Phosphosulfate Reductase Research Articles

Expression patterns of mRNAs for methanotrophy and thiotrophy in symbionts of the hydrothermal vent mussel Bathymodiolus puteoserpentis.

The hydrothermal vent mussel Bathymodiolus puteoserpentis (Mytilidae) from the Mid-Atlantic Ridge hosts symbiotic sulfur- and methane-oxidizing bacteria in its gills. In this study, we investigated the activity and distribution of these two symbionts in juvenile mussels from the Logatchev hydrothermal vent field (14°45'N Mid-Atlantic Ridge). Expression patterns of two key genes for chemosynthesis were examined: pmoA (encoding subunit A of the particulate methane monooxygenase) as an indicator for methanotrophy, and aprA (encoding the subunit A of the dissimilatory adenosine-5'-phosphosulfate reductase) as an indicator for thiotrophy. Using simultaneous fluorescence in situ hybridization (FISH) of rRNA and mRNA we observed highest mRNA FISH signals toward the ciliated epithelium where seawater enters the gills. The levels of mRNA expression differed between individual specimens collected in a single grab from the same sampling site, whereas no obvious differences in symbiont abundance or distribution were observed. We propose that the symbionts respond to the steep temporal and spatial gradients in methane, reduced sulfur compounds and oxygen by modifying gene transcription, whereas changes in symbiont abundance and distribution take much longer than regulation of mRNA expression and may only occur in response to long-term changes in vent fluid geochemistry.

The key enzyme of sulfate assimilation, adenosine 5′‐phosphosulfate reductase, is regulated by HY5 in Arabidopsis

Plant sulfate assimilation is regulated by demand for reduced sulfur, as is its key enzyme, adenosine 5'-phosphosulfate reductase (APR). In a genetic screen for mutants lacking this regulation, we identified the bZIP transcription factor LONG HYPOCOTYL 5 (HY5) as a necessary component of the regulatory circuit. Regulation of APR activity by the inhibitor of glutathione synthesis, buthionine sulfoximine, or by the precursor of cysteine, O-acetylserine, was disrupted in the hy5 mutant. When dark-adapted plants were re-illuminated, the rapid induction of APR1 and APR2 mRNA levels was attenuated in hy5 seedlings, but APR3 regulation was not affected. Chromatin immunoprecipitation revealed that HY5 binds directly to the APR1 and APR2 promoters but not to the APR3 promoter. Accordingly, the regulation of APR1 and APR2 by O-acetylserine was disturbed in hy5 roots. HY5 is also important for the coordination of nitrogen and sulfur assimilation, as, unlike the wild-type, hy5 mutants do not undergo a reduction in sulfate uptake and APR activity during nitrogen starvation. Altogether, these data show that HY5 plays an important role in regulation of APR gene expression and plant sulfate assimilation.

Geometric and Electrostatic Study of the [4Fe-4S] Cluster of Adenosine-5′-Phosphosulfate Reductase from Broken Symmetry Density Functional Calculations and Extended X-ray Absorption Fine Structure Spectroscopy

Adenosine-5'-phosphosulfate reductase (APSR) is an iron-sulfur protein that catalyzes the reduction of adenosine-5'-phosphosulfate (APS) to sulfite. APSR coordinates to a [4Fe-4S] cluster via a conserved CC-X(~80)-CXXC motif, and the cluster is essential for catalysis. Despite extensive functional, structural, and spectroscopic studies, the exact role of the iron-sulfur cluster in APS reduction remains unknown. To gain an understanding into the role of the cluster, density functional theory (DFT) analysis and extended X-ray fine structure spectroscopy (EXAFS) have been performed to reveal insights into the coordination, geometry, and electrostatics of the [4Fe-4S] cluster. X-ray absorption near-edge structure (XANES) data confirms that the cluster is in the [4Fe-4S](2+) state in both native and substrate-bound APSR while EXAFS data recorded at ~0.1 Å resolution indicates that there is no significant change in the structure of the [4Fe-4S] cluster between the native and substrate-bound forms of the protein. On the other hand, DFT calculations provide an insight into the subtle differences between the geometry of the cluster in the native and APS-bound forms of APSR. A comparison between models with and without the tandem cysteine pair coordination of the cluster suggests a role for the unique coordination in facilitating a compact geometric structure and "fine-tuning" the electronic structure to prevent reduction of the cluster. Further, calculations using models in which residue Lys144 is mutated to Ala confirm the finding that Lys144 serves as a crucial link in the interactions involving the [4Fe-4S] cluster and APS.

