-phosphoadenosine-5'-phosphosulfate), the high-energy donor of sulfate, is required for sulfation and high-affinity recognition of HEV sialomucins GlyCAM-1 and CD34 by the lymphocyte homing receptor L-selectin. Here, we report the molecular characterization of a novel 2.5 kb human cDNA from MECA-79+ HEV-derived endothelial cells that encodes the target of chlorate, PAPS synthetase, a multifunctional enzyme containing domains for both ATP sulfurylase and adenosine-5'-phosphosulfate kinase. Functional expression of the isolated cDNA in Chinese hamster ovary cells results in high levels of PAPS synthesis, which is abolished by treatment of the transfected cells with chlorate. Northern blot analysis reveals a wide tissue distribution of PAPS synthetase mRNA in the human body, suggesting that human PAPS synthetase may be important for sulfation not only of HEV sialomucins, but also of many other molecules, including mucins such as the P-selectin ligand PSGL-1, proteoglycans, hormones, neurotransmitters, drugs, and xenobiotics.

Regulating energy transfer in the ATP sulfurylase-GTPase system.

ATP sulfurylase, isolated from Escherichia coli K-12, is a GTPase-target complex that catalyzes and links the energetics of GTP hydrolysis to the synthesis of activated sulfate (APS). When the GTP concentration is saturating and held fixed with a regenerating system, the APS reaction reaches a steady state in which its mass ratio is shifted (5.4 x 10(6))-fold toward the product by the hydrolysis of GTP. If GTP is not regenerated, the shift toward the product is transient, producing a pulse-shaped progress curve. The mechanistic basis of this transience is the subject of this paper. The product transient is caused by the binding of GDP to the enzyme which establishes a catalytic pathway that allows the chemical potential that had been transferred to the APS reaction to "leak" into the chemical milieu. The system leaks because the E.GDP complex catalyzes the uncoupled APS reaction. The addition of phosphate to the leaky GDP.E.APS.PPi complex converts it into the central Pi.GDP.E.APS.PPi complex which catalyzes the energy-transfer reaction. Thus, Pi binding directs the system through the coupled mechanism, "plugging" the leak. GMPPNP, which also causes a leak, is used to demonstrate that the mass ratio of the APS reaction can be "tuned" by adjusting flux through the coupled and uncoupled pathways. This energy-coupling mechanism provides a means for controlling the quantity of chemical potential transferred to the APS reaction. This versatile linkage might well be used to the cell's advantage to avoid the toxicity associated with an excess of activated sulfate.

Cloning and bacterial expression of adenosine-5'-triphosphate sulfurylase from the enteric protozoan parasite Entamoeba histolytica.

A gene encoding adenosine-5'-triphosphate sulfurylase (AS) was cloned from the enteric protozoan parasite Entamoeba histolytica by polymerase chain reaction using degenerate oligonucleotide primers corresponding to conserved regions of the protein from a variety of organisms. The deduced amino acid sequence of E. histolytica AS revealed a calculated molecular mass of 47925 Da and an unusual basic pI of 9.38. The amebic protein sequence showed 23-48% identities with AS from bacteria, yeasts, fungi, plants, and animals with the highest identities being to Synechocystis sp. and Bacillus subtilis (48 and 44%, respectively). Four conserved blocks including putative sulfate-binding and phosphate-binding regions were highly conserved in the E. histolytica AS. The upstream region of the AS gene contained three conserved elements reported for other E. histolytica genes. A recombinant E. histolytica AS revealed enzymatic activity, measured in both the forward and reverse directions. Expression of the E. histolytica AS complemented cysteine auxotrophy of the AS-deficient Escherichia coli strains. Genomic hybridization revealed that the AS gene exists as a single copy gene. In the literature, this is the first description of an AS gene in Protozoa.

Sulfur availability and the SAC1 gene control adenosine triphosphate sulfurylase gene expression in Chlamydomonas reinhardtii.

A Chlamydomonas reinhardtii adenosine triphosphate (ATP) sulfurylase cDNA clone (pATS1) was selected by complementing a mutation in the ATP sulfurylase gene (cysD) of Escherichia coli. E. coli cysD strains harboring pATS1 grow on medium containing sulfate as the sole sulfur source and exhibit ATP sulfurylase activity. The amino acid sequence of the C. reinhardtii ATP sulfurylase, derived from the nucleotide sequence of the complementing gene (ATS1), is 25 to 40% identical to that of ATP sulfurylases in other eukaryotic organisms and has a putative transit peptide at its amino terminus. ATP sulfurylase mRNA was present when cells were grown in sulfur-replete medium, but accumulated to higher levels when the cells were exposed to sulfur-limiting conditions. Furthermore, sulfur-stress-induced accumulation of the ATS1 transcript was reduced in a strain defective in SAC1, a gene that is critical for acclimation to sulfur-limited growth.

