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At least 37 records · Page 2Linked to original sources
PubMed2001

Application of oxygen vectors to Claviceps purpurea cultivation.

The application of a two-phase fermentation system for the production of ergot peptide alkaloids by Claviceps purpurea is described. Perfluorocarbons (PFC) are used as oxygen vectors in Claviceps fermentation for the first time. In shake-flask cultivations, the inclusion of PFC in the medium brings about a five-fold increase in the total alkaloid production and a six-fold increase in the pharmaceutically important component, ergotamine. This rise cannot be correlated with the concentration of the added PFC and it is thought that the enhancement is due to a combination of factors, including the influence of PFC. Other oxygen vectors, such as several hydrocarbons, prove to be poor oxygen carriers in our study. Cultivations with PFC in a bioreactor are reproducible, the maximum total alkaloid and ergotamine production being attained on the 11th and 9th days, respectively. The relatively lower increase in the total alkaloid production in the bioreactor as compared to the shake-flasks is attributed to the unequal oxygen availability in the reactor. Processes with PFC offer the operational advantage of a five-fold reduction in aeration rate.

Alkaloids↗
PubMed1999

Evidence for an ergot alkaloid gene cluster in Claviceps purpurea.

A gene (cpd1) coding for the dimethylallyltryptophan synthase (DMATS) that catalyzes the first specific step in the biosynthesis of ergot alkaloids, was cloned from a strain of Claviceps purpurea that produces alkaloids in axenic culture. The derived gene product (CPD1) shows only 70% similarity to the corresponding gene previously isolated from Claviceps strain ATCC 26245, which is likely to be an isolate of C. fusiformis. Therefore, the related cpd1 most probably represents the first C. purpurea gene coding for an enzymatic step of the alkaloid biosynthetic pathway to be cloned. Analysis of the 3'-flanking region of cpd1 revealed a second, closely linked ergot alkaloid biosynthetic gene named cpps1, which codes for a 356-kDa polypeptide showing significant similarity to fungal modular peptide synthetases. The protein contains three amino acid-activating modules, and in the second module a sequence is found which matches that of an internal peptide (17 amino acids in length) obtained from a tryptic digest of lysergyl peptide synthetase 1 (LPS1) of C. purpurea, thus confirming that cpps1 encodes LPS1. LPS1 activates the three amino acids of the peptide portion of ergot peptide alkaloids during D-lysergyl peptide assembly. Chromosome walking revealed the presence of additional genes upstream of cpd1 which are probably also involved in ergot alkaloid biosynthesis: cpox1 probably codes for an FAD-dependent oxidoreductase (which could represent the chanoclavine cyclase), and a second putative oxidoreductase gene, cpox2, is closely linked to it in inverse orientation. RT-PCR experiments confirm that all four genes are expressed under conditions of peptide alkaloid biosynthesis. These results strongly suggest that at least some genes of ergot alkaloid biosynthesis in C. purpurea are clustered, opening the way for a detailed molecular genetic analysis of the pathway.

Alkyl and Aryl Transferases↗
PubMed2005

The ergot alkaloid gene cluster in Claviceps purpurea: extension of the cluster sequence and intra species evolution.

The genomic region of Claviceps purpurea strain P1 containing the ergot alkaloid gene cluster [Tudzynski, P., Hölter, K., Correia, T., Arntz, C., Grammel, N., Keller, U., 1999. Evidence for an ergot alkaloid gene cluster in Claviceps purpurea. Mol. Gen. Genet. 261, 133-141] was explored by chromosome walking, and additional genes probably involved in the ergot alkaloid biosynthesis have been identified. The putative cluster sequence (extending over 68.5kb) contains 4 different nonribosomal peptide synthetase (NRPS) genes and several putative oxidases. Northern analysis showed that most of the genes were co-regulated (repressed by high phosphate), and identified probable flanking genes by lack of co-regulation. Comparison of the cluster sequences of strain P1, an ergotamine producer, with that of strain ECC93, an ergocristine producer, showed high conservation of most of the cluster genes, but significant variation in the NRPS modules, strongly suggesting that evolution of these chemical races of C. purpurea is determined by evolution of NRPS module specificity.