Crystallization of Adenylylsulfate Reductase from Desulfovibrio gigas: A Strategy Based on Controlled Protein Oligomerization

Adenylylsulfate reductase (adenosine 5′-phosphosulfate reductase, APS reductase or APSR, E.C.1.8.99.2) catalyzes the conversion of APS to sulfite in dissimilatory sulfate reduction. APSR was isolated and purified directly from massive anaerobically grown Desulfovibrio gigas, a strict anaerobe, for structure and function investigation. Oligomerization of APSR to form dimers–α_2β_2, tetramers–α_4β_4, hexamers–α_6β_6, and larger oligomers was observed during purification of the protein. Dynamic light scattering and ultracentrifugation revealed that the addition of adenosine monophosphate (AMP) or adenosine 5′-phosphosulfate (APS) disrupts the oligomerization, indicating that AMP or APS binding to the APSR dissociates the inactive hexamers into functional dimers. Treatment of APSR with β-mercaptoethanol decreased the enzyme size from a hexamer to a dimer, probably by disrupting the disulfide Cys156—Cys162 toward the C-terminus of the β-subunit. Alignment of the APSR sequences from D. gigas and A. fulgidus revealed the largest differences in this region of the β-subunit, with the D. gigas APSR containing 16 additional amino acids with the Cys156—Cys162 disulfide. Studies in a pH gradient showed that the diameter of the APSR decreased progressively with acidic pH. To crystallize the APSR for structure determination, we optimized conditions to generate a homogeneous and stable form of APSR by combining dynamic light scattering, ultracentrifugation, and electron paramagnetic resonance methods to analyze the various oligomeric states of the enzyme in varied environments.

Diversity and function in microbial mats from the Lucky Strike hydrothermal vent field

Diversity and function in microbial mats from the Lucky Strike hydrothermal vent field (Mid-Atlantic Ridge) were investigated using molecular approaches. DNA and RNA were extracted from mat samples overlaying hydrothermal deposits and Bathymodiolus azoricus mussel assemblages. We constructed and analyzed libraries of 16S rRNA gene sequences and sequences of functional genes involved in autotrophic carbon fixation [forms I and II RuBisCO (cbbL/M), ATP-citrate lyase B (aclB)]; methane oxidation [particulate methane monooxygenase (pmoA)] and sulfur oxidation [adenosine-5'-phosphosulfate reductase (aprA) and soxB]. To gain new insights into the relationships between mats and mussels, we also used new domain-specific 16S rRNA gene primers targeting Bathymodiolus sp. symbionts. All identified archaeal sequences were affiliated with a single group: the marine group 1 Thaumarchaeota. In contrast, analyses of bacterial sequences revealed much higher diversity, although two phyla Proteobacteria and Bacteroidetes were largely dominant. The 16S rRNA gene sequence library revealed that species affiliated to Beggiatoa Gammaproteobacteria were the dominant active population. Analyses of DNA and RNA functional gene libraries revealed a diverse and active chemolithoautotrophic population. Most of these sequences were affiliated with Gammaproteobacteria, including hydrothermal fauna symbionts, Thiotrichales and Methylococcales. PCR and reverse transcription-PCR using 16S rRNA gene primers targeted to Bathymodiolus sp. symbionts revealed sequences affiliated with both methanotrophic and thiotrophic endosymbionts.

Spectroscopic Studies on the [4Fe-4S] Cluster in Adenosine 5′-Phosphosulfate Reductase from Mycobacterium tuberculosis

