Energy metabolism study of Fusidium coccineum strains with varying levels of antibiotic formation

Energy parameters of the two strains of the inperfect fungus Fusidium coccineum with different levels of antibiotic production were studied comparatively. It was shown that the respiration activity of the both strains was almost completely suppressed by KCN+ salycyl hydroxomate. It provided a supposition that the respiration activity was almost selectively connected with the mitochondria. The analysis of the energy parameters and especially the efficiency of the substrate consumption indicated that the system of oxidative phospholiration was dominant in the energy supply in both the strains. The differences between the strains were observed in the growth rate and the level of KCN-stable respiration. A change in the respiration of the active strain to the alternative KCN-stable path was observed by the end of the 6-8 day fermentation process when significant amounts of fusidin accumulated in the fermentation broth. Correlation between the level of KCN-stability and the antibiotic production was found.     

Relationship between intracellular pH and antibiotic biosynthesis in Fusidium coccineum

A correlation between the value of the intracellular pH and the biosynthesis of fusidic acid was studied by ³¹P-NMR spectroscopy in two strains of the fungus Fusidium coccineum. One of the strains was highly active and the other strain had low activity with respect to the antibiotic production. The position of the orthophosphate resonance in the ³¹P-NMR spectra was considered as a measure of the intracellular pH. In the cells of the highly active strain pH was in the range 7.0–7.5 in the cytoplasm and 6.1–6.25 in the vacuoles. In the cells of the strain with low activity was in the range 7.3–7.9 in the cytoplasm and 6.0–6.2 in the vacuoles. During high antibiotic productivity, the intracellular pH in the highly active strain full sharply, while in the less active strain it effectively did not change. This suggested that the change in the intracellular pH was responsible for the action of the enzymes in the cells and could be a factor defining the function of the cyanide-resistant respiration pathway and consequently the synthesis of fusidic acid in F. coccineum. *** DIRECT SUPPORT *** AG903062 00009     

Sporogenesis, antibiotic biosynthesis and the systematic position of Fusidium coccineum

Cytomorphological study of the wild and isogenic improved strains of F. coccineum revealed that with increasing of the antibiotic potency the character of the strain sporulation changed towards predominant formation of the spore "heads" which is characteristic of the sporogenesis of the genus Acremonium. These data allow one to revise the systematic position of the strains of F. coccineum and to classify them according to Gamsas belonging to Acremonium designated as Acremonium fusidioides. The criteria of the highly potent strains of this organisms are the following: formation of the spore "heads", changes in the event sequence during sporulation, decreasing of the spore index L/B with an increase in the strain potency. These criteria may be used in further improvement on the organism.     

Characteristics of fucidin biosynthesis by a Fusidium coccineum 257 A strain

The physiological features of Fusidium coccineum, strain 257 A, an organism producing fusidin were studied. It was found that increased concentrations of the carbon sources in the medium stimulated production of fusidin, while an increase in the content of various forms of nitrogen differently affected the level of the antibiotic viosynthesis: high concentrations of the amino acid-peptide form of nitrogen of corn-steep liquor decreased, while the protein form of nitrogen was associated with consumption of the significant part of carbon in the medium for formation of the fungus mycelium. Therefore, the concentration of the easily mobilizing forms of nitrogen may be considered as a regulator of the growth process of F. coccineum 257 A and production of fusidin by it.     

Respiratory system of Fusidium coccineum and regulation of biosynthesis of fusidic acid and sporogenesis

Summary The respiratory system, sporulation, and dynamics of alkaline protease formation were studied in three strains of the fungus Fusidium coccineum, differing in their ability to make antibiotics.Oxidative phosphorylation provided most of the energy in high and low activity strains and their respiratory activity was exclusively related to mitochondria functioning.In inactive and low activity strains, the terminal oxidation of reduced equivalents proceeds mainly by the respiratory chain with cytochrome oxidase as the terminal component. In the high activity strain there is a cyanide-resistant alternative pathway which is parallel to the classical cytochrome chain. The complete transition to the use of this pathway coincides with the stage of maximum antibiotic biosynthesis. The induction of the alternative pathway in the high activity strain was not concerned with the inhibition of the cytochrome site of the respiratory chain by fusidic acid. It was shown that the quantity of the antibiotic synthesized and the character of cellular differentiation can be altered by changing the oxidation pathwats used with inhibitors such as chloramphenicol and salicyl hydroxamate.We suggest that there must be common regulation of antibiotic formation, sporulation and induction of the alternative oxidation pathway.     