Amino Acid Sequence↗
PubMed1998

Nonradioactive in situ hybridization for detection of hydrophobin mRNA in the phytopathogenic fungus Claviceps purpurea during infection of rye.

Hydrophobins are unique fungal extracellular proteins that produce amphipathic films at interfaces, mediate contact to hydrophobic surfaces and are known to be important in phytopathogenicity. In the pathogenic ascomycete Claviceps purpurea, causing ergot disease in grasses and cereals and ergotism in livestock, a gene encoding an extraordinary type of hydrophobin has been detected, which appeared to be induced during alkaloid synthesis in axenic culture of an ergot-alkaloid producing strain of Claviceps (V. Garre and P. Tudzynski, pers. communication; Arntz and Tudzynski, 1997, Curr. Genet. 31, 357-360). To elucidate presence and function of this hydrophobin during infection of rye, the nonradioactive in situ hybridization technique was successfully adapted to the fungal organism and optimized in the pathogenic interaction system. Semithin cryosections proved to be suitable for microscopical gene expression analysis using immune-mediated alkaline-phosphatase staining for detection of digoxigenin-labeled cRNA probes. Specific hybridization of the prepared antisense riboprobe to hydrophobin mRNA was confirmed in nonradioactive Northern blots. While permeabilization by proteinase K had only a minor effect, the inclusion of detergent into the hybridization solutions enhanced specific RNA-RNA hybridization under maximum stringency. Hydrophobin mRNA was found in fungal cells, growing in axenic culture. In the disease cycle, hydrophobin transcripts were localized in abundance during vegetative fructification in conidiophores that actively produced conidia. No signals were observed in sclerotial hyphae during formation of the alkaloid-containing ergots, although they fluoresced intensely during total RNA detection using acridine orange. Notably, in situ hybridization experiments resulted in specific signals during early infection and colonization phases in the external mycelia and in hyphae penetrating the host epidermal layer. The presumed role of the hydrophobin gene product in ergot pathogenicity is discussed with respect to the described spatio-temporal distribution of the hydrophobin transcripts.

Claviceps↗
PubMed1997

Mechanism of alkaloid cyclopeptide synthesis in the ergot fungus Claviceps purpurea.

BACKGROUND: Previous analyses of the biosynthesis of the alkaloid cyclopeptides from the ergot fungus Claviceps purpurea were hampered by a lack of suitable systems for study in vitro, and this led to conflicting results concerning the mechanism of alkaloid cyclopeptide formation. Recently, D-lysergyl peptide synthetase (LPS) of the ergot fungus Claviceps purpurea, which assembles the non-cyclol precursors of the ergopeptines, has been partially purified and shown to consist of two polypeptide chains of 370 kDa (LPS 1) and 140 kDa (LPS 2); these contain all the sites necessary for the assembly of the D-lysergyl peptide backbone. The mechanism of D-lysergyl peptide synthesis remained unclear, however. RESULTS: We have identified the obligatory peptidic intermediates in D-lysergyl peptide synthesis and the sequential order of their formation. The two LPS subunits catalyze the formation of D-lysergyl mono-, di-, and tripeptides as enzyme-thioester intermediates, the formation of which appears to be irreversible. Peptide synthesis starts when D-lysergic acid binds to the LPS 2 subunit, which most probably occurs after the previous round of synthesis has been completed by the release of the end product from the LPS enzyme. CONCLUSIONS: We have shown that the mechanism of D-lysergyl peptide synthesis is an ordered process of successive acyl transfers on a multienzyme complex. This knowledge opens the way for enzymatic and genetic investigations into the formation of novel alkaloid cyclopeptides.

Alkaloids↗
PubMed2002

Rediscovery of Claviceps sorghi (Ascomycotina: Clavicipitaceae) in India.