Mycobacterium tuberculosis adenosine 5'-phosphosulfate reductase (MtAPR) is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. The enzyme harbors a [4Fe-4S](2+) cluster that is coordinated by four cysteinyl ligands, two of which are adjacent in the amino acid sequence. The iron-sulfur cluster is essential for catalysis; however, the precise role of the [4Fe-4S] cluster in APR remains unknown. Progress in this area has been hampered by the failure to generate a paramagnetic state of the [4Fe-4S] cluster that can be studied by electron paramagnetic resonance spectroscopy. Herein, we overcome this limitation and report the EPR spectra of MtAPR in the [4Fe-4S](+) state. The EPR signal is rhombic and consists of two overlapping S = ½ species. Substrate binding to MtAPR led to a marked increase in the intensity and resolution of the EPR signal and to minor shifts in principle g values that were not observed among a panel of substrate analogs, including adenosine 5'-diphosphate. Using site-directed mutagenesis, in conjunction with kinetic and EPR studies, we have also identified an essential role for the active site residue Lys-144, whose side chain interacts with both the iron-sulfur cluster and the sulfate group of adenosine 5'-phosphosulfate. The implications of these findings are discussed with respect to the role of the iron-sulfur cluster in the catalytic mechanism of APR.

Real-Time PCR Quantification and Diversity Analysis of the Functional Genes aprA and dsrA of Sulfate-Reducing Prokaryotes in Marine Sediments of the Peru Continental Margin and the Black Sea

Sulfate-reducing prokaryotes (SRP) are ubiquitous and quantitatively important members in many ecosystems, especially in marine sediments. However their abundance and diversity in subsurface marine sediments is poorly understood. In this study, the abundance and diversity of the functional genes for the enzymes adenosine 5′-phosphosulfate reductase (aprA) and dissimilatory sulfite reductase (dsrA) of SRP in marine sediments of the Peru continental margin and the Black Sea were analyzed, including samples from the deep biosphere (ODP site 1227). For aprA quantification a Q-PCR assay was designed and evaluated. Depth profiles of the aprA and dsrA copy numbers were almost equal for all sites. Gene copy numbers decreased concomitantly with depth from around 108/g sediment close to the sediment surface to less than 105/g sediment at 5 mbsf. The 16S rRNA gene copy numbers of total bacteria were much higher than those of the functional genes at all sediment depths and used to calculate the proportion of SRP to the total Bacteria. The aprA and dsrA copy numbers comprised in average 0.5–1% of the 16S rRNA gene copy numbers of total bacteria in the sediments up to a depth of ca. 40 mbsf. In the zone without detectable sulfate in the pore water from about 40–121 mbsf (Peru margin ODP site 1227), only dsrA (but not aprA) was detected with copy numbers of less than 104/g sediment, comprising ca. 14% of the 16S rRNA gene copy numbers of total bacteria. In this zone, sulfate might be provided for SRP by anaerobic sulfide oxidation. Clone libraries of aprA showed that all isolated sequences originate from SRP showing a close relationship to aprA of characterized species or form a new cluster with only distant relation to aprA of isolated SRP. For dsrA a high diversity was detected, even up to 121 m sediment depth in the deep biosphere.

Characterization of Amino Acid and S-alk(en)yl-L-cysteine Sulfoxide Production in Japanese Bunching Onion Carrying an Extra Chromosome of Shallot

Eight Japanese bunching onion (Allium fistulosum L.)—shallot (Allium cepa L. Aggregatum group) monosomic addition lines (MALs, FF + 1A–FF + 8A) were used to reveal the effects of single alien chromosomes of A. cepa on the production of amino acids and S-alk(en)yl-L-cysteine sulfoxides (ACSOs) in leaf tissues of A. fistulosum. Amino acid and ACSO contents in MAL leaf blades were determined once every 3 months from August 2005 to May 2006. The amino acid found in the greatest amount in all of the MALs throughout the year was cysteine, except for FF + 7A accumulated glycine as the greatest amount of amino acid (Nov. 2005). All of the MALs contained three ACSOs in varying amounts and proportions, and all accumulated S-1-propenyl CSO as a major ACSO but hardly produced S-2-propenyl CSO throughout the year. FF + 3A showed greatly increased proportions of S-methyl CSO in total ACSO, suggesting that anonymous genes controlling S-methyl CSO production are located on chromosome 3A of shallot. High accumulation of total ACSOs in the monosomic additions FF + 3A, FF + 4A, FF + 5A, and FF + 8A was observed during different growth periods. Using PCR-based marker analysis, sulfate transporter, adenosine 5'-phosphosulfate reductase (APSR), serine acetyltransferase, O-acetylserine thiol-lyase, glutamylcysteine synthase, glutathione synthase, and γ-glutamyl transpeptidase candidate genes related to sulfur assimilation and ACSO biosynthesis were allocated to chromosomes 7A, 2A, 7A, 7A, 2A, 7A, and 4A, respectively. This result showed little association between the chromosomal locations of these genes and ACSO accumulation, suggesting that anonymous genes controlling ACSO accumulation were dispersed on the 2A, 3A, 4A, 5A, 7A, and 8A chromosomes of shallot. APSR gene expression was inhibited by 5A chromosome additions, suggesting that one of the regulatory genes was located on a 5A chromosome and inhibited APSR expression.

