Community profiling and gene expression of fungal assimilatory nitrate reductases in agricultural soil

Although fungi contribute significantly to the microbial biomass in terrestrial ecosystems, little is known about their contribution to biogeochemical nitrogen cycles. Agricultural soils usually contain comparably high amounts of inorganic nitrogen, mainly in the form of nitrate. Many studies focused on bacterial and archaeal turnover of nitrate by nitrification, denitrification and assimilation, whereas the fungal role remained largely neglected. To enable research on the fungal contribution to the biogeochemical nitrogen cycle tools for monitoring the presence and expression of fungal assimilatory nitrate reductase genes were developed. To the ∼100 currently available fungal full-length gene sequences, another 109 partial sequences were added by amplification from individual culture isolates, representing all major orders occurring in agricultural soils. The extended database led to the discovery of new horizontal gene transfer events within the fungal kingdom. The newly developed PCR primers were used to study gene pools and gene expression of fungal nitrate reductases in agricultural soils. The availability of the extended database allowed affiliation of many sequences to known species, genera or families. Energy supply by a carbon source seems to be the major regulator of nitrate reductase gene expression for fungi in agricultural soils, which is in good agreement with the high energy demand of complete reduction of nitrate to ammonium.     

Chlamydomonas reinhardtii strains expressing nitrate reductase under control of the cabII-1 promoter: isolation of chlorate resistant mutants and identification of new loci for nitrate assimilation

The Chlamydomonas reinhardtii strain Tx11-8 is a transgenic alga that bears the nitrate reductase gene (Nia1) under control of the CabII-1 gene promoter (CabII-1-Nia1). Approximately nine copies of the chimeric CabII-1-Nia1 gene were found to be integrated in this strain and to confer a phenotype of chlorate sensitivity in the presence of ammonium. We have used this strain for the isolation of spontaneous chlorate resistant mutants in the presence of ammonium that were found to be defective at loci involved in MoCo metabolism and light-dependent growth in nitrate media. Of a total of 45 mutant strains analyzed first, 44 were affected in the MoCo activity (16 Nit⁻, unable to grow in nitrate, and 28 Nit⁺, able to grow in nitrate). All the Nit⁻ strains lacked MoCo activity. Diploid complementation of Nit⁻, MoCo⁻ strains with C. reinhardtii MoCo mutants and genetic analysis indicated that some strains were defective at known loci for MoCo biosynthesis, while three strains were defective at two new loci, hereafter named Nit10 and Nit11. The other 28 Nit⁺ strains showed almost undetectable MoCo activity or activity was below 20% of the parental strain. Second, only one strain (named 23c⁺) showed MoCo and NR activities comparable to those in the parental strain. Strain 23c⁺ seems to be affected in a locus, Nit12, required for growth in nitrate under continuous light. It is proposed that this locus is required for nitrate/chlorate transport activity. In this work, mechanisms of chlorate toxicity are reviewed in the light of our results.     

Cloning of the nitrate reductase gene (niaD) of Aspergillus nidulans and its use for transformation of Fusarium oxysporum

An heterologous transformation system for the phytopathogenic fungus Fusarium oxysporum has been developed based on the use of the Aspergillus nidulans nitrate reductase gene (niaD). F. oxysporum nia- mutants were easily selected by chlorate resistance. The A. nidulans niaD gene was isolated from a gene library by complementation of an A. nidulans niaD mutant. The cloned gene is capable of transforming F. oxysporum nia- mutants at a frequency of up to ten transformants per microgram of DNA. Southern analysis of the DNA of the F. oxysporum transformants showed that transformation resulted in integration of one or more copies of the vector DNA into the genome.     

Molecular approaches to the analysis of nitrate assimilation

Abstract. The application of molecular approaches such as mutant analysis and recombinant DNA technology, in conjunction with immunology, are set to revolutionize our understanding of the nitrate assimilation pathway. Mutant analysis has already led to the identification of genetic loci encoding a functional nitrate reduction step and is expected to lead ultimately to the identification of genes encoding nitrate uptake and nitrite reduction. Of particular significance would be identification of genes whose products contribute to regulatory networks controlling nitrogen metabolism. Recombinant DNA techniques are particularly powerful and have already allowed the molecular cloning of the genes encoding the apoprotein of nitrate reductase and nitrite reductase. These successes allow for the first lime the possibility to study directly the role of environmental factors such as type of nitrogen source (NO₃⁻ or NH₄⁺) available to the plant, light, temperature water potential and CO₂ and O₂ tensions on nitrate assimilation gene expression and its regulation at the molecular level. This is an important advance since our current understanding of the regulation of nitrate assimilation is based largely on changes of activity of the component steps. The availability of mutants, cloned genes, and gene transfer systems will permit attempts to manipulate the nitrate assimilation pathway.     

