Heterologous transformation ofClaviceps purpurea

Summary A transformation system based on dominant selection markers was established for an industrialClaviceps purpurea strain. Transformants could be obtained by using plasmid pAN 7-1 carrying a bacterial gene for hygromycin (hph) resistance fused to a fungal promoter or by plasmid p3SR2 which carries the acetamidase gene (amdS) fromAspergillus nidulans.     

DNA mediated transformation of the filamentous fungus Curvularia lunata using a dominant selectable marker

Summary A DNA mediated transformation system was developed for the filamentous fungus Curvularia lunata. Incubation of protoplasts with a plasmid (pAN7-1) which contains the Escherichia coli hygromycin B phosphotransferase gene (hph) fused to an Aspergillus nidulans promoter resulted in the formation of hygromycin B resistant transformants. DNA-DNA hybridization experiments revealed that plasmid pAN7-1 became integrated into the fungal genome. All transformants analyzed so far proved to be stable even after vegetative growth for three months without selective pressure.Dedicated to Professor Dr. Dr. h. c. Esser on the occasion of his 65th birthday     

Glucose catabolism during alkaloid production in a strain ofClaviceps purpurea

Badiorespirometric experiments with glucose labelled in positions 1; 2; 3,4 and 6 were carried out inClaviceps purpurea strain pepty 695. The pattern of¹⁴CO₂ evolution from 5 – 50 mM glucose indicated the operation of the pentose phosphate cycle during alkaloid production. The authors thank Mrs. H. Pechfelder for her skilled technical assistance and Prof. Dr. H. Reinbothe and Dr. C. Wasternack for helpful discussions.     

Emergence and phytopathological properties of a new strain of Claviceps purpurea (Fr.) Tul. on rye

The emergence of a new stable strain of Claviceps purpurea is described. The strain formed the sphacelial stage very weakly on rye, and was unlikely therefore to be able to initiate an epidemic under field conditions, but the sclerotia contained chanoclavine as the major alkaloid in contrast to the ergotamine component of the parent strain. Isolates of C. purpurea derived from ryegrass ergots were also shown to parasitize rye.     

Gene cloning and transformation in the basidiomycete fungus Coprinus cinereus: Isolation and expression of the isocitrate lyase gene (acu-7)

Summary The cloned isocitrate lyase structural gene of Aspergillus nidulans (acuD) was shown to hybridize under reduced stringency conditions to unique sequences in genomic DNA digests of the basidiomycete fungus Coprinus cinereus. A gene library of C. cinereus was constructed in the lambda replacement vector L47 and screened for sequences hybridizing to the A. nidulans gene. A recombinant phage was isolated which contained the hybridizing sequence on a 5.6-kb BamHI fragment. This fragment was subcloned into pUC13 to give plasmid pHIONA1 and shown to contain a functional C. cinereus isocitrate lyase gene (acu-7) by transformation of an acu-7 mutant. Direct selection for Acu⁺ transformants was not possible because of the toxicity of the acetate selection medium. Acu⁺ transformants were obtained as cotransformants by transforming an acu-7 trp-1 double mutant, having mutations in both the isocitrate lyase and tryptophan synthetase structural genes, with a plasmid containing the trp-1 gene and either pHIONA1 or the original lambda clone. Up to 47.5% of the selected Trp⁺ transformants were cotransformed to Acu⁺. A physical analysis of 40 Acu⁺ transformants showed that the acu-7 gene had integrated at non-homologous and often multiple sites in the genome. Meiotic stability of the integrated gene was demonstrated by genetic crosses.     

The Alkaloids of Fungi

Examination of different species of local fungi, grown on two nutritive solutions of different composition, for alkaloid formation was investigated. The formation of alkaloids was confined to four species, namely: Geotrichum candidum, Mucor hiemalis, Aspergillus flavus and Rhizopus nigricans. A comparative study of the growth as well as the formation of alkaloids by these species and by Claviceps purpurea NRRL was carried out. Methods were also described with which the different alkaloids produced by the experimental strains were identified. Peptides as well as clavine type alkaloids were detected in all cases except with Mucor hiemalis where a compound corresponding to ergosine was the only alkaloid present.     