Mechanisms and evolution of oxidative sulfur metabolism in green sulfur bacteria.

Green sulfur bacteria (GSB) constitute a closely related group of photoautotrophic and thiotrophic bacteria with limited phenotypic variation. They typically oxidize sulfide and thiosulfate to sulfate with sulfur globules as an intermediate. Based on genome sequence information from 15 strains, the distribution and phylogeny of enzymes involved in their oxidative sulfur metabolism was investigated. At least one homolog of sulfide:quinone oxidoreductase (SQR) is present in all strains. In all sulfur-oxidizing GSB strains except the earliest diverging Chloroherpeton thalassium, the sulfide oxidation product is further oxidized to sulfite by the dissimilatory sulfite reductase (DSR) system. This system consists of components horizontally acquired partly from sulfide-oxidizing and partly from sulfate-reducing bacteria. Depending on the strain, the sulfite is probably oxidized to sulfate by one of two different mechanisms that have different evolutionary origins: adenosine-5′-phosphosulfate reductase or polysulfide reductase-like complex 3. Thiosulfate utilization by the SOX system in GSB has apparently been acquired horizontally from Proteobacteria. SoxCD does not occur in GSB, and its function in sulfate formation in other bacteria has been replaced by the DSR system in GSB. Sequence analyses suggested that the conserved soxJXYZAKBW gene cluster was horizontally acquired by Chlorobium phaeovibrioides DSM 265 from the Chlorobaculum lineage and that this acquisition was mediated by a mobile genetic element. Thus, the last common ancestor of currently known GSB was probably photoautotrophic, hydrogenotrophic, and contained SQR but not DSR or SOX. In addition, the predominance of the Chlorobium–Chlorobaculum–Prosthecochloris lineage among cultured GSB could be due to the horizontally acquired DSR and SOX systems. Finally, based upon structural, biochemical, and phylogenetic analyses, a uniform nomenclature is suggested for sqr genes in prokaryotes.

The Sulfate-Rich and Extreme Saline Sediment of the Ephemeral Tirez Lagoon: A Biotope for Acetoclastic Sulfate-Reducing Bacteria and Hydrogenotrophic Methanogenic Archaea

Our goal was to examine the composition of methanogenic archaea (MA) and sulfate-reducing (SRP) and sulfur-oxidizing (SOP) prokaryotes in the extreme athalassohaline and particularly sulfate-rich sediment of Tirez Lagoon (Spain). Thus, adenosine-5′-phosphosulfate (APS) reductase α (aprA) and methyl coenzyme M reductase α (mcrA) gene markers were amplified given that both enzymes are specific for SRP, SOP, and MA, respectively. Anaerobic populations sampled at different depths in flooded and dry seasons from the anoxic sediment were compared qualitatively via denaturing gradient gel electrophoresis (DGGE) fingerprint analysis. Phylogenetic analyses allowed the detection of SRP belonging to Desulfobacteraceae, Desulfohalobiaceae, and Peptococcaceae in ∂-proteobacteria and Firmicutes and SOP belonging to Chromatiales/Thiotrichales clade and Ectothiorhodospiraceae in γ-proteobacteria as well as MA belonging to methylotrophic species in Methanosarcinaceae and one hydrogenotrophic species in Methanomicrobiaceae. We also estimated amino acid composition, GC content, and preferential codon usage for the AprA and McrA sequences from halophiles, nonhalophiles, and Tirez phylotypes. Even though our results cannot be currently conclusive regarding the halotolerant strategies carried out by Tirez phylotypes, we discuss the possibility of a plausible “salt-in” signal in SRP and SOP as well as of a speculative complementary haloadaptation between salt-in and salt-out strategies in MA.

