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PubMed1981

Crystallization and partial characterization of dimethylallyl pyrophosphate: L-tryptophan dimethylallyltransferase from Claviceps sp. SD58.

Dimethylallyl pyrophosphate: L-tryptophan dimethylallyltransferase (dimethylallyl tryptophan synthetase) has been purified from Claviceps strain SD58 to a homogeneous crystalline form. The enzyme is pure as judged by polyacrylamide gel electrophoresis and contains two similar subunits of 34,000 molecular weight as shown by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Since the enzyme was determined to have a molecular weight of 70,000 by gel exclusion chromatography, it is dimeric. The substrates dimethylallyl pyrophosphate and tryptophan each have mixed (negative to positive) cooperativity with a minimum Hill coefficient of 0.37 and 0.73, respectively. Calcium ion is a positive allosteric effector and, at 20 mM concentration, deregulates the enzyme. The deregulated enzyme has Km values of 7.2 microM for dimethylallyl pyrophosphate and 8.8 microM tryptophan. The Vmax under deregulated conditions is 953 nmol/min/mg, giving a turnover number of 14. Ca2+ has a S[0.5] of 4.5 mM. The kinetic mechanism is random sequential.

Alkyl and Aryl Transferases↗
PubMed1996

A tremorgenic mycotoxicosis in cattle caused by Paspalum distichum (l.) infected by Claviceps paspali.

This is the first reported outbreak of paspalum staggers in cattle following ingestion of Paspalum distichum (L) infected by Claviceps paspali in South Africa. A day after starting to graze on an ergotised paspalum field, 43 animals exhibited various degrees of generalised tremors and ataxia. Four animals became paretic and went into sternal recumbency. All affected animals recovered. The condition was reproduced by feeding ergotised grass to a calf. Signs of intoxication were observed 48 h after ingestion of c. 30 g/kg of the semi-dry plant material. The calf developed tremors and a slight ataxia, which became more pronounced following light exercise.

Animals↗
PubMed1987

The concept of specificity in the procercoid-copepod system: Bothriocephalus claviceps (Cestoda) a parasite of the eel (Anguilla anguilla).

This paper describes experimental work on parasite specificity in the copepod-procercoid system of Bothriocephalus claviceps. Two criteria were used to characterize five potential host species Macrocyclops albidus, Macrocyclops fuscus, Eucyclops serrulatus, Acanthocyclops robustus and Macrocyclops viridis viridis. The first criterion involves susceptibility of the copepods to infection. The results show random or aggregative distributions and variable susceptibilities according to the species. We observed an ethological barrier to infection in M. viridis viridis and M. fuscus. The second criterion involves the growth and development of the procercoid. Three factors modify the profiles of the growth curves: host species, sex and intensity of infection. The growth of the procercoids is density-dependent, whereas their development is independent of density. This last characteristic is interpreted as a factor favouring the aggregation of procercoid populations. The two most susceptible hosts are M. albidus and A. robustus.

Anguilla↗
PubMed1997

Life-cycle of Bothriocephalus claviceps, a specific parasite of eels.

The life-cycle of the tapeworm Bothriocephalus claviceps (Goeze, 1782) (Cestoda: Pseudophyllidea), a specific parasite of eels (Anguilla spp.), was studied under experimental conditions. It was demonstrated that the parasite completed its development within 4 months at the temperature range of 22-24 degrees C. Embryonic development was finished in 2 days at this temperature, when coracidia spontaneously hatched. The rate of their development was controlled by water temperature, with delayed formation of coracidia at lower temperatures (8 days at 10-12 degrees C). At 2-4 and 6 degrees C, development did not take place but the eggs remained viable; at 33 degrees C the eggs died. The procercoids developed in copepods of the species Macrocyclops albidus, M. fuscus, Megacyclops viridis, Cyclops strenuus, C. vicinus and Acanthocyclops vernalis (Copepoda: Cyclopidae); their development was finished after 8-12 days at 22-24 degrees C. The definitive hosts, eels, acquired infection after ingestion of infected copepods; the prepatent period of the parasite was more than three months. Perch (Perca fluviatilis) and guppies (Poecilia reticulata) were shown to serve as paratenic hosts of the parasite; in guppies, the tapeworms survived up to 14 days after exposure and they developed similarly to tapeworms in the definitive host.

