Effect of glycerin on the biosynthesis of heliomycin by Streptomyces olivocinereus

Glycerol as the sole carbon source was added to the medium or biosynthesis of heliomycin by Streptomyces olivocinereus and the effect of its concentration on the culture growth and antibiotic production was studied. The culture growth and the amount of the antibiotic synthesized per 1 unit of the fermentation broth were limited by glycerol added in quantities of 0.05 to 1 per cent. Further increasing of the glycerol concentration had no significant effect on the culture growth and antibiotic biosynthesis. The amount of the antibiotic synthesized per 1 unit of the mycelial mass relatively slightly depended on the glycerol concentration. The rate of glycerol consumption by the young 24-hour culture in batch fermentations markedly exceeded that of glycerol consumption by the 48-hour culture. The younger mycelium significantly increased its rate of glycerol consumption when the initial concentration was increased whereas the rate of glycerol consumption by the more mature mycelium did not depend on the initial concentration of the carbon source (within 0.5-2 per cent). The rate of heliomycin biosynthesis practically slightly depended on the initial concentration of glycerol.     

Study of the carbohydrate content in the mycelia of an active strain producing oxytetracycline and in an inactive mutant]

The content of carbohydrates in the mycelium of the active strain and inactive mutant of the oxytetracycline-producing organism under conditions favourable (starch medium) and unfavourable (glucose medium) for the antibiotic biosynthesis was studied. The mycelium of both organisms was fractionated and carbohydrate distribution according to the mycelium fractions and carbohydrate content in every fraction were investigated. No significant differences were observed between the active strain and inactive mutant with respect to the characteristics studied. The carbon source in the medium had the dominating effect on the chemical composition of the mycelium. The mycelium of both strains grown on the starch medium contained much more carbohydrates than that grown on the glucose medium. The carbohydrates of the mycelium grown on the starch medium were mainly found in fraction III and must be represented by polysaccharides.     

Selection of the seeding media and the age and quantity of mycelia providing simultaneous biosynthesis of an antibiotic and pigment by the fungus Hypomyces rosellus

The authors observed maximum simultaneous biosynthesis of antibiotic and pigment in the microphilic fungus with using 48-hour seed mycelium having the specific growth rate of 0.008-0.011 h-1 in an amount of 5-7 per cent (v). The Balling 4 degrees wart and Chapek medium with 1 per cent of soybean flower and 1 per cent of corn steep liquor may be used for growing the seed mycelium. No significant effect of the seed medium composition and seed mycelium age on the pigment production was observed.     

Inhibition of oleandomycin synthesis by glucoses added during fermentation

Oleandomycin biosynthesis by Streptomyces antibioticus is repressed by glucose added to the growth medium in the process of fermentation. Phosphotransferase involved in the synthesis of acetyl CoA and propionyl CoA (the precursors of the antibiotic macrolactone ring) is neither inhibited nor repressed, and the substrate specificity of the enzyme does not change. The content of cAMP in the mycelium of S. antibioticus does not change significantly when either glucose or sucrose is added to the medium 24 h after the inoculation whereas the content of exogenous cAMP rises abruptly 24 h after glucose addition. At the same time, the medium becomes much more acidic and the content of protein in the mycelium rises noticeably. Consequently, cAMP may be involved in the regulation of the culture growth.     

Effect of organic acids on the biosynthesis of macrotetralide antibiotics by an Actinomyces chrysomallus var. carotenoides strain

The biosynthesis of macrotetrolides by Actinomyces chrysomalus var. carotenoides was stimulated by acetic, succinic, propionic, oxalic, malic, tartaric, citric, pyruvic, alpha-ketoglutaric and fumaric acids. Incorporation of 14C-acetate into the molecule of the antibiotic and the data on dependence of the stimulating effect upon the quantitative ratio and time of the organic acid addition were indicative of the role of acetic, succinic and propionic acids as precursors of macrotetrolides. The other organic acids increased the biosynthesis of macrotetolides when added to the culture within wide time ranges of the culture development and prolonged the period of the mycelium productive state.     

Precursor formation and biosynthesis of the macrolide antibiotic a 6599 (turimycin) by streptomyces hygroscopicus JA 6599]

The possible role of some metabolic systems producing acetyl-CoA, and methylmalonyl-CoA as initial precursors in the biosynthesis of the macrolide antibiotic A 6599 by Streptomyces hygroscopicus JA 6599 was studied. The activities of pyruvate decarboxylase exceeded