Novel groups of Gammaproteobacteria catalyse sulfur oxidation and carbon fixation in a coastal, intertidal sediment

The oxidation of hydrogen sulfide is essential to sulfur cycling in marine habitats. However, the role of microbial sulfur oxidation in marine sediments and the microorganisms involved are largely unknown, except for the filamentous, mat-forming bacteria. In this study we explored the diversity, abundance and activity of sulfur-oxidizing prokaryotes (SOP) in sulfidic intertidal sediments using 16S rRNA and functional gene sequence analyses, fluorescence in situ hybridization (FISH) and microautoradiography. The 16S rRNA gene analysis revealed that distinct clades of uncultured Gammaproteobacteria are important SOP in the tidal sediments. This was supported by the dominance of gammaproteobacterial sequences in clone libraries of genes encoding the reverse dissimilatory sulfite reductase (rDSR) and the adenosine phosphosulfate reductase (APR). Numerous sequences of all three genes grouped with uncultured autotrophic SOP. Accordingly, Gammaproteobacteria accounted for 40-70% of all ¹⁴CO₂ -incorporating cells in surface sediments as shown by microautoradiography. Furthermore, phylogenetic analysis of all three genes consistently suggested a discrete population of SOP that was most closely related to the sulfur-oxidizing endosymbionts of the tubeworm Oligobrachia spp. FISH showed that members of this population (WS-Gam209 group) were abundant, reaching up to 1.3 × 10⁸ cells ml⁻¹ (4.6% of all cells). Approximately 25% of this population incorporated CO₂, consistent with a chemolithoautotrophic metabolism most likely based on sulfur oxidation. Thus, we hypothesize that novel, gammaproteobacterial SOP attached to sediment particles may play a more important role for sulfide removal and primary production in marine sediments than previously assumed.

Enhanced Glutathione Metabolism Is Correlated with Sulfur-Induced Resistance in Tobacco mosaic virus–Infected Genetically Susceptible Nicotiana tabacum Plants

Sulfur-induced resistance, also known as sulfur-enhanced defense (SIR/SED) was investigated in Nicotiana tabacum cv. Samsun nn during compatible interaction with Tobacco mosaic virus (TMV) in correlation with glutathione metabolism. To evaluate the influence of sulfur nutritional status on virus infection, tobacco plants were treated with nutrient solutions containing either sufficient sulfate (+S) or no sulfate (-S). Sufficient sulfate supply resulted in a suppressed and delayed symptom development and diminished virus accumulation over a period of 14 days after inoculation as compared with -S conditions. Expression of the defense marker gene PR-1a was markedly upregulated in sulfate-treated plants during the first day after TMV inoculation. The occurrence of SIR/SED correlated with a higher level of activity of sulfate assimilation, cysteine, and glutathione metabolism in plants treated with sulfate. Additionally, two key genes involved in cysteine and glutathione biosynthesis (encoding adenosine 5'-phosphosulfate reductase and γ-glutamylcysteine synthetase, respectively) were upregulated within the first day after TMV inoculation under +S conditions. Sulfate withdrawal from the soil was accelerated at the beginning of the infection, whereas it declined in the long term, leading to an accumulation of sulfur in the soil of plants grown with sulfate. This observation could be correlated with a decrease in sulfur contents in TMV-infected leaves in the long term. In summary, this is the first study that demonstrates a link between the activation of cysteine and glutathione metabolism and the induction of SIR/SED during a compatible plant-virus interaction in tobacco plants, indicating a general mechanism behind SIR/SED.

Discovery of a novel adenosine 5′-phosphosulfate (APS) reductase from the methanarcheon Methanocaldococcus jannaschii

Here we report the first discovery of adenosine 5′-phosphosulfate reductase (APR), a key enzyme of the sulfate reduction pathway, in the methanarchaeon Methanocaldococcus jannaschii. While the sulfate reduction pathway is present in other organisms, it is not expected to exist in methanarchaea because their habitats often already possess an abundance of reduced sulfur, particularly H 2S. However, the gene product of open reading frame (ORF) Mj0973 in M. jannaschii possesses sequence similarities with known APRs and 3′-phosphoadenosine-5′-phosphosulfate reductases (PAPRs) from various organisms. In order to further investigate this ORF, we expressed the gene Mj0973 from M. jannaschii and purified the resulting protein. Kinetic studies revealed that the purified protein is able to reduce APS with Escherichia coli thioredoxin (Trx) supplied as the electron donor, but is unable to reduce PAPS. The apparent K m, V max, and k cat/ K m values at pH 8.0 and 30 °C were 0.29 μM, 0.079 μM mg −1 min −1, and 299,655 M −1 s −1, respectively. This observation of APR activity strongly indicates the presence of an APS-utilizing sulfate reduction pathway in the methanarchaeon M. jannaschii.

