Truncating variants in PAPSS2 gene: A cause of early prenatal onset brachyolmia?

Brachyolmia is a rare form of skeletal dysplasia characterized by a wide genetic and clinical heterogeneity. This condition is usually diagnosed postnatally, and very few cases of prenatal diagnosis have been described so far. Here, we report a case of a pregnant woman at 20 weeks' gestation referred to our center because of fetal short long bones. On targeted ultrasound, mild bowing of the femurs and fibulae and mild micrognathia were also observed. Exome sequencing analysis showed the presence in compound heterozygosity of two pathogenic variants-both truncating variants-in the 3-prime-phosphoadenosine 5-prime-phosphosulfate synthase 2 (PAPSS2) gene, known to cause brachyolmia type 4 (OMIM #612847). Of note, all of the few cases reported prenatally have indeed truncating variants. Hence, we speculate this kind of variant is likely responsible for a complete loss of function of the protein leading to an earlier and more severe phenotype.

Identification of ascorbate- and salicylate-responsive miRNAs and verification of the spectral control of miR395 in Arabidopsis.

We assumed that miRNAs might regulate the physiological and biochemical processes in plants through their effects on the redox system and phytohormones. To check this hypothesis, the transcriptome profile of wild-type Arabidopsis and lines with decreased ascorbate (Asc), glutathione (GSH), or salicylate (Sal) levels were compared. GSH deficiency did not influence the miRNA expression, whereas lower levels of Asc and Sal reduced the accumulation of 9 and 44 miRNAs, respectively, but only four miRNAs were upregulated. Bioinformatics analysis revealed that their over-represented target genes are associated with the synthesis of nitrogen-containing and aromatic compounds, nucleic acids, and sulphate assimilation. Among them, the sulphate reduction-related miR395 - ATP-sulfurylase couple was selected to check the assumed modulating role of the light spectrum. A greater induction of the Asc- and Sal-responsive miR395 was observed under sulphur starvation in far-red light compared to white and blue light in wild-type and GSH-deficient Arabidopsis lines. Sal deficiency inhibited the induction of miR395 by sulphur starvation in blue light, whereas Asc deficiency greatly reduced it independently of the spectrum. Interestingly, sulphur starvation decreased only the level of ATP sulfurylase 4 among the miR395 target genes in far-red light. The expression level of ATP sulfurylase 3 was higher in far-red light than in blue light in wild-type and Asc-deficient lines. The results indicate the coordinated control of miRNAs by the redox and hormonal system since 11 miRNAs were affected by both Asc and Sal deficiency. This process can be modulated by light spectrum, as shown for miR395.

Intestinal Sulfation Is Essential to Protect Against Colitis and Colonic Carcinogenesis.

BACKGROUND & AIMS: Sulfation is a conjugation reaction essential for numerous biochemical and cellular functions in mammals. The 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase 2 (PAPSS2) is the key enzyme to generate PAPS, which is the universal sulfonate donor for all sulfation reactions. The goal of this study was to determine whether and how PAPSS2 plays a role in colitis and colonic carcinogenesis. METHODS: Tissue arrays of human colon cancer specimens, gene expression data, and clinical features of cancer patients were analyzed. Intestinal-specific Papss2 knockout mice (Papss2ΔIE) were created and subjected to dextran sodium sulfate-induced colitis and colonic carcinogenesis induced by a combined treatment of azoxymethane and dextran sodium sulfate or azoxymethane alone. RESULTS: The expression of PAPSS2 is decreased in the colon cancers of mice and humans. The lower expression of PAPSS2 in colon cancer patients is correlated with worse survival. Papss2ΔIE mice showed heightened sensitivity to colitis and colon cancer by damaging the intestinal mucosal barrier, increasing intestinal permeability and bacteria infiltration, and worsening the intestinal tumor microenvironment. Mechanistically, the Papss2ΔIE mice exhibited reduced intestinal sulfomucin content. Metabolomic analyses revealed the accumulation of bile acids, including the Farnesoid X receptor antagonist bile acid tauro-ß-muricholic acid, and deficiency in the formation of bile acid sulfates in the colon of Papss2ΔIE mice. CONCLUSIONS: We have uncovered an important role of PAPSS2-mediated sulfation in colitis and colonic carcinogenesis. Intestinal sulfation may represent a potential diagnostic marker and PAPSS2 may serve as a potential therapeutic target for inflammatory bowel disease and colon cancer.

