Strain improvement ofArthrobacter simplex by protoplast fusion

Anl-tryptophan auxotroph and milky mutants were derived from an inducible cholesterol oxidase-producing bacterium,Arthrobacter simplex USA18, via UV-mutagenesis. Protoplasts of these mutants and a constitutive cholesterol oxidase producer, strain US3011, were prepared by growing cells in the presence of ampicillin (20g ml^–1) followed by digestion with lysozyme. Protoplast fusion between tested strains with complementary characteristics was achieved in the presence of 20–40% polyethylene glycol 6000. The fusion frequency was about 1.5–1.7×10^–3. The cholesterol oxidase activity of four fusants in a cholesterol-containing medium was 20–60% higher than that of parental strains. This study demonstrated that protoplast fusion is applicable to strain improvement ofArthrobacter strains for enzyme production.     

Activation of ergot alkaloid biosynthesis in prototrophic isolates by Claviceps purpurea protoplast fusion.

Intraspecific protoplast fusions were carried out with active ergocornine-ergokryptine and inactive ergocristine Claviceps purpurea strains and vice versa. The isolated prototrophic strains from both types of crosings produced all three alkaloid types, showing that biosynthesis of distinct alkaloid was activated in an inactive partner strain. The prototrophic isolates were stable on minimal medium but they segregated by subculturing on complete medium. In comparison with the original partner strains, differences in morphological and cytological characteristics were also established.     

Intraspecific heterokaryon and fruit body formation in Coprinus macrorhizus by protoplast fusion of auxotrophic mutants

Summary Fusion of protoplasts of Coprinus macrorhizus mutants with different amino acid requirements resulted in the production of prototrophic clones at frequencies of 1–4% of the protoplasts surviving the fusion treatment. The frequencies were at least 200 times higher than those of the appearance of revertants. Few prototrophic colonies appeared also when the mutant protoplasts were individually subjected to fusion treatment, or when they were mixedly cultured without fusion treatment. It was thus concluded that intraspecific heterokaryons were formed by protoplast fusion.The auxotrophic mutants did not form fruit bodies when cultured singly or mixedly with each other. In contrast, the heterokaryons produced by protoplast fusion between the mutants of compatible mating types developed into fruit bodies with intermediate morphology of those of the strains from which the mutants were derived. Heterokaryons were also formed by fusion of mutant protoplasts with identical mating genotype, but they failed to form fruit bodies.Abbreviations PEG polyethyleneglycol - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid     

Complementation by intraspecific protoplast fusion of auxotrophic cell lines of Datura innoxia P. Mill.

It was determined using electrophoresis in polyacrylamide gels containing native DNA or RNA that sugar non-specific nuclease active at pH 5.2 was expressed in tobacco callus. The nuclease had a relative molecular mass of about 34.6 kDaltons and degraded substrates in the following order of decreasing rate: denaturated DNA>poly dA>UV-irradiated native DNA>native DNA>alkylated native DNA>apurinated native DNA>poly dGpoly dC. The nuclease activity changed during callus growth and plant regeneration, but no developmental changes in electrophoretic patterns were detected. The increase in specific DNAse activity of nuclease was maximal in the exponential phase of callus growth on both growth and regeneration media, except for activity in the cytokinin-independent cell strain grown on growth medium. The specific DNAse activity of nuclease decreased during the bud formation period, while total DNAse activity calculated per mg of dry weight was slightly higher in vegetative buds (9.1U) than in undifferentiated tissue of callus (8.5U). Specific DNAse activity was, on the average, several hundred-fold lower in the vegetative tissues of flowering tobacco plants than in calluses in the exponential phase of growth.     

Semicontinuous cultivation of immobilized Claviceps purpurea

Summary Mycelia of Claviceps purpurea CBS 164.59 were immobilized in 2%, 4%, and 8% calcium alginate. Alkaloid production by free cells declined after 60 days, while immobilized cells retained their activity for 200 days. The cumulative alkaloid production for all fermentation cycles using 8% calcium alginate immobilized mycelia was 25 times higher than that from free cells. The best yields of the ergopeptide ergometrine were reached with 4% gel immobilized mycelia, while higher gel concentrations caused a shift in the alkaloid biosynthesis towards high clavine alkaloid production.Beginning with the third cycle of reincubation the immobilized mycelia showed a marked tendency to fragmentize into vacuolated arthrosporoid-like structures and produced violet-black pigments so that the beads recalled sclerotial structures of parasitically living Claviceps.Dedicated to Prof. Dr. K. Esser to his 60th birthday     

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.     

Model of growth and ergot alkaloid production by Claviceps purpurea

A growth model for Claviceps purpurea in submerged batch culture is presented. In developing the model, the basic principles of the growth and the morphological properties of C. purpurea are considered. The growth of C. purpurea is assumed to occur in a three-step manner; the first step involves the assimilation and the growth of cells; the second one involves cell division, and the third one involves transformation of the mature cells to a state where they have no ability to divide but do have the ability to produce ergot alkaloids and then they gradually die. Inorganic phosphate is assumed to be the limiting substrate for the first and the second steps in conditions of carbon source being in excess. The model constants are determined by model simulation and graphical searching techniques to find the minimum value of the absolute difference between the experimental and the simulated curves for biomass, alkaloids, and sucrose.     