Anguilla↗
PubMed1972

A -D-fructofuranosidase from Claviceps purpurea.

Evidence suggests that sucrose is the main carbon source for growth of Claviceps spp. in the parasitic condition. The sucrose acts as substrate for an active beta-fructofuranosidase, produced by the fungus, which in the first instance converts the disaccharide into glucose and an oligofructoside. In this way, 50% of the glucose, supplied as sucrose, is made available to the parasite for assimilation. Subsequent action of the enzyme on both sucrose and the oligofructoside leads to the release of more glucose and the formation of additional oligosaccharides. The structures of the main oligosaccharides formed have been elucidated and the interactions of each compound studied. In experiments with purified enzyme in vitro the interaction of the oligosaccharides is rapid but in culture they are assimilated only slowly; in each case some free fructose is liberated. Free fructose is not assimilated in the presence of glucose and, further, inhibits growth at concentrations which might be expected to occur in the parasitic condition. A dual role has been suggested for the enzyme, with sucrose as substrate, in which glucose is made available to the growing parasite, while at the same time transfer of the fructose to form oligosaccharides prevents it from accumulating at inhibitory concentrations. Ultimately, when glucose becomes limiting, the fungus will adapt to fructose assimilation.

Adaptation, Physiological↗
PubMed1973

Biosynthesis of ergotamine by Claviceps purpurea (Fr.) Tul.

High-yielding strains of Claviceps purpurea (Fr.) Tul, grown on a defined medium, have been used for a study of the biosynthesis of the peptide ergot alkaloid, ergotamine. l-[U-(14)C]tryptophan, dl-[2-(14)C]mevalonic acid lactone, sodium [2-(14)C]acetate, sodium [(14)C]formate and the methyl group of l-[methyl-(14)C]methionine were efficiently incorporated into the peptide alkaloids and specifically labelled the ergoline moiety of ergotamine. These results are the same as previously found for the biosynthesis of other ergot alkaloids. Time-course incubation experiments demonstrated that l-[U-(14)C]phenylalanine, l-[U-(14)C]proline and l-[U-(14)C]alanine were incorporated into the peptide ergot alkaloids. Chemical degradation of the radioactive alkaloid derived from additional precursor incubation experiments showed that phenylalanine and proline function as the most efficient precursors, and specifically label the constitutive side-chain phenylalanyl and prolyl moieties of the alkaloid. The evidence obtained from l-[U-(14)C]alanine-incorporation experiments was inconclusive. However, degradation of ergotamine isolated after incubation with dl-[1-(14)C]alanine, showed that the carboxyl group of the labelled amino acid was specifically incorporated into the alpha-hydroxy-alpha-amino acid residue of the alkaloid. This, in conjunction with the l-[U-(14)C]alanine-incorporation results, showed conclusively that all three carbon atoms of alanine were incorporated as a biosynthetic unit into the alpha-hydroxy-alpha-amino acid moiety of ergotamine.

Acetates↗
PubMed1986

[Experimental approach to the role of the paratenic host in the circulation of the parasite Bothriocephalus claviceps Goeze, 1782 (Cestoda, Pseudophyllidea)].

This article concerns an experimental work monitored in 5 species of fishes in the order to evaluate their value of paratenic hosts, in the cycle of Bothriocephalus claviceps, a parasite of the eel, Anguilla anguilla. The intestinal localisation and the lack of development of parasites are the two principal characters of this parasitism. The second character is sometimes contradicted. The effective evolution has been observed during 20 days. The more important losses of parasites coincide with the invasion of fishes. After that stage, the decrease of effectives is slower. The authors are led to consider that two modes of circulation are required for the parasite: the direct transmission and the transmission with intercalation of paratenic hosts.