Structural basis for the substrate recognition mechanism of ATP-sulfurylase domain of human PAPS synthase 2.

Sulfation is an essential modification on biomolecules in living cells, and 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is its unique and universal sulfate donor. Human PAPS synthases (PAPSS1 and 2) are the only enzymes that catalyze PAPS production from inorganic sulfate. Unexpectedly, PAPSS1 and PAPSS2 do not functional complement with each other, and abnormal function of PAPSS2 but not PAPSS1 leads to numerous human diseases including bone development diseases, hormone disorder and cancers. Here, we reported the crystal structures of ATP-sulfurylase domain of human PAPSS2 (ATPS2) and ATPS2 in complex with is product 5'-phosphosulfate (APS). We demonstrated that ATPS2 recognizes the substrates by using family conserved residues located on the HXXH and PP motifs, and achieves substrate binding and releasing by employing a non-conserved phenylalanine (Phe550) through a never observed flipping mechanism. Our discovery provides additional information to better understand the biological function of PAPSS2 especially in tumorigenesis, and may facilitate the drug discovery against this enzyme.

Gene Identification, expression analysis and molecular docking of ATP sulfurylase in the selenization pathway of Cardamine hupingshanensis.

BACKGROUND: ATP sulfurylase (ATPS) is a crucial enzyme for the selenate assimilation pathway in plants. RESULTS: In this study, genome-wide and comparative analyses of ATPS in Cardamine hupingshanensis, including sequence and structural analyses, were performed. The expression of ChATPS gene family members in C. hupingshanensis under selenium (Se) stress was also investigated, and our results suggest that ChATPS1-2 play key roles in the response to Se stress. Nine ATPS genes were found from C. hupingshanensis, which share highly conserved sequences with ATPS from Arabidopsis thaliana. In addition, we performed molecular docking of ATP sulfurylase in complex with compounds ATP, selenate, selenite, sulfate, and sulfite. ChAPS3-1 was found to have stronger binding energies with all compounds tested. Among these complexes, amino acid residues Arg, Gly, Ser, Glu, and Asn were commonly present. CONCLUSION: Our study reveals the molecular mechanism of C. hupingshanensis ATP sulfurylase interacting with selenate, which is essential for understanding selenium assimilation. This information will guide further studies on the function of the ChATPS gene family in the selenium stress response and lay the foundation for the selenium metabolic pathway in higher plants.

PoMet3 and PoMet14 associated with sulfate assimilation are essential for conidiogenesis and pathogenicity in Pyricularia oryzae.

Pyricularia oryzae is the causal agent of blast disease on staple gramineous crops. Sulphur is an essential element for the biosynthesis of cysteine and methionine in fungi. Here, we targeted the P. oryzae PoMET3 encoding the enzyme ATP sulfurylase, and PoMET14 encoding the APS (adenosine-5'-phosphosulphate) kinase that are involved in sulfate assimilation and sulphur-containing amino acids biosynthesis. In P. oryzae, deletion of PoMET3 or PoMET14 separately results in defects of conidiophore formation, significant impairments in conidiation, methionine and cysteine auxotrophy, limited invasive hypha extension, and remarkably reduced virulence on rice and barley. Furthermore, the defects of the null mutants could be restored by supplementing with exogenous cysteine or methionine. Our study explored the biological functions of sulfur assimilation and sulphur-containing amino acids biosynthesis in P. oryzae.

Genome-wide analyses of ATP sulfurylase (ATPS) genes in higher plants and expression profiles in sorghum (Sorghum bicolor) under cadmium and salinity stresses.