During last twenty years, endemic Claviceps sorghi was largely replaced in India by an introduced species Claviceps africana. Recently, C. sorghi was found and isolated from Sorghum bicolor (Gulbarga, Karnataka). Macroconidia were smaller than in the type description, 7.6-14.5 (avg. 11.4) x 3.8-6.1 (avg. 4.8) microm; microconidia were rounded to oval, 2.5-3 microm in diameter. When plated, only the macroconidia underwent secondary conidiation. Sporulation was maintained only in cultures grown on sucrose-asparagine medium T2. Mycelium was reduced to sparse hyphae with numerous short conidiophores soon obscured by yeastlike conidial mass. RAPD patterns of isolates GUL, MH74 (Maharashtra), NAP7 and NAP5 (Andhra Pradesh) with 6 primers were almost identical and distinct from those of C. africana. Sequence of rDNA containing ITS1-5.8S-ITS2 region for isolate GUL (AJ306621) corresponded to that of the authentic material of C. sorghi (AJ242869) confirming thus the presence of C. sorghi in Central India.

Claviceps↗
PubMed2002

Agalactia in mares fed with grain contaminated with Claviceps purpurea.

This article reports an outbreak of intoxication of female horses with Claviceps purpurea in southern Brazil. The outbreak affected twelve pregnant mares which were fed with black oat (Avena strigosa) during the pre-delivery period. Underdevelopment of the mammary gland in the pre-delivery period resulting in post-delivery agalactia was the most pronounced finding. These mares delivered weak and unviable foals, which showed no suckling reflex and died within a few hours of birth. Laboratory analysis of oat samples fed to the animals resulted in the identification of Claviceps purpurea sclerotia. The fungus was identified in 0.22% of the examined seeds.

Animals↗
PubMed2000

The use of biomagnetic separation to recover DNA suitable for PCR from Claviceps species.

DNA analysis of agriculturally important fungi using polymerase chain reaction (PCR)-based methods is becoming routine in research and for diagnostic purposes. Rapid, small-scale DNA isolation methods that take advantage of the sensitivity, speed and automation potential of PCR technology are needed for timely analysis of important plant pathogens. DNA isolated from Claviceps africana (causal agent of ergot of sorghum) using several standard DNA extraction protocols was found to be unamplifiable using PCR. The standard methods apparently failed to separate DNA from substances inhibitory to the Taq polymerase enzyme. We obtained DNA amenable to PCR analysis using a novel method involving magnetic beads and high salt extraction buffer. The biomagnetic purification method allowed us to obtain reliable PCR amplification of the internal transcribed spacer (ITS) regions of rDNA of Claviceps africana, making genetic comparisons possible.

Claviceps↗
PubMed1961

Glutamic decarboxyiase of ergot, Claviceps purpurea.

Anderson, John A. (Oregon State University, Corvallis), Vernon H. Cheldelin, and Tsoo E. King. Glutamic decarboxylase of ergot, Claviceps purpurea. J. Bacteriol. 82:354-358. 1961.-l-Glutamic acid is the only naturally occurring amino acid which can be decarboxylated by cell-free extracts of Claviceps purpurea. This decarboxylase was partially purified and the properties of the enzyme studied. The specific activity of the purified preparation was 111 muliters per 10 min per mg of protein. The products formed, stability, inhibition, stimulation of activity with pyridoxal phosphate, and pH activity curve were typical of l-glutamic decarboxylase in Escherichia coli and other microorganisms. The substrate constants at pH 4.6, 5.25, and 5.65 were 0.0169 m, 0.0174 m, and 0.0139 m, respectively. The respective maximal velocities at these pH values were 104, 104, and 90 muliters per 10 min. The pH optimum was 4.8 to 5.2. The enzyme was unstable below pH 4.5 and it was suggested that the fall in activity at the lower end of the pH curve was due to inactivation of the enzyme. The decrease in activity above pH 5.2 did not appear to be due to a change in affinity of enzyme for substrate but to a change of the enzyme-substrate complex into an inactive form.

Amino Acids↗
PubMed2004

Pleomorphic conidiation in Claviceps.