Characterization of the pyruvate kinase-encoding gene (pki1) of Trichoderma reesei

The pyruvate kinase-encoding gene (pki1) from Trichoderma reesei was isolated by hybridization to the corresponding Aspergillus nidulans pkiA gene. The 1614-bp nucleotide (nt) sequence of the cloned gene codes for a 538-amino-acid protein. The coding sequence contains a single intron of 246 nt at a position identical to that of intron E in the A. nidulans gene. The PKI protein shows extensive homology to the PKIs of A. nidulans and A. niger (67%) and Saccharomyces cerevisiae (59%). The 5' non-coding sequence contains a number of motifs typical for yeast glycolytic genes, but so far only rarely found in filamentous fungi.     

兰科植物黄花白及Bletilla ochracea内生真菌多样性分析

黄花白及Bletilla ochracea是一种地生兰科植物,也是中国传统中药材之一。以黄花白及为实验材料,对其叶和根组织中内生真菌类群组成及多样性进行分析。结果表明,从黄花白及植株的叶片和根组织块(段)中分离到可培养内生真菌140株,根据形态特征将它们鉴定为16个分类单元,其中包括10种子囊菌和6种担子菌。从叶片组织中分离的内生真菌有6种,其中刺盘孢属Colletotrichum真菌为叶片组织中的优势种类;从根组织中分离内生真菌有10种,瘤菌根菌属Epulorhiza和腊壳菌属Sebacina真菌构成了根组织内生真菌的优势类群。根组织可培养内生真菌的多样性(H′=1.968)要高于叶片组织(H′=1.459)。     

Cloning and characterization of the gene cluster encoding type 3 (MR/K) fimbriae of Klebsiella pneumoniae

Abstract The gene cluster encoding the type 3 fimbriae of a Klebsiella pneumoniae isolate was cloned using the cosmid-cloning technique. Escherichia coli transformants, expressing type 3 fimbriae, were selected by reactivity with a monoclonal antibody directed against an epitope of the purified type 3 fimbriae. The phenotypic expression of type 3 fimbriae by transformants possessing the parental plasmid was dependent upon the host strain used. However, subcloning of this plasmid resulted in the construction of a chimeric molecule which imparted a stable phenotype regardless of the host strain. In addition, subcloning of the parental recombinant plasmid suggested that the minimal size of DNA necessary for production and expression of fimbriae was approximately 5.5 kb.     

Selection for mutants with low nitrate uptake ability in rice (Oryza sativa)

Screening for mutants deficient in the high affinity system of nitrate uptake was performed using mutagenized M₂ population of rice ( Oryza sativa , cv. Nipponbare or Kinmaze). For selecting mutants, M₂ seedlings were transferred individually to 10 ml solution containing 250 μ M potassium nitrate and 500 μ M calcium sulphate at 20 or 28°C. After 6 or 24 h, nitrate concentration of the solution was determined with a nitrate selective electrode and the seedlings showing impaired nitrate uptake were selected as nitrate uptake deficient variants. Of 74 variants, three were confirmed to be mutants with low nitrate uptake ability in the M₃ generation. Potassium uptake ability also decreased in the mutants. Three mutants were divided into two groups based on the analysis of nitrate reductase (NR, EC 1.6.6.1) activity and chlorate resistance. Two, NUE13 and NUE36 , had a lower level of NR activity than the original cultivar and were not resistant to chlorate, while the seedlings of NUE50 had the same level of NR activity as the original cultivar and were more resistant to chlorate than the original cultivar. All mutants were resistant to cesium, a toxic ion analogue for potassium, suggesting that the decreased levels of both nitrate and potassium uptake were coupled to the change of plasma membrane H⁺-ATPase activity.     

Cell-free conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine into an antibiotic with the properties of isopenicillin N in Cephalosporium acremonium.

Cell-free extracts of antibiotic-negative mutants of Cephalosporium acremonium converted delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (LLD-tripeptide) into an antibiotic that was destroyed by penicillinase. The enzymic activity of the extracts was destroyed by boiling, but was not inhibited by cycloheximide. LLL-Tripeptide was totally inactive as substrate. The product resembled isopenicillin N, but not penicillin N, in its antibacterial spectrum. We propose that isopenicillin N is the first product of cyclization of LLD-tripeptide.     

First steps towards biological control of the pear gall midge (Contarinia pyrivora) with the insect pathogenic fungus Metarhizium brunneum

Gall midges are important pests in many crops. In fruit, they are difficult to control due to their life cycle, which takes place partially within the fruit. Here, we provide the first successful laboratory experiment to infect pear gall midge (Contarinia pyrivora) with the insect pathogenic fungus Metarhizium brunneum. We developed a procedure for sampling larvae, maintaining them in the laboratory and subjecting them to the fungus. We demonstrated that dipping larvae in a fungus suspension or adding a fungus suspension to the soil result in significant fungus induced mortality of the pear gall midge. An immune response in treated larvae was recorded proving that there was a real pathogenesis. Finally, we discuss next steps and a strategy for field experiments.     