ATP sulfurylase (ATPS, EC: 2.7.7.4) is a crucial enzyme for sulfate assimilation pathway in both plastids and cytosol in plants. In this study, genome-wide and comparative analyses of ATPSs in 11 higher plant species, including sequence and structural analyses have been performed. Expression of ATPS genes in sorghum under cadmium (Cd) and salinity (NaCl) stresses were also investigated to provide a model experimental data for the regulation of ATPS genes under stress conditions. Thirty-one ATPS genes from 11 plant species were found. It showed that ATPSs from different species have high sequence divergences, which cause structural differences among them. Phylogenetic analysis has shown that there are two major types of ATPSs evolved in dicots while monocots were evolved to have one type of ATPs. Finally, expression analysis of ATPS genes revealed tissue and stress dependent expression pattern, which indicates expressions of ATPS genes are tightly regulated.

Specific inhibitors of respiratory sulfate reduction: towards a mechanistic understanding.

Microbial sulfate reduction (SR) by sulfate-reducing micro-organisms (SRM) is a primary environmental mechanism of anaerobic organic matter mineralization, and as such influences carbon and sulfur cycling in many natural and engineered environments. In industrial systems, SR results in the generation of hydrogen sulfide, a toxic, corrosive gas with adverse human health effects and significant economic and environmental consequences. Therefore, there has been considerable interest in developing strategies for mitigating hydrogen sulfide production, and several specific inhibitors of SRM have been identified and characterized. Specific inhibitors are compounds that disrupt the metabolism of one group of organisms, with little or no effect on the rest of the community. Putative specific inhibitors of SRM have been used to control sulfidogenesis in industrial and engineered systems. Despite the value of these inhibitors, mechanistic and quantitative studies into the molecular mechanisms of their inhibition have been sparse and unsystematic. The insight garnered by such studies is essential if we are to have a more complete understanding of SR, including the past and current selective pressures acting upon it. Furthermore, the ability to reliably control sulfidogenesis - and potentially assimilatory sulfate pathways - relies on a thorough molecular understanding of inhibition. The scope of this review is to summarize the current state of the field: how we measure and understand inhibition, the targets of specific SR inhibitors and how SRM acclimatize and/or adapt to these stressors.

PAPSS2-related brachyolmia: Clinical and radiological phenotype in 18 new cases.

Brachyolmia is a skeletal dysplasia characterized by short spine-short stature, platyspondyly, and minor long bone abnormalities. We describe 18 patients, from different ethnic backgrounds and ages ranging from infancy to 19 years, with the autosomal recessive form, associated with PAPSS2. The main clinical features include disproportionate short stature with short spine associated with variable symptoms of pain, stiffness, and spinal deformity. Eight patients presented prenatally with short femora, whereas later in childhood their short-spine phenotype emerged. We observed the same pattern of changing skeletal proportion in other patients. The radiological findings included platyspondyly, irregular end plates of the elongated vertebral bodies, narrow disc spaces and short over-faced pedicles. In the limbs, there was mild shortening of femoral necks and tibiae in some patients, whereas others had minor epiphyseal or metaphyseal changes. In all patients, exome and Sanger sequencing identified homozygous or compound heterozygous PAPSS2 variants, including c.809G>A, common to white European patients. Bi-parental inheritance was established where possible. Low serum DHEAS, but not overt androgen excess was identified. Our study indicates that autosomal recessive brachyolmia occurs across continents and may be under-recognized in infancy. This condition should be considered in the differential diagnosis of short femora presenting in the second trimester.

Tissue-specific transcriptional regulation of seven heavy metal stress-responsive miRNAs and their putative targets in nickel indicator castor bean (R. communis L.) plants.