Types of asexual sporulation in 17 Claviceps species and the closely related Corallocytostroma ornicopreoides were revised in relation to the phylogeny of clavicipitaceous fungi. We observed: (1) enteroblastic conidiation from branched phialidic conidiophores typical of the genus (anamorph Sphacelia) in all species including Corallocytostroma; (2) widespread and often sequential formation of terminal holoblastic secondary conidia on tapering hyphae arising from sphacelial macroconidia: and (3) in addition to sphacelial conidiation, sympodial holoblastic conidiation of the Ephelis-type in cultures of C. zizaniae and in both the culture and sphacelial tissue of C. citrina. Secondary conidiation was not found in C. purpurea, C. citrina and C. sorghicola. During sphacelial fructification, most species produced macroconidia and microconidia. Only macroconidia formed in planta underwent secondary conidiation whereas microconidia did not germinate at all. In C. phalaridis, the formation of holoblastic 2-3 celled appendaged conidia was observed, similar to that of Aciculosporium and Neoclaviceps. In dendrograms based on ITS1-5.8S rDNA-ITS2 sequences, genera and species with appendaged conidia grouped on a highly supported clade with ancestral Corallocytostroma. The clade was placed inside a group of tropical species of Claviceps, without any relationship to Balansiae.

Claviceps↗
PubMed2025

Computational identification of potential antifungal targets against Claviceps purpurea via MD simulation and MM/GBSA.

Ergot alkaloids produced by the fungus Claviceps purpurea pose significant risks to agriculture and human health. This study systematically investigates the pathogenicity of C. purpurea, analyzing five strains for their proteomic profiles, which revealed genetic variability in size and GC content. We identified proteins localized in various cellular compartments, contributing to our understanding of essential cellular processes. A focus on potential drug targets led to the identification of Alpha-N-acetylglucosaminidase, a hydrolase with significant mass and functional relevance, despite not matching a UniProt entry. The 3D structure prediction confirmed its integrity, making it a suitable target for further analysis. Molecular docking identified ligands CID:51,535,944 and CID:145,242,255 with strong binding affinities to Alpha-N-acetylglucosaminidase, highlighting interactions with key residues like TRP138, ARG651. Molecular docking interactions were validated and showed consistency through MD simulation analyses with greater RMSD, RMSF and PL contacts. This research enhances our understanding of C. purpurea, offering insights into its genetic diversity and cellular mechanisms while identifying promising therapeutic targets. The findings contribute to strategies for mitigating the economic and health impacts of C. purpurea infections, paving the way for innovative interventions in sustainable agriculture.

Molecular Docking Simulation↗
PubMed2004

Preparation of nanoparticles by electrocoagulation from soluble exopolysaccharide produced by Claviceps viridis.

Electrocoagulation is an evolving technology that has been effectively applied for wastewater treatment but its applications in biotechnology and nanotechnology are very limited. This method was applied for the preparation of nanoparticles from soluble exopolysaccharide (EPS) produced by Claviceps viridis in a submerged batch culture. A cathode/anode pair electrode (Al or Fe) system was used for determination of the separation rates of electrocoagulation and the yields of EPS nanoparticles production. The separation rates of 0.170 +/- 0.003 mg EPS/sec (Fe electrodes) and 0.250 +/- 0.003 mg EPS/sec (Al electrodes) were calculated for voltage gradient 1 V/1 cm of electrodes distance and were constant during experiments. The specific yield of EPS nanoparticles production based on the consumed electric power was dependent on the material of the electrodes and its value was determined as 0.71 +/- 0.01 mg EPS/W for Fe electrodes and 0.91 +/- 0.01 mg EPS/W for Al electrodes, respectively.

Chemical Precipitation↗
PubMed1978

Fine structure of imbibed sclerotial cells of Claviceps purpurea (Fr.) Tul revealed by freeze-etching.

The fine structure of the cortex of the natural sclerotium of Claviceps purpurea was studied. The cell wall of sclerotial cells is thickened due to overproduction of the fibrillar component of the wall. The intracellular spaces of the cortex tissue form a continuous system which is apparently instrumental in mediating communication between the growing sclerotium and the external milieu. The cytoplasmic membrane of imbibed sclerotial cells carriers abundant signs of secretory activity. Secretion vesicles, the content of which is discharged into external space, apparently contain exo 1,3-glucanase. Cytoplasmic vesicles migrating toward the plugged pores of the thickened septa apparently involve hydrolase secretion too. Spherosomes with lipid content are a predominant component of the cytoplasm of sclerotial cells. The activity of the membrane systems of imbibed cells indicates that the mobilization of lipids sets in only after activation of the hydrolases. Findings of phagocytosis of lipid granules by vacuoles are relatively frequent so that lipolysis might proceed in the vacuoles. Alkaloids could not be detected with the aid of freeze-etching.