Assimilation of nitrate and ammonium by the Scots pine (Pinus sylvestris) seedling under conditions of high nitrogen supply

Seedlings of Scots pine ( Pinus sylvestris L.) were grown on perlite for 21 days under controlled conditions. Apart from the water control, KNO₃ (15 m M ), (NH₄)₂SO₄ (7.5 m M ), and NH₄NO₃ (15 m M ) were offered to study the effects of a high nitrogen supply on nitrogen assimilation. In some experiments 1.3 m M potassium was added to the basic ammonium solutions. In labelling studies nitrate and ammonium were 2.3 atom%¹⁵N-enriched. It was found that over the 21-day period approximately three times more ammonium-N was taken up than nitrate-N. However, nitrate and ammonium, applied simultaneously, were taken up to the same extent as if they were applied separately (additivity). The presence of K⁺ in the medium did not affect N-uptake. Among the soluble N-containing compounds nitrate, ammonium and 8 amino acids were quantified. It was found that assimilation of nitrate can cope with the uptake of NO⁻₃ under all circumstances. Neither free nitrate nor ammonium or amino acids accumulated to an extent exceeding the values of water-grown seedlings. On the other hand, in case of high ammonium supply considerably more nitrogen was taken up than could be incorporated into nonsoluble N-containing substance ('protein'). The remaining nitrogen was found to accumulate in intermediary storage pools (free NH₄⁺, glutamine, asparagine, arginine). Part of this accumulated N could be incorporated into protein when potassium was offered in the nutrient solution. It is concluded that potassium is a requirement for a high rate of protein synthesis not only in crop plants but also in conifers.     

Characterization of the Ferredoxin-Gogat gene (OsGog2 clone) expression in rice

Ferredoxin-dependent glutamate synthase (Fd-Gogat; EC 1.4.7.1) in leaf and root plastids is the last enzyme involved in the pathway of nitrate assimilation in higher plants. Arabidopsis thaliana expresses two different genes: the first, light regulated, specific of green tissues and the second expressed in other tissues. In this work, we investigated whether in our clone, OsGog2 AC Y12595, this gene is up-regulated by light or it is expressed under darkness. Fd-Gogat specific activity, protein and mRNA increased after light treatment in rice shoots. In roots, the activity and the protein content remained constant, whereas the mRNA is repressed by light treatment. The results obtained using a specific probe, situated in the 3′ untranslated region of the OsGog2 cDNA, indicated that OsGog2 gene is up-regulated by light and that its expression is tissue specific and suggested that a dark expressed Fd-Gogat gene could be present in rice similarly as in Arabidopsis.     

Efficient integrative transformation of the phytopathogenic fungus Alternaria alternata mediated by the repetitive rDNA sequences

An attempt was made to transform Alternaria alternata protoplasts using a plasmid vector, pDH25, bearing the Escherichia coli hygromycin B (Hy) phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter. Transformants arose on a selective medium containing 100 μg Hy/ml. There were two types of transformants, forming large and small colonies on the selective medium. Transformation with one μg of the vector produced an average of 4.5 large colonies and 600 small ones. In large-colony transformants, the vector often integrated into the recipient chromosome in the form of highly rearranged tandem arrays. To increase transformation efficiency, fragments of the highly repetitive ribosomal RNA gene cluster (rDNA) of A. alternata were used to construct four new vectors for homologous recombination system. Use of these vectors gave higher transformation efficiency than the original plasmid. The best vector, pDH25r1a, gave rise to large-colony transformants at a frequency 20 times higher than pDH25. Transformation events in A. alternata with pDH25r1a occured by homologous recombination as a single crossover between the plasmid-borne rDNA segment and its homologue in the chromosome, often giving rise to tandemly repeated vector DNA.     

Effects of norflurazon (San 9789) on light-increased extractable nitrate reductase activity in soybean [Glycine max (L.) Merr.]seedlings

Abstract. The effects of norflurazon (San 9789) on light-increased extractable NADH nitrate reductase activity (NRA) in soybean seedlings were studied. Continuous white light (W) increased NRA steadily in root and cotyledonary tissues over a 5 d period. Morflurazon, a pyridazinone herbicide which causes chlorophyll bleaching in W, reduced the initial NRA induction rate in roots and cotyledons. However, in cotyledons of norfiurazon-treated plants NRA increased at a more rapid rate than in the control after 24 h of W, with activity levels reaching three times those of control seedlings after 5 d. NRA induced by W in control and norflurazon-treated cotyledons was fluence-rate dependent. Continuous FR induced equal amounts of NRA in control and norflurazontreated tissues, suggesting that the superinduceable NRA of norflurazon-treated plants under W is not phytochrome induced. The FR-induced NRA of control and norflurazon-treated cotyledons had pH optima of 6.6, but during development under W the pH optimum of control cotyledons changed from 6.3 to between 6.6 and 7.1. The pH optimum of the norflurazon-induced NRA of the cotyledon under W was about 7.5. The NADH/NADPH NRA ratio after 4 d of W was 1.3 in control and 2.5 in norflurazontreated cotyledons. These data indicate that photosynthelic pigments are involved only secondarily in light-induction of NRA in this system.