R.communis L. has high capability to accumulate nickel which is a trace nutrient for higher plants and also an environmental contaminant causes toxicity related symptoms at higher concentrations. MicroRNAs (miRNAs) are known to be important modulators of responses against heavy metal stress for detoxification of the metal. In this study, we experimentally measured and validated the transcript levels of the seven heavy metal stress response-related miRNAs and the expression levels of target genes in both leaf and root tissues of R. communis L. subjected to three different concentrations of nickel stress via qRT-PCR quantification. The results demonstrated differential regulations of heavy metal stress-responsive miRNAs and their putative targets in both tissues in same stress treatments. This dynamic regulation suggest that regulatory processes differ between the tissues under nickel stress. Our data suggest that, miR838 was the most responsive to the Ni2+ stress. miR398 target gene Cu-Zn/SOD was found to be up-regulated in both root and leaf tissues. The relations between TCP and expression levels of miR159 and miR319 were also found statistically significant exclusive to leaf tissues. In leaf tissue, changes in miR395 level and its putative target genes, sulphate transporter and sulphate adenyltransferase gene were found in relation whereas, only expression level of sulphate transporter represented a statistically significant relation in root tissue. The sharp decrease in transcript levels of 2r3 myb gene at lower nickel dose suggest to investigate the role of r2r3 myb and the all MYB family members in primary and secondary metabolisms against nickel stress.

ATP Sulfurylase is Essential for the Utilization of Sulfamate as a Sulfur Source in the Yeast Komagataella pastoris (syn. Pichia pastoris).

The methylotrophic yeast Komagataella pastoris (syn. Pichia pastoris) is one of the few known yeasts that can utilize sulfamate ([Formula: see text]) as a sulfur source. The biochemical pathway responsible for the catabolism of sulfamate has yet to be identified. The present study sought to investigate whether sulfamate catabolism proceeds through either of the inorganic sulfur intermediates sulfate ([Formula: see text]) or sulfite ([Formula: see text]) before its assimilation and subsequent incorporation into sulfur-containing amino acids and their derivatives. Two key genes in the K. pastoris inorganic sulfur assimilation pathway were deleted separately and the ability of each deletion mutant to utilize sulfamate and other selected sulfur sources was studied. Deletion of the MET3 gene (which encodes the enzyme ATP sulfurylase) did not affect growth on L-methionine, sulfite, methanesulfonate, or taurine but completely abolished growth on sulfate, methyl sulfate and sulfamate. Deletion of the MET5 gene (which encodes the ß subunit of the enzyme sulfite reductase) abolished growth on all tested sulfur sources except L-methionine. These results suggest that the catabolism of sulfamate proceeds through a sulfate intermediate before its assimilation.

Sulfotransferase-1A1-dependent bioactivation of aristolochic acid I and N-hydroxyaristolactam I in human cells.

Aristolochic acids (AA) are implicated in the development of chronic renal disease and upper urinary tract carcinoma in humans. Using in vitro approaches, we demonstrated that N-hydroxyaristolactams, metabolites derived from partial nitroreduction of AA, require sulfotransferase (SULT)-catalyzed conjugation with a sulfonyl group to form aristolactam-DNA adducts. Following up on this observation, bioactivation of AA-I and N-hydroxyaristolactam I (AL-I-NOH) was studied in human kidney (HK-2) and skin fibroblast (GM00637) cell lines. Pentachlorophenol, a known SULT inhibitor, significantly reduced cell death and aristolactam-DNA adduct levels in HK-2 cells following exposure to AA-I and AL-I-NOH, suggesting a role for Phase II metabolism in AA activation. A gene knockdown, siRNA approach was employed to establish the involvement of selected SULTs and nitroreductases in AA-I bioactivation. Silencing of SULT1A1 and PAPSS2 led to a significant decrease in aristolactam-DNA levels in both cell lines following exposure to AA-I, indicating the critical role for sulfonation in the activation of AA-I in vivo Since HK-2 cells proved relatively resistant to knockdown with siRNAs, gene silencing of xanthine oxidoreductase, cytochrome P450 oxidoreductase and NADPH:quinone oxidoreductase was conducted in GM00637 cells, showing a significant increase, decrease and no effect on aristolactam-DNA levels, respectively. In GM00637 cells exposed to AL-I-NOH, suppressing the SULT pathway led to a significant decrease in aristolactam-DNA formation, mirroring data obtained for AA-I. We conclude from these studies that SULT1A1 is involved in the bioactivation of AA-I through the sulfonation of AL-I-NOH, contributing significantly to the toxicities of AA observed in vivo.

pDGO100, a type 1 IncC plasmid from 1981 carrying ARI-A and a Tn1696-like transposon in a novel integrating element.