Heterologous expression and biochemical characterization of novel pyranose 2-oxidases from the ascomycetes <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> and <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

A gene encoding a pyranose 2-oxidase (POx; pyranose/oxygen 2-oxidoreductase; glucose 2-oxidase; EC 1.1.3.10) was identified in the genome of the ascomycete Aspergillus nidulans. Attempts to isolate POx directly from A. nidulans cultures or to homologously overexpress the native POx (under control of the constitutive gpdA promoter) in A. nidulans were unsuccessful. cDNA encoding POx was synthesized from mRNA and expressed in Escherichia coli, and the enzyme was subsequently purified and characterized. A putative pyranose 2-oxidase-encoding gene was also identified in the genome of Aspergillus oryzae. The coding sequence was synthetically produced and was also expressed in E. coli. Both purified enzymes were shown to be flavoproteins consisting of subunits of 65 kDa. The A. nidulans enzyme was biochemically similar to POx reported in literature. From all substrates, the highest catalytic efficiency was found with D-glucose. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones and 2,6-dichloroindophenol. As judged by the catalytic efficiencies (k _cat/k _m), some of these quinone electron acceptors are better substrates for pyranose oxidase than oxygen. The enzyme from A. oryzae was physically similar but showed lower kinetic constants compared to the enzyme from A. nidulans. Distinct differences in the stability of the two enzymes may be attributed to a deletion and an insertion in the sequence, respectively.     

Initiation of the dikaryon in Claviceps microcephala (Wallr.) Tul.

Summary An account of the nuclear behaviour in the initiation of the dikaryon inClaviceps microcephala (Wallr.)Tul. inciting the ergot of bajri in India is given. The pattern follows closely the mode of dikaryotization reported byKillian (1919) forClaviceps purpurea (Fr.)Tul., characterised by functional antheridium, absence of trichogyne, the Claussentype of nuclear fusion and hook-formation.     

Über den Einfluss verschiedener Nährmedien auf den Polymorphismus und die Sporulation von Claviceps purpurea-Stämmen

Summary In this paper we examined the influence of various natural and synthetic mediums upon several morphological and physical characters ofClaviceps purpurea, upon its sporulation and alcaloids formation. It was shown that the composition and choice of the medium has a very essential influence on the morphological and physical characters, on the sporulation and alcaloids formation. The results obtained may be useful to gather a great quantity ofClaviceps purpurea spores for an artificial infection of corn-ears.     

Effect of culture temperature on the heterologous expression of <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> versatile peroxidase in <Emphasis Type="Italic">Aspergillus</Emphasis> hosts

Production of recombinant versatile peroxidase in Aspergillus hosts was optimized through the modification of temperature during bioreactor cultivations. To further this purpose, the cDNA encoding a versatile peroxidase of Pleurotus eryngii was expressed under control of the alcohol dehydrogenase (alcA) promoter of Aspergillus nidulans. A dependence of recombinant peroxidase production on cultivation temperature was found. Lowering the culture temperature from 28 to 19 °C enhanced the level of active peroxidase 5.8-fold and reduced the effective proteolytic activity twofold. Thus, a maximum peroxidase activity of 466 U L^-1 was reached. The same optimization scheme was applied to a recombinant Aspergillus niger that bore the alcohol dehydrogenase regulator (alcR), enabling transformation with the peroxidase cDNA under the same alcA promoter. However, with this strain, the peroxidase activity was not improved, while the effective proteolytic activity was increased between 3- and 11-fold compared to that obtained with A. nidulans.     