Anguilla↗
PubMed1983

Highly Efficient Mutagenesis of Claviceps purpurea by Using Protoplasts.

Claviceps purpurea ATCC 20102, which is aconidial under laboratory conditions, was grown in submerged culture in the presence of mutagens and various nutritional additives. Protoplasts from such cultures were prepared and regenerated on solid medium to obtain colonies from single cell units. Frequencies of auxotrophs and high alkaloid producers were on the order of 1 to 2%. Some of the auxotrophic mutants derived from strain ATCC 20102 were constantly segregating prototrophs. High-alkaloid-producing derivatives showed sclerotia-like morphology and violet-brown pigmentation, in contrast to the parent strain; some of them also showed segregation sectors when grown as giant colonies. Mutagenesis of strain 1029, isolated during this study and having an increased level of alkaloid synthesis and sclerotia-like cell morphology, was done in the same fashion as with the original parent strain, ATCC 20102. Mutants obtained from this strain were all stable with respect to their genotypes. However, a large proportion of colonies derived from regenerated protoplasts, even in the mutagen-free controls, showed a lowered level of alkaloid production and were morphologically more similar to the original wild type, ATCC 20102. The influence of protoplast preparation or regeneration or both on the stability of genes involved in differentiation is discussed.

Journal Article↗
PubMed1984

Isolation and Characterization of Vacuoles from the Ergot Fungus Claviceps purpurea.

Protoplasts of Claviceps purpurea were prepared by treatment of mycelium with a lytic mixture of snail gut enzyme and cellulase from Trichoderma viride. Such protoplasts could be efficiently lysed by Triton X-100 treatment at high osmotic pressure without Ca or Mg, allowing the release of intact vacuoles in high yields. Vacuoles obtained from cells grown in modified Vogel medium (vegetative-type cells not producing alkaloids) were isolated and purified by centrifugation from a 5% Ficoll 400 (wt/vol) phase into the interphase between two layers, one containing 0.25 M each of mannitol and sucrose, and one containing 0.5 M mannitol. Vacuoles derived from cells grown in a medium favoring ergot alkaloid synthesis (sclerotia-like cells) were isolated by gentle centrifugation of filtered protoplast lysates without addition of Ficoll 400. Biochemical analyses of the vacuole fraction isolated from either kind of cell revealed their function as compartments harboring several hydrolytic enzymes. However, the enrichment of free amino acids in vacuoles of sclerotia-like cells was less pronounced than that in vacuoles of vegetative-type cells, indicating a difference in metabolic compartmentation in the two types of cells.

Journal Article↗
PubMed1993

Long-Term Production of Ergot Peptides by Immobilized Claviceps purpurea in Semicontinuous and Continuous Culture.

The semicontinuous and continuous production of pharmaceutically useful ergot peptides with immobilized Claviceps purpurea could be demonstrated. A key aspect was the presence of high concentrations of CaCl(2) (96.9 mM) to give marked prolongation of the productive phase, and cultivation in a bubble column reactor became possible. Restriction of the phosphate supply avoided an otherwise problematic massive increase of outgrowing hyphae.

Journal Article↗
PubMed1967

Heterokaryosis and alkaloid production in Claviceps purpurea.