Most A/C plasmids sequenced to date were recovered in the last two decades. To gain insight into the evolution of this group, the IncC plasmid pDGO100, found in a multiply antibiotic-resistant Escherichia coli strain isolated in 1981, was sequenced. pDGO100 belongs to the type 1 lineage and carries an ARI-A antibiotic resistance island but not an ARI-B island. The A/C2 backbone of pDGO100 has a deletion in the rhs1 gene previously found in pRMH760 and differs by only six single base pair substitutions from pRMH760, recovered at the same hospital 16years later. This confirms that the separation of type 1 and type 2 IncC plasmids is long standing. The ARI-A islands are also closely related, but pRMH760 contains Tn4352B in tniA of Tn402, while in pDGO100, Tn4352 has inserted into merA of pDUmer. pDGO100 also carries an additional 46kb insertion that includes a Tn1696-like transposon with the dfrB3 gene cassette. This insertion was identified as a novel integrating element, with an int gene at one end, and also includes the fec iron uptake operon that has been acquired from the E. coli chromosome. Related integrating elements carrying the same int gene were found in A/C2, IncHI1, and IncHI2 plasmids, and in the chromosomes of Enterobacter cloacae, Klebsiella oxytoca, and Cronobacter sakazakii isolates. In the Enterobacteriaceae chromosomes, these integrating elements appear to target a gene encoding a radical SAM superfamily protein. In the A/C2, IncHI1, and IncHI2 plasmids, genes encoding a phosphoadenosine phosphosulfate reductase were interrupted. The extremities of the integrating element are highly conserved, whilst the internal gene content varies. The detection of integrative elements in plasmids demonstrates an increased range of locations into which this type of mobile element can integrate and insertion in plasmids is likely to assist their spread.

Rex (encoded by DVU_0916) in Desulfovibrio vulgaris Hildenborough is a repressor of sulfate adenylyl transferase and is regulated by NADH.

Although the enzymes for dissimilatory sulfate reduction by microbes have been studied, the mechanisms for transcriptional regulation of the encoding genes remain unknown. In a number of bacteria the transcriptional regulator Rex has been shown to play a key role as a repressor of genes producing proteins involved in energy conversion. In the model sulfate-reducing microbe Desulfovibrio vulgaris Hildenborough, the gene DVU_0916 was observed to resemble other known Rex proteins. Therefore, the DVU_0916 protein has been predicted to be a transcriptional repressor of genes encoding proteins that function in the process of sulfate reduction in D. vulgaris Hildenborough. Examination of the deduced DVU_0916 protein identified two domains, one a winged helix DNA-binding domain common for transcription factors, and the other a Rossman fold that could potentially interact with pyridine nucleotides. A deletion of the putative rex gene was made in D. vulgaris Hildenborough, and transcript expression studies of sat, encoding sulfate adenylyl transferase, showed increased levels in the D. vulgaris Hildenborough Rex (RexDvH) mutant relative to the parental strain. The RexDvH-binding site upstream of sat was identified, confirming RexDvH to be a repressor of sat. We established in vitro that the presence of elevated NADH disrupted the interaction between RexDvH and DNA. Examination of the 5' transcriptional start site for the sat mRNA revealed two unique start sites, one for respiring cells that correlated with the RexDvH-binding site and a second for fermenting cells. Collectively, these data support the role of RexDvH as a transcription repressor for sat that senses the redox status of the cell.