Effect of the deletion of qmoABC and the promoter-distal gene encoding a hypothetical protein on sulfate reduction in Desulfovibrio vulgaris Hildenborough.

The pathway of electrons required for the reduction of sulfate in sulfate-reducing bacteria (SRB) is not yet fully characterized. In order to determine the role of a transmembrane protein complex suggested to be involved in this process, a deletion in Desulfovibrio vulgaris Hildenborough was created by marker exchange mutagenesis that eliminated four genes putatively encoding the QmoABC complex and a hypothetical protein (DVU0851). The Qmo (quinone-interacting membrane-bound oxidoreductase) complex is proposed to be responsible for transporting electrons to the dissimilatory adenosine-5'-phosphosulfate reductase in SRB. In support of the predicted role of this complex, the deletion mutant was unable to grow using sulfate as its sole electron acceptor with a range of electron donors. To explore a possible role for the hypothetical protein in sulfate reduction, a second mutant was constructed that had lost only the gene that codes for the DVU0851 protein. The second constructed mutant grew with sulfate as the sole electron acceptor; however, there was a lag that was not present with the wild-type or complemented strain. Neither deletion strain was significantly impaired for growth with sulfite or thiosulfate as the terminal electron acceptor. Complementation of the Delta(qmoABC-DVU0851) mutant with all four genes or only the qmoABC genes restored its ability to grow by sulfate respiration. These results confirmed the prediction that the Qmo complex is in the electron pathway for sulfate reduction and revealed that no other transmembrane complex could compensate when Qmo was lacking.

Regulation of sulfate assimilation in Physcomitrella patens: mosses are different!

Sulfur is an essential nutrient, taken up as sulfate from soil, reduced and incorporated into bioorganic compounds in plant cells. The pathway of sulfate assimilation is highly regulated in a demand-driven manner in seed plants. To test the evolutionary conservation of the regulatory mechanisms, we analyzed regulation of the pathway in the model for basal plants, the moss Physcomitrella patens. While in Arabidopsis the key enzyme of sulfate assimilation, adenosine 5'-phosphosulfate reductase (APR), is feedback repressed by thiols and induced by reduced levels of glutathione, in P. patens such regulation does not occur. The control of the pathway was not moved to other components as these conditions affected neither mRNA accumulation of other genes of sulfate assimilation nor sulfate uptake. Other treatments known to regulate APR, O-acetylserine, cadmium and sulfur deficiency affected APR transcript levels, but not enzyme activity. It appears that the sulfate assimilation pathway in P. patens is much more robust than in seed plants. Thus, the regulatory networks controlling the pathway have probably evolved only later in the evolution of the seed plants after separation of the bryophytes.

Exogenous application of 5-aminolevulinic acid increases the transcript levels of sulfur transport and assimilatory genes, sulfate uptake, and cysteine and glutathione contents in Arabidopsis thaliana

5-Aminolevulinic acid (ALA), a key precursor of porphyrin biosynthesis, promotes plant growth and crop yields. Although ALA is known to promote carbon fixation and nitrogen assimilation in plants, the effects of ALA on sulfur assimilation have not been determined. In the present study, we analyzed the effect of ALA on sulfur assimilation. We used a fusion gene construct consisting of a promoter region of the high-affinity sulfate transporter SULTR1;2 from Arabidopsis and green fluorescent protein ([GFP] P SULTR1;2 -GFP) to determine whether ALA treatment influences the expression of the sulfur transport gene. The GFP levels in P SULTR1;2 -GFP plants were significantly increased by 0.3 and 1 mmol L−1 ALA under both sulfur-sufficient and sulfur-deficient conditions. Real-time reverse transcription-polymerase chain reaction experiments revealed that these concentrations of ALA also increased the mRNA levels of other key sulfur transport and assimilatory genes, such as SULTR, adenosine 5′-phosphosulfate reductases and serine acetyl transferase. Sulfate uptake was enhanced by ALA treatment under sulfur-sufficient conditions. In addition, ALA treatment increased the accumulation of cysteine and glutathione, particularly in the shoot. Our data demonstrated that exogenously applied ALA increases the transcript levels of some sulfur assimilatory genes, sulfate uptake, and the contents of cysteine and glutathione. We propose a new role for ALA in regulating the sulfur assimilatory pathway.