Cotransformation of Aspergillus nidulans: a tool for replacing fungal genes

Summary When a non-selected DNA sequence was added during the transformation of amdS320 deletion strains of Aspergillus nidulans with a vector containing the wild-type amdS gene the AmdS⁺ transformants were cotransformed at a high frequency. Cotransformation of an amdS320, trpC801 double mutant strain showed that both the molar ratio of the two vectors and the concentration of the cotransforming vector affected the cotransformation frequency. The maximum frequency obtained was defined by the gene chosen as selection marker for transformation. Cotransformation was used to induce a gene replacement in A. nidulans. An amdS320 strain was transformed to AmdS⁺ and cotransformed with a DNA fragment containing a fusion between a non-functional A. nidulans trpC gene and the Escherichia coli lacZ gene. Ten AmdS⁺, LacZ⁺ transformants with a Trp^– mutant phenotype were selected. All of these strains could be transformed with a functional copy of the A. nidulans trpC gene, but only two strains yielded TrpC⁺ transformants which, with a low frequency, had a LacZ^– phenotype. These latter transformants had also lost the AmdS⁺ phenotype. Southern blotting analysis of DNA from these transformants confirmed the inactivation of the wild-type trpC gene, but revealed that amdS vector sequences were also involved in the gene replacement events.     

Depletion of Aspergillus nidulans cotA causes a severe polarity defect which is not suppressed by the nuclear migration mutation nudA2

The Aspergillus nidulans homologue of Neurospora crassa cot-1, cotA, encoding a member of the NDR protein kinase family, has been cloned and expressed under the control of the conditional alcA promoter. Depletion of CotA by repression of the alcA promoter led to a severe growth defect accompanied by loss of polarity. Germlings show greatly enlarged volume of the spores and hyphae, accompanied by an increase in number of nuclei per compartment, though the nucleus/volume ratio is not significantly altered. The depleted CotA phenotype was not suppressed by a nuclear migration mutation nudA2. Double mutants showed an additive, defective phenotype, unlike the suppression of the cot-1 ts mutation by ropy mutations seen in N. crassa, suggesting a different relationship between nuclear migration and the cot signalling pathway in A. nidulans. A functional CotA–GFP fusion protein was found in punctate regions of fluorescence similar to the distribution reported for human NDR2, and as a cap at the hyphal tip.     

Gel mobility shift scanning of the acetate-inducible promoters fromNeurospora crassa reveals a common co-inducible DNA-binding protein

The promoter regions of four acetate-inducible genes ofNeurospora crassa, acu-3, acu-5, acu-8 andacu-9, have been sequenced. Using a scanning gel mobility shift assay particular DNA regions in each promoter have been shown specifically to bind partially purified protein extracted from acetate-induced mycelia. The protein-binding regions so defined have common sequence motifs, elements of which are similar to those required for acetate induction inAspergillus nidulans.     

Regulation of the xylanase gene, cgxA, from Chaetomium gracile by transcriptional factors, XlnR and AnRP

The roles of XlnR and AnRP in regulating the expression of the xylanase gene, cgxA, from Chaetomium gracile were investigated using Aspergillus nidulansas an intermediate host. The XlnR consensus binding sequence –GGCTAA– in the promoter region was functional in vivo. The cgxA gene was induced when xylan was used as a carbon source but this inducibility was abolished when the XlnR binding sequence was mutated. Furthermore, the induction by xylan was increased when the AnRP binding sequence –TTGACAAAT– was mutated. Electrophoretic mobility shift assays using partially purified AnRP and an Aspergillus oryzae XlnR fusion protein, MalE-AoXlnR, provided evidence that the binding of the two proteins to their respective sites in the cgxA promoter region was mutually exclusive.     

Cell-free conversion of 4-γ,γ-dimethylallyltryptophan to 4-[4-hydroxy-3-methyl-Δ2-butenyl]-tryptophan in Claviceps purpurea PRL 1980

The conversion of 4-γ,γ-dimethylallyltryptophan to 4-[4-hydroxy-3-methyl-Δ²-butenyl]-tryptophan was catalyzed by the 60–80% ammonium sulphate fraction from Claviceps purpurea PRL 1980. The conversion was stimulated by NADPH. Two major unidentified products in the incubation mixture were not significantly incorporated into elymoclavine when they were added to cultures of C. purpurea PRL 1980.     