Redox signaling mediates the expression of a sulfate-deprivation-inducible microRNA395 in Arabidopsis.

MicroRNA395 (miR395) is a conserved miRNA that targets a low-affinity sulfate transporter (AST68) and three ATP sulfurylases (APS1, APS3 and APS4) in higher plants. In this study, At2g28780 was confirmed as another target of miR395 in Arabidopsis. Interestingly, several dicots contained genes homologous to At2g28780 and a cognate miR395 complementary site but possess a gradient of mismatches at the target site. It is well established that miR395 is induced during S deprivation in Arabidopsis; however, the signaling pathways that mediate this regulation are unknown. Several findings in the present study demonstrate that redox signaling plays an important role in induction of miR395 during S deprivation. These include the following results: (i) glutathione (GSH) supplementation suppressed miR395 induction in S-deprived plants (ii) miR395 is induced in Arabidopsis seedlings exposed to Arsenate or Cu(2+) , which induces oxidative stress (iii), S deprivation-induced oxidative stress, and (iv) compromised induction of miR395 during S deprivation in cad2 mutant (deficient in GSH biosynthesis) that is defective in glutaredoxin-dependent redox signaling and ntra/ntrb (defective in thioredoxin reductases a and b) double mutants that are defective in thioredoxin-dependent redox signaling. Collectively, these findings strongly support the involvement of redox signaling in inducing the expression of miR395 during S deprivation in Arabidopsis.

Upregulation of UGT2B4 Expression by 3'-Phosphoadenosine-5'-Phosphosulfate Synthase Knockdown: Implications for Coordinated Control of Bile Acid Conjugation.

During cholestasis, the bile acid-conjugating enzymes, SULT2A1 and UGT2B4, work in concert to prevent the accumulation of toxic bile acids. To understand the impact of sulfotransferase deficiency on human hepatic gene expression, we knocked down 3'-phosphoadenosine-5'-phosphosulfate synthases (PAPSS) 1 and 2, which catalyze synthesis of the obligate sulfotransferase cofactor, in HepG2 cells. PAPSS knockdown caused no change in SULT2A1 expression; however, UGT2B4 expression increased markedly (∼41-fold increase in UGT2B4 mRNA content). Knockdown of SULT2A1 in HepG2 cells also increased UGT2B4 expression. To investigate the underlying mechanism, we transfected PAPSS-deficient HepG2 cells with a luciferase reporter plasmid containing ∼2 Kb of the UGT2B4 5'-flanking region, which included a response element for the bile acid-sensing nuclear receptor, farnesoid X receptor (FXR). FXR activation or overexpression increased UGT2B4 promoter activity; however, knocking down FXR or mutating or deleting the FXR response element did not significantly decrease UGT2B4 promoter activity. Further evaluation of the UGT2B4 5'-flanking region indicated the presence of distal regulatory elements between nucleotides -10090 and -10037 that negatively and positively regulated UGT2B4 transcription. Pulse-chase analysis showed that increased UGT2B4 expression in PAPSS-deficient cells was attributable to both increased mRNA synthesis and stability. Transfection analysis demonstrated that the UGT2B4 3'-untranslated region decreased luciferase reporter expression less in PAPSS-deficient cells than in control cells. These data indicate that knocking down PAPSS increases UGT2B4 transcription and mRNA stability as a compensatory response to the loss of SULT2A1 activity, presumably to maintain bile acid-conjugating activity.

Differential expression of sulfur assimilation pathway genes in Acidithiobacillus ferrooxidans under Cd²âº stress: evidence from transcriptional, enzymatic, and metabolic profiles.