Biosynthesis of ergotamine by Claviceps purpurea (Fr.) Tul

1. Partially purified ceramide trihexoside alpha-galactosidase from human liver was studied by using ceramide trihexoside specifically tritiated in the terminal galactose. 2. The hydrolysis of ceramide trihexoside was absolutely dependent on a mixture of sodium taurocholate and Triton X-100 and was markedly inhibited by human serum albumin and by NaCl. 3. The Lineweaver-Burk plot for ceramide trihexoside hydrolysis was upward curving. Ceramide lactoside inhibited hydrolysis of all concentrations of ceramide trihexoside. Ceramide digalactoside stimulated hydrolysis of low concentrations of ceramide trihexoside, but inhibited hydrolysis of high concentrations of the lipid. 4. alpha-Galactosidase activity assayed with the synthetic substrate 4-methylumbelliferyl alpha-d-galactopyranoside fractionated together with activity assayed with the natural substrate ceramide trihexoside. Both activities had identical heat-inactivation kinetics. 5. Characteristics of the hydrolysis of the synthetic substrate differed considerably from those of the natural substrate, including pH optimum, shape of the Lineweaver-Burk plot, and differential effects of inhibitors and activators. Mutual inhibition of hydrolysis between the synthetic and natural substrates was predominantly non-competitive. 6. These results are discussed in the light of special problems involved in the hydrolysis of lipids in an aqueous milieu.     

Chemoraces and Habitat Specialization of Claviceps purpurea Populations

We studied genetic variability of 100 isolates of Claviceps purpurea by using randomly amplified polymorphic DNA (RAPD), an EcoRI restriction site polymorphism in the 5.8S ribosomal DNA (rDNA), the alkaloids produced, and conidial morphology. We identified three groups: (i) group G1 from fields and open meadows (57 isolates), (ii) group G2 from shady or wet habitats (41 isolates), and (iii) group G3 from Spartina anglica from salt marshes (2 isolates). The sclerotia of G1 isolates contained ergotamines and ergotoxines; G2 isolates produced ergosine and ergocristine along with small amounts of ergocryptine; and G3 isolates produced ergocristine and ergocryptine. The conidia of G1 isolates were 5 to 8 μm long, the conidia of G2 isolates were 7 to 10 μm long, and the conidia of G3 isolates were 10 to 12 μm long. Sclerotia of the G2 and G3 isolates floated on water. In the 5.8S rDNA analysis, an EcoRI site was found in G1 and G3 isolates but not in G2 isolates. The host preferences of the groups were not absolute, and there were host genera that were common to both G1 and G2; the presence of members of different groups in the same locality was rare. Without the use of RAPD or rDNA polymorphism, it was not possible to distinguish the three groups solely on the basis of phenotype, host, or habitat. In general, populations of C. purpurea are not host specialized, as previously assumed, but they are habitat specialized, and collecting strategies and toxin risk assessments should be changed to reflect this paradigm shift.     

Colonization of Claviceps purpurea sclerotia on Triticale by Fusaria in 1985

During 1985 60% of Claviceps purpurea sclerotia collected from Triticale heads were colonized by Fusarium acuminatum Ell. et Ev. and F. heterosporum Nees.     

Chemoraces and habitat specialization of Claviceps purpurea populations

We studied genetic variability of 100 isolates of Claviceps purpurea by using randomly amplified polymorphic DNA (RAPD), an EcoRI restriction site polymorphism in the 5.8S ribosomal DNA (rDNA), the alkaloids produced, and conidial morphology. We identified three groups: (i) group G1 from fields and open meadows (57 isolates), (ii) group G2 from shady or wet habitats (41 isolates), and (iii) group G3 from Spartina anglica from salt marshes (2 isolates). The sclerotia of G1 isolates contained ergotamines and ergotoxines; G2 isolates produced ergosine and ergocristine along with small amounts of ergocryptine; and G3 isolates produced ergocristine and ergocryptine. The conidia of G1 isolates were 5 to 8 microm long, the conidia of G2 isolates were 7 to 10 microm long, and the conidia of G3 isolates were 10 to 12 microm long. Sclerotia of the G2 and G3 isolates floated on water. In the 5.8S rDNA analysis, an EcoRI site was found in G1 and G3 isolates but not in G2 isolates. The host preferences of the groups were not absolute, and there were host genera that were common to both G1 and G2; the presence of members of different groups in the same locality was rare. Without the use of RAPD or rDNA polymorphism, it was not possible to distinguish the three groups solely on the basis of phenotype, host, or habitat. In general, populations of C. purpurea are not host specialized, as previously assumed, but they are habitat specialized, and collecting strategies and toxin risk assessments should be changed to reflect this paradigm shift.