Biochemical characterization of the inoculum of Claviceps purpurea for submerged production of ergot alkaloids

Summary Biochemical and morphological changes during the growth and development of Claviceps purpurea seed cultures under submerged conditions were established. The fall in RNA content at about the middle of the exponential phase of growth has been found to be associated with the transition from sphacelial to sclerotial growth and may be considered the major indicator of changes in the metabolism of the fungus. It may also serve as a biochemical indicator for the optimal activity of Claviceps purpurea seed cultures.     

Comparison of Ergot Alkaloid Biosynthesis Gene Clusters in Claviceps Species Indicates Loss of Late Pathway Steps in Evolution of C. fusiformis

The grass parasites Claviceps purpurea and Claviceps fusiformis produce ergot alkaloids (EA) in planta and in submerged culture. Whereas EA synthesis (EAS) in C. purpurea proceeds via clavine intermediates to lysergic acid and the complex ergopeptines, C. fusiformis produces only agroclavine and elymoclavine. In C. purpurea the EAS gene (EAS) cluster includes dmaW (encoding the first pathway step), cloA (elymoclavine oxidation to lysergic acid), and the lpsA/lpsB genes (ergopeptine formation). We analyzed the corresponding C. fusiformis EAS cluster to investigate the evolutionary basis for chemotypic differences between the Claviceps species. Other than three peptide synthetase genes (lpsC and the tandem paralogues lpsA1 and lpsA2), homologues of all C. purpurea EAS genes were identified in C. fusiformis, including homologues of lpsB and cloA, which in C. purpurea encode enzymes for steps after clavine synthesis. Rearrangement of the cluster was evident around lpsB, which is truncated in C. fusiformis. This and several frameshift mutations render CflpsB a pseudogene (CflpsB^Ψ). No obvious inactivating mutation was identified in CfcloA. All C. fusiformis EAS genes, including CflpsB^Ψ and CfcloA, were expressed in culture. Cross-complementation analyses demonstrated that CfcloA and CflpsB^Ψ were expressed in C. purpurea but did not encode functional enzymes. In contrast, CpcloA catalyzed lysergic acid biosynthesis in C. fusiformis, indicating that C. fusiformis terminates its EAS pathway at elymoclavine because the cloA gene product is inactive. We propose that the C. fusiformis EAS cluster evolved from a more complete cluster by loss of some lps genes and by rearrangements and mutations inactivating lpsB and cloA.     

Origin of sclerotia-like cells in submerged Claviceps purpurea producing clavine alkaloids

Ultrathin sectioning of submerged mycelium of Claviceps purpurea Tul. producing clavine alkaloids revealed yeast-like budding resulting in asexual sporesblastospores. These deciduous spores were born by extended hyphal cells and retained the same ultrastructure of cell organelles. Both the extended hyphae and the blastospores resembled the cells of ergot sclerotial tissue. A surface culture of C. purpurea Tul. producing no alkaloids was used as a reference.     

PepJ is a new extracellular proteinase of <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>

Under carbon starvation, Aspergillus nidulans released a metallo-proteinase with activities comparable to those of PrtA, the major extracellular serine proteinase of the fungus. The relative molar mass of the enzyme was 19 kDa as determined with both denaturing and renaturing SDS PAGE, while its isoelectric point and pH and temperature optima were 8.6, 5.5 and 65 °C, respectively. The enzyme was stable at pH 3.5–10.5 and was still active at 95 °C in the presence of azocasein substrate. MALDI-TOF MS analysis demonstrated that the proteinase was encoded by the pepJ gene (locus ID AN7962.3), and showed high similarity to deuterolysin from Aspergillus oryzae. The size of the mature enzyme, its EDTA sensitivity and heat stability also supported the view that A. nidulans PepJ is a deuterolysin-type metallo-proteinase.