Cell Wall↗
PubMed1981

[Effect of amitrol on Claviceps fusiformis, strain SD 58].

The influence of amitrole (3-amino-1,2,4-triazole) on growth, alkaloid formation and on some aromatic as well as ergoline pathway specific enzymes were studied in Claviceps strain, SD 58. In a phosphate-rich medium the addition of amitrole can partially reverse the alkaloid-depressing effect of phosphate. Using a typical fermentation medium amitrole reduces the activities of ergoline specific enzymes but increases drastically tryptophan synthase activity and to a lesser extent transaminase of aromatic amino acids.

Alkyl and Aryl Transferases↗
PubMed1982

Changes in cytoplasmic ultrastructure during submerged cultivation of a peptide alkaloids-producing strain of Claviceps purpurea (Fr.) Tul.

A strain of Claviceps purpurea, designated Pepty 695/S produces ergotoxine alkaloids under particular conditions of fermentation. The onset of alkaloid synthesis occurs around the second day of cultivation. Alkaloid formation is connected with morphological and ultrastructural changes. In the first 3-5 days of cultivation short thickened, septated hyphae, organized in plectenchymatic pellets as well as large single cells are formed. The hyphae are ultrastructurally characterized by increasing number of lipid droplets, deposits of glycogen and by extended ER membranes, which apparently may form numerous vesicles. The correlations between lipid and alkaloid synthesis are discussed.

Cell Nucleus↗
PubMed1975

Conversion of agroclavine to setoclavine and isosetoclavine in cell-free extracts from Claviceps sp. SD 58 and in a thioglycolate-iron (II) system.

Agroclavine was converted to setoclavine and isosetoclavine in crude extracts from Claviceps sp. SD 58. The ratio of setoclavine to isosetoclavine was 0.95. The conversion with boiled crude extract was 68% of the conversion with unboiled extract. In a thioglycolate-iron (II) system at 40 degrees for 5 hr, 45.5% of agroclavine was converted to setoclavine and isosetoclavine. At 70 degrees for 4 hr in the thioglycolate-iron (II) system, 4% of 4-dimethylallyltryptophan was converted to clavicipitic acid.

Alkaloids↗
PubMed1995

The Claviceps purpurea gene encoding dimethylallyltryptophan synthase, the committed step for ergot alkaloid biosynthesis.

The first pathway-specific step of ergot alkaloid biosynthesis in the fungus, Claviceps purpurea, is catalyzed by the prenyltransferase, 4-(gamma,gamma-dimethylallyl)tryptophan synthase. Partial sequence information was obtained for the purified enzyme and a degenerate oligonucleotide mixture was used to identify and amplify segments of the gene, dmaW. The complete gene and near-full-length cDNA were cloned and sequenced. The cDNA was cloned in a yeast expression vector in sense and antisense orientations relative to the inducible GAL1 promoter. Extracts of yeast transformants with the sense constructs, but not antisense constructs or cloning vector, catalyzed production of 4-(gamma,gamma-dimethylallyl)tryptophan. The sequence of dmaW and its cDNA indicated that it encoded a 455 amino acid polypeptide with a predicted molecular mass of 51,824 Da and a putative prenyl diphosphate binding motif.

Alkyl and Aryl Transferases↗
PubMed1993

pClK1 and pClT5--two linear mitochondrial plasmids from unrelated Claviceps purpurea strains: a comparison.

pClT5, a linear mitochondrial (mt) plasmid from Claviceps purpurea, strain T5, was sequenced and compared to pClK1, a linear mt plasmid from an unrelated C. purpurea strain. Both plasmids have terminal proteins (TPs) at their inverted terminal repeats (TlR). The TlRs of both plasmids show short conserved sequences, which are probably involved in plasmid transcription and replication. The coding capacity of pClT5 and pClK1 is similar: there are two large ORFs (ORF1 and ORF2) homologous to the DNA and RNA polymerase ORFs of pClK1 and several small hydrophobic ORFs. ORF3 shows homology to a small ORF of the Neurospora crassa mt plasmid maranhar and is transcribed. ORF6 of pClT5 is homologous to ORF4 of pClK1; both are transcribed and are possible candidates for the TP encoding ORF.

Base Sequence↗