Strain 275 FI of Claviceps purpurea, which produces large amounts of peptide alkaloids in submerged culture, is a heterokaryon; after several generations on agar media, it segregates the single components. These components (by us labeled V, C, and W) are stable and produce almost no alkaloids under described conditions of submerged culture. The mycelium of strain 275 FI consists of hyphae with multinucleate cells and does not produce conidia. Strains V, C, and W form numerous anastomoses when grown together on agar. By combining strains V and C, a heterokaryon similar to 275 FI in appearance has been obtained. This new strain produces amounts of alkaloids much larger than those produced by V and C separately or in associated submerged culture. We conclude, therefore, that in strain 275 FI the heterokaryotic condition is favorable to the production of alkaloids. Several conidiaproducing cultures of C. purpurea of various origins, as well as sclerotia of the same species, have been examined. The results demonstrated that the heterokaryotic condition is rare in the cultures, but it is frequent in the mycelium from sclerotia. Since it is known that the production of alkaloids is typical of the sclerotial phase in C. purpurea, it is suggested that this capacity is related to the heterokaryosis of the producing strains.

Alkaloids↗
PubMed1967

Production of Lysergic Acid Derivatives in Submerged Culture: IV. Inorganic Nutrition Studies with Claviceps paspali.

The inorganic requirements for growth and alkaloid production by submerged cultures of a lysergic acid alkaloid-producing isolate of Claviceps paspali were determined in a defined medium. The requirement for peak growth was essentially the same as the requirement for maximal alkaloid production in the case of potassium and magnesium. With phosphorus, however, maximal growth was reached at lower levels than those required for maximal alkaloid production. Sulfur was required in smaller amounts for peak alkaloid production than for maximal growth. In the case of iron and zinc, the requirement for maximal alkaloid production was greater than for growth. Manganese and copper could be sufficiently depleted to demonstrate their essentiality for alkaloid production, but their requirements for growth could not be demonstrated.

Journal Article↗
PubMed1969

Production of peptide ergot alkaloids in submerged culture by three isolates of Claviceps purpurea.

Three strains of Claviceps purpurea (Fr.) Tul., isolated from sclerotia grown on rye, produce under submerged conditions ergocryptine and ergotamine, ergocornine and ergosine, and ergocristine, respectively. All of the strains either lacked the ability to produce conidia or formed them sparingly, but they accumulated large quantities of lipids and sterols. The fermentations are typically divided into two phases. The first is characterized by the rapid utilization and exhaustion of the phosphate contained in the medium, rapid uptake of ammonium nitrogen and of citric acid, rapid growth, and low alkaloid production; the second phase is characterized by slower growth and by a marked accumulation of lipids, sterols, and alkaloids.

Ascomycota↗
PubMed1971

Correlation of certain alterations in metabolic activity with alkaloid production by submerged Claviceps.

Alkaloid biosynthesis in Claviceps paspali MG-6 was favored by unbalanced growth. A positive correlation between the rate of protein turnover and alkaloid formation was noted. The pattern of the orthophosphate content in the mycelium resembled that in the ripening sclerotia of the parasitic strains. Alkaloids were revealed as potentially effective in energy metabolism. Reduced adenosine triphosphate (ATP) utilization and an increase in the ATP pool were found to be favorable for alkaloid production. Acetyl-coenzyme A carboxylase activity and the level of cell lipids were directly related to the intensity of alkaloid biosynthesis. An inverse relationship was observed between the activities of the tricarboxylic acid and glycoxylate cycles and the rate of alkaloid formation.

Adenine Nucleotides↗
PubMed1969

Role of glycols and tweens in the production of ergot alkaloids by Claviceps paspali.

Several glycols and Tweens markedly stimulated the production of ergot alkaloids in submerged cultures of Claviceps paspali. The role of these compounds was investigated in shake flasks and bench-scale fermentors. 2,3-Butanediol was not utilized by the fungus, and 1,2-propanediol-1-(14)C was not incorporated into the alkaloids. Glycols and Tweens lowered the surface tension of the basal medium and promoted the utilization of metabolites. In the presence of glycols and Tweens, an increased uptake of labeled sorbitol and succinic acid took place, whereas the specific radioactivity of the alkaloids was not affected. These results indicated that glycols and Tweens are not involved directly in the biosynthetic process; they apparently acted as surface-active agents, facilitating transport of metabolites into the cells.

Ascomycota↗