Cloning of a cDNA encoding ATP sulfurylase from Arabidopsis thaliana by functional expression in Saccharomyces cerevisiae.

ATP sulfurylase, the first enzyme in the sulfate assimilation pathway of plants, catalyzes the formation of adenosine phosphosulfate from ATP and sulfate. Here we report the cloning of a cDNA encoding ATP sulfurylase (APS1) from Arabidopsis thaliana. APS1 was isolated by its ability to alleviate the methionine requirement of an ATP sulfurylase mutant strain of Saccharomyces cerevisiae (yeast). Expression of APS1 correlated with the presence of ATP sulfurylase enzyme activity in cell extracts. APS1 is a 1748-bp cDNA with an open reading frame predicted to encode a 463-amino acid, 51,372-D protein. The predicted amino acid sequence of APS1 is similar to ATP sulfurylase of S. cerevisiae, with which it is 25% identical. Two lines of evidence indicate that APS1 encodes a chloroplast form of ATP sulfurylase. Its predicted amino-terminal sequence resembles a chloroplast transit peptide; and the APS1 polypeptide, synthesized in vitro, is capable of entering isolated intact chloroplasts. Several genomic DNA fragments that hybridize with the APS1 probe were identified. The APS1 cDNA hybridizes to three species of mRNA in leaves (1.85, 1.60, and 1.20 kb) and to a single species of mRNA in roots (1.85 kb).

Isolation and characterization of two cDNA clones encoding ATP-sulfurylases from potato by complementation of a yeast mutant.

Sulfur plays an important role in plants, being used for the biosynthesis of amino acids, sulfolipids and secondary metabolites. After uptake sulfate is activated and subsequently reduced to sulfide or serves as donor for sulfurylation reactions. The first step in the activation of sulfate in all cases studied so far is catalyzed by the enzyme ATP-sulfurylase (E.C. 2.7.7.4.) which catalyzes the formation of adenosine-5'-phosphosulfate (APS). Two cDNA clones from potato encoding ATP-sulfurylases were identified following transformation of a Saccharomyces cerevisiae mutant deficient in ATP-sulfurylase activity with a cDNA library from potato source leaf poly(A)+ RNA cloned in a yeast expression vector. Several transformants were able to grow on a medium with sulfate as the only sulfur source, this ability being strictly linked to the presence of two classes of cDNAs. The clones StMet3-1 and StMet3-2 were further analyzed. DNA analysis revealed an open reading frame encoding a protein with a molecular mass of 48 kDa in the case of StMet3-1 and 52 kDa for StMet3-2. The deduced polypeptides are 88% identical at the amino acid level. The clone StMet3-2 has a 48 amino acid N-terminal extension which shows common features of a chloroplast transit peptide. Sequence comparison of the ATP-sulfurylase Met3 from Saccharomyces cerevisiae with the cDNA StMet3-1 (StMet3-2) reveals 31% (30%) identity at the amino acid level. Protein extracts from the yeast mutant transformed with the clone StMet3-1 displayed ATP-sulfurylase activity. RNA blot analysis demonstrated the expression of both genes in potato leaves, root and stem, but not in tubers.(ABSTRACT TRUNCATED AT 250 WORDS)

The sulfate activation locus of Escherichia coli K12: cloning, genetic, and enzymatic characterization.

The sulfate activation locus of Escherichia coli K12 has been cloned by complementation. The genes and gene products of this locus have been characterized by correlating the enzyme activity, complementation patterns, and polypeptides associated with subclones of the cloned DNA. The enzymes of the sulfate activation pathway, ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) and APS kinase (ATP:adenosine-5'-phosphosulfate 3'-phosphotransferase, EC 2.7.1.25) have been overproduced approximately 100-fold. Overproduction of ATP sulfurylase requires the expression of both the cysD gene, encoding a 27-kDa polypeptide, and a previously unidentified gene, denoted cysN, which encodes a 62-kDa polypeptide. Purification of ATP sulfurylase to homogeneity reveals that the enzyme is composed of two types of subunits which are encoded by cysD and cysN. Insertion of a kanamycin resistance gene into plasmid or chromosomal cysN prevents sulfate activation and decreases expression of the downstream cysC gene. cysC appears to be the APS kinase structural gene and encodes a 21-kDa polypeptide. The genes are adjacent and are transcribed counterclockwise on the E. coli chromosome in the order cysDNC. cysN and cysC are within the same operon and cysDNC are not in an operon containing cysHIJ.