Genes of primary sulfate assimilation are part of the glucosinolate biosynthetic network inArabidopsis thaliana

Glucosinolates are plant secondary metabolites involved in responses to biotic stress. The final step of their synthesis is the transfer of a sulfo group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) onto a desulfo precursor. Thus, glucosinolate synthesis is linked to sulfate assimilation. The sulfate donor for this reaction is synthesized from sulfate in two steps catalyzed by ATP sulfurylase (ATPS) and adenosine 5'-phosphosulfate kinase (APK). Here we demonstrate that R2R3-MYB transcription factors, which are known to regulate both aliphatic and indolic glucosinolate biosynthesis in Arabidopsis thaliana, also control genes of primary sulfate metabolism. Using trans-activation assays we found that two isoforms of APK, APK1, and APK2, are regulated by both classes of glucosinolate MYB transcription factors; whereas two ATPS genes, ATPS1 and ATPS3, are differentially regulated by these two groups of MYB factors. In addition, we show that the adenosine 5'-phosphosulfate reductases APR1, APR2, and APR3, which participate in primary sulfate reduction, are also activated by the MYB factors. These observations were confirmed by analysis of transgenic lines with modulated expression levels of the glucosinolate MYB factors. The changes in transcript levels also affected enzyme activities, the thiol content and the sulfate reduction rate in some of the transgenic plants. Altogether the data revealed that the MYB transcription factors regulate genes of primary sulfate metabolism and that the genes involved in the synthesis of activated sulfate are part of the glucosinolate biosynthesis network.

Composition and dynamics of sulfate-reducing bacteria during the waterflooding process in the oil field application

The composition and dynamics of sulfate-reducing bacteria (SRB) during the waterflooding process of Daqing Oilfield were investigated in this study. PCR-DGGE analysis indicated that the microbial communities were significantly different in each treatment unit, and the dominant members were mainly close to Clostridium sp., Thauera sp., Hydrogenophaga sp., Pseudomonas sp., Eubacterium sp. and Arcobacter sp. However, the members belonging to SRB were relatively few and mainly consisted of Desulfovibrio sp. and Desulfovibrio profundus. According to APS (adenosine-5'-phosphosulfate reductase) gene clone library and sequence analysis, it was found that SRB mainly belonged to Proteobacteria and Deltaproteobacteria. In the library, the genus Desulfovibrio of Deltaproteobacteria was about 65.2% while genus Desulfomonas, Desulfomicrobium, Desulfohalobium, Desulfonatronum, Desulfbulbus, Desulforhopalus, Desulfnema and Desulforhopalus were occupied nearly 34.8%. It was also proved that most of SRB in the oil field were enriched in the ground treatment unit and they are not in the liquid of oil well.

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.

Identification of Critical Ligand Binding Determinants in Mycobacterium tuberculosis Adenosine-5′-phosphosulfate Reductase

Mycobacterium tuberculosis adenosine-5'-phosphosulfate (APS) reductase is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. To facilitate the development of potent and specific inhibitors of APS reductase, we have probed the molecular determinants that underlie binding and specificity through a series of substrate and product analogues. Our study highlights the importance of specific substitutent groups for substrate binding and provides functional evidence for ligand-specific conformational states. An active site model has been developed for M. tuberculosis APS reductase that is in accord with the results presented here as well as prior structural data reported for Pseudomonas aeruginosa APS reductase and related enzymes. This model illustrates the functional features required for the interaction of APS reductase with a ligand and provides a pharmacological roadmap for the rational design of small molecules as potential inhibitors of APS reductase present in human pathogens, including M. tuberculosis.