Morphofunctional characteristics of the development of strains of Fusidium coccineum differing in antibiotic activity in stab cultivation

The object of the work was to study the morphological and functional characteristics of Fusidium coccineum strains producing fusidic acid and differing in the antibiotic activity. The high metabolic activity of the culture is accompanied by the following morphological characteristics: the cells are rich in ribosomes and mitochondria, they have early vacuolisation, are filled with lipid granules, and then the mycelium undergoes autolysis. As strains with a high activity grow, the structure of the cells changes, the number of ribosomes and mitochondria falls down, and the latter are destroyed. For a long time, the cells contain electron-dense granular structures limited with the membrane and capable of transformation into lipid granules and membranous structures. As was shown by cytochemical studies, the structures have not merely proteins and lipids, but also phosphorus compounds. Their functional role in the fungal metabolism is discussed. As soon as super-synthesis of fusidic acid commences, the cells of the highly active strains are filled with lipid granules associated possibly with the steroid antibiotic. These formations are released from the cell during local lysis of the cell wall and the cytoplasmic membrane.     

Carbohydrate and pyruvic acid degradation pathways in Fusidium coccineum strains with varying levels of antibiotic synthesis]

A number of enzymes and reactions of glycolysis, pentose-phosphate cycle and degradation of pyruvic acid in strains of F. coccineum with various levels of antibiotic production was studied comparatively. The experiments showed that highly productive strains were characterized by higher activity of the NADP-deficient enzymes of the pentoze-phosphate cycle as compared to the low active strains. The activity levels of glycolytic enzymes, such as fructose-diphosphate-aldolase and 3-phosphoglycerolaldehydehydrogenase did not practically differ. Significant differences were found in the reactions of puryvic acid degradation: the activity of cytoplasmic pyruvatedecarboxylase in the mutant with high antibiotic production level was lower than that in the low productive strain, while oxidation of the pyruvate of the mitochondrial fraction was on the contrary more intensive than in the highly productive strain. Therefore, metabilism in the strains studied was characterized by ever-increasing biochemical changes with an increase in their antibiotic productivity. Lowering of the growth rate of the mutants as their capacity for antibiotic supersynthesis increased and subsequently the anabolic processes became more intensive was accompanied by increasing derepression of the key enzymes of carbohydrate metabolism and in particular NADR-deficient dehydrogenase of the pentose cycle and pyruvatedehydrogenase, significant for fusidin biosynthesis and providing production of the antibiotic of steroid nature by cofactor NADP-H and acetyl-KoA, the primary precursor.     

Optimization of protoplast formation,regeneration, and viability in Microsporum gypseum

Factors affecting high yields, regeneration frequencies, and viability of protoplasts from clonal cultures of Microsporum gypseum were investigated. Maximum yields of protoplasts were obtained after 6 hrs digestion of 2–4 days old mycelium with Novozyme 234 using CaCl₂ (0.4 M) as an osmotic stabilizer and glycine + HCl (pH 4.5) as the buffer system. Mercaptoethanol + dithiothreitol (0.01 M) proved to be the best pretreatment of mycelium prior to digestion with enzyme. A regeneration frequency of 94.4% was obtained using the top agar method with complete medium (pH 6.5) containing 0.5% agar and 0.4 M CaCl₂ as an osmoticum. Colonies from regenerated protoplasts on medium containing CaCl₂ were pigmented and completely powdery with high sporulation. Protoplast viability was studied in osmotic stabilizer supplemented with glucose or glutamine. After 24 hrs, glucose (2%) and glutamine (2%) enhanced protoplast viability by 22% and 23%, respectively. Protein synthesis, as measured by ³H-lysine uptake, matched the viability profile determined by fluorescence microscopy.     

Factors affecting protoplast formation and regeneration by four species of Streptomyces

Four species of Streptomyces, Streptomyces canescens, S. limosus, S. griseus and S. griseolus , were used to study the effects of glycerine and gelatin on the formation and regeneration of protoplasts. For each species efficient protoplast formation with high protoplast concentrations and low levels of non-protoplast units was obtained with mycelia grown in medium without glycerine. The low regeneration frequencies of protoplasts of S. canescens and S. limosus on R2 medium were increased substantially by the addition of 1% gelatin. The use of single colonies, rather than spores, to establish mycelial cultures was found routinely to produce good protoplast preparations.     

无花果曲霉原生质体形成与再生条件的探讨

根据正交试验得出无花果曲霉原生质体形成的最佳条件,用１％的混合酶液（０．５％纤维素酶＋０．２５％蜗牛酶＋０．２５％溶菌酶）作用无花果曲霉菌体细胞,原生质体产量达３．２×１０７个·ｍｌ－１,渗透压稳定剂为０．６ｍｏｌ·Ｌ－１ＫＣｌ于０．２ｍｏｌ·Ｌ－１ＰＯ３＋４（ｐＨ５．８）中,酶解时间和酶解温度分别为３．０ｈ、３０℃．比较不同酶解时间、再生稳定剂和碳源等因素对原生质体再生的影响,可确定最佳再生条件,再生率达３０％以上．     

Cytologic control of protoplast formation and regeneration in Streptomyces erythraeus, strain BTCC-2

Experiments on protoplast formation and regeneration in S. erythraeus, strain BTCC-2 (Saccharopolyspora erythrae) were performed under microscopic control at all the stages. It was shown that the highest protoplast titer was provided by the mycelium grown in one step in the absence of glycine. For characterizing the protoplasts formed by the mycelium grown under different conditions, their regeneration capacity was estimated by microscopic examination of the protoplasts after 15-20-hour growth in microchambers and evaluation of the regeneration efficiency 7-10 hours later. Of interest was the fact of spontaneous development of colonies consisting of the protoplast-like cells (L-cells) in 15-20 hours. Such colonies were formed only by the protoplasts grown from the mycelium incubated in one step in the absence of glycine or in the presence of 0.1 per cent of glycine. Such conditions provided also the maximum efficiency of the protoplast regeneration. The long-term storage of protoplasts led to a decrease in their viability.     

A simple method for protoplast formation and improvement of protoplast regeneration from various fungi using an enzyme from Trichoderma harzianum

Summary An enzyme from Trichoderma harzianum dissolved the cell walls of a wide range of filamentous fungi belonging to Basidiomycotina, Ascomycotina, Deuteromycotina, and Zygomycotina and so could be used to make protoplasts. A lyophilized preparation of the Trichoderma enzyme had about 0.3 units/mg -1,3-glucanase activity and 0.36 units/mg chitinase activity. About twice as many protoplasts were produced from different species of fungi by a single treatment with this enzyme than with combined commercial enzymes. The greatest number of protoplasts could be produced from most of the fungi by incubation for about 2 h t 30°C, but the number was decreased by incubation for more than 4 h or by use of a higher dose of the enzyme. An enzyme prepared by bentonite treatment from the original Trichoderma enzyme had less proteinase activity and protoplasts were fairly stable with this product during incubation for 8 h. Protoplasts produced by the proteinase-reduced preparation of the Trichoderma enzyme from three fungi regenerated at about 1.8 times the rate of those produced by the original enzyme.     

Cauliflower inflorescence protoplast culture and plant regeneration

A yield of 2–4×10⁶ protoplasts/g F.W. could be obtained when fresh cauliflower inflorescence segments were digested with 2% cellulase Onozuka R-10, 1% cellulase RS and 0.4% Macerozyme R-10 in CPW18S for 7 to 10 h. Purified protoplasts were cultured in K8p liquid and agarose medium. Although protoplasts in liquid medium divided earlier than in agarose, protoplast-derived cells in liquid culture could not avoid browning. With agarose culture, sustained division and callus formation could be achieved. After 20 days, calli were transferred onto B5 agar medium with ZT 1.5 mg l^-1, BA 0.5 mg l^-1 and IAA 0.1 mg l^-1 for shoot formation. The frequency of bud formation varied from 56.7% for calli of 1mm in size to 5.6% for 5mm calli. The shoots formed were rooted in B5 medium containing 0.5 mg l^-1 IBA, and the regenerated plants were transplanted to pots and grew normally. It took about two months from protoplasts to the regenerated plants.Abbreviations Ade adenine - BA 6-benzyl aminopurine - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4,-dichlorophenoxyacetic acid - GA₃ gibberellic acid - Gln glutamine - NAA -naphthylacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - ZT zeatin     

Mycelial protoplast isolation and regeneration of Lentinus lepideus

Generation of fungal protoplast is essential for fusion and transformation systems. Protoplast fusion offers great potential for the improvement of industrially important microorganisms. To establish conditions for the protoplast isolation and regeneration of the mycelia of Lentinus lepideus, various enzymes and osmotic stabilizers were examined. To investigate suitable medium for the culture of L. lepideus, the mycelia were grown in ten different media at 28 degrees C for 10 days. Among them potato dextrose agar (PDA) medium was found to be the best for colony growth. When Novozym 234, cellulase and beta-glucuronidase were added to the mycelia in combination or alone, Novozym 234 alone at the concentration of 10 mg/ml was the most effective for the protoplast yield. Purified spherical protoplasts of the mycelia were osmotically hypersensitive and further incubation of the mycelia with the lytic enzyme resulted in the older parts of the hyphae swollen. When we applied various osmotic stabilizers at the fixed concentration of 0.6 M on the protoplasts, the yields of protoplasts were increased until 4-hr incubation. However application of sucrose or MgSO4 led to further protection of protoplasts after that time and reached a plateau on 5- and 7-hr incubations, respectively. The suitable incubation time and optimal pH with the lytic enzyme for the maximum release of protoplasts were 6 hrs of incubation and pH 5, respectively. When we examined various osmotic stabilizers for the regeneration of the protoplast, the complete medium containing 0.6 M sucrose induced highest hyphal growth with regeneration frequency of 3.28%.     

Plant regeneration and protoplast culture of Browollia speciosa

Explants from hypcotyls and cotyledons of Browalia speciosa were shown to regenerate plantlets.Protoplasts were isolated from etiolated cotyledon material, and, although callus was readily obtained, plantlet regeneration was not observed using numerous hormone regimes.Abbreviations M Mannitol - 2,4-D Dichlorophenoxyacetic acid - NAA Naphthalene-acetic acid - BAP Benzylaminopurine - MS medium Murashige and Skoog (1962) medium - UM medium Uchimiya and Murashige (1974) medium - COT cotyledon - SH shoot - R root     

Cell wall composition and protoplast regeneration in Candida albicans

The transition of blastospores to the mycelial phase in Candida albicans was induced after the blastospores were kept at 4 degrees C for several hours and then transferred to a fresh medium prewarmed at 37 degrees C. Glucan was the most abundant polymer in the wall in the two morphogenetic forms but the amount of chitin was higher in the mycelial form than in blastospores. Efficient protoplasting required reducing agents and proteases together with beta-glucanases (zymolyase). Protein synthesis in regenerating protoplasts was initiated after about 30 min. Chitin synthetase, initially very low, was incorporated in important amounts into cell membranes mainly in a zymogenic state. After a few hours chitin was the most abundant polymer found in the aberrant wall of the regenerating protoplast.     

Effect of protoplast formation and regeneration on the production of nebramycin complex in Streptomyces cremeus subsp. tobramycini

Production of the nebramycin complex in Streptomyces cremeus subsp. tobramycini before and after the protoplast formation and regeneration was comparatively studied. The antibiotic production was estimated by the total activity and component composition of the nebramycin complex. It was found that formation and regeneration of the protoplast led to lowering of the activity and changing of the complex component composition. Strains mainly synthesizing each one of the three basic components of the nebramycin complex were isolated. The strains proved to be unstable by the antibiotic production property and after three subcultures lost the differences in the complex component composition.