Acidithiobacillus ferrooxidans is a heavy metal-tolerant acidophilic chemolithotroph found in acidic mine effluent and is used commercially in the bioleaching of sulfide ores. In this work, we investigated the interplay between divalent cadmium (Cd(2+)) resistance and expression of genes involved in the sulfur assimilation pathway (SAP). We also investigated the response of the thiol-containing metal-chelating metabolites, cysteine and glutathione(GSH), to increasing Cd(2+) concentrations. During growth in the presence of 30 mM Cd(2+), the concentrations of mRNA for 5 genes in the SAP pathway increased more than fourfold: these encode ATP sulfurylase (ATPS), adenosine 5'-phosphosulfate (APS) reductase, sulfite reductase (SiR), serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL). Increased transcription was also reflected in increased enzyme activities: those of SAT and adenosylphosphosulfate reductase (APR) reached a peak of 26- and 15.8-fold, respectively, compared to the control culture in the presence of 15 mM Cd(2+). In contrast, the activity of OAS-TL, which is responsible for the biosynthesis of cysteine, was diminished. At the metabolite level, the intracellular cysteine and GSH contents nearly doubled. These results suggested that Cd(2+) induced transcription of SAP genes, while directly inhibiting the activities of some enzymes (e.g., OAS-TL). Overall, these results are consistent with a detoxification/resistance mechanism involving enhanced sulfur uptake and sequestration of Cd(2+) by cysteine and glutathione.

L-Methionine repressible promoters for tuneable gene expression in Trichoderma reesei.

BACKGROUND: Trichoderma reesei is the main producer of lignocellulolytic enzymes that are required for plant biomass hydrolysis in the biorefinery industry. Although the molecular toolbox for T. reesei is already well developed, repressible promoters for strain engineering and functional genomics studies are still lacking. One such promoter that is widely employed for yeasts is that of the L-methionine repressible MET3 gene, encoding ATP sulphurylase. RESULTS: We show that the MET3 system can only be applied for T. reesei when the cellulase inducing carbon source lactose is used but not when wheat straw, a relevant lignocellulosic substrate for enzyme production, is employed. We therefore performed a transcriptomic screen for genes that are L-methionine repressible in a wheat straw culture. This analysis retrieved 50 differentially regulated genes of which 33 were downregulated. Among these, genes encoding transport proteins as well as iron containing DszA like monooxygenases and TauD like dioxygenases were strongly overrepresented. We show that the promoter region of one of these dioxygenases can be used for the strongly repressible expression of the Aspergillus niger sucA encoded extracellular invertase in T. reesei wheat straw cultures. This system is also portable to other carbon sources including D-glucose and glycerol as demonstrated by the repressible expression of the Escherichia coli lacZ encoded ß-galactosidase in T. reesei. CONCLUSION: We describe a novel, versatile set of promoters for T. reesei that can be used to drive recombinant gene expression in wheat straw cultures at different expression strengths and in an L-methionine repressible manner. The dioxygenase promoter that we studied in detail is furthermore compatible with different carbon sources and therefore applicable for manipulating protein production as well as functional genomics with T. reesei.

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.

Use of the cysteine-repressible HpMET3 promoter as a novel tool to regulate gene expression in Hansenula polymorpha.

OBJECTIVES: The promoter of HpMET3, encoding an ATP sulfurylase, was evaluated for its potential as a repressible promoter to downregulate the expression of target genes in the thermotolerant, methylotrophic yeast Hansenula polymorpha. RESULTS: The expression of lacZ under the control of the 0.6 kb HpMET3 promoter was efficiently downregulated by cysteine, but not by methionine or sulfate. The HpMET3 promoter was used to generate a conditional mutant of the HpPMT2 gene encoding an O-mannosyltransferase, which is involved in post-translational protein modification. The addition of 0.5 mM cysteine adversely affected the growth of the conditional HpMET3(p)-Hppmt2 mutant strain by downregulating transcription of HpPMT2 to approx. 40 % of the normal levels, indicating that the HpPMT2 gene is essential for cell viability. However, the HpMET3 promoter was neither induced nor repressed in the heterologous host Saccharomyces cerevisiae. CONCLUSION: Our results reveal that the cysteine-repressible HpMET3 promoter is a useful tool that downregulates the expression of various genes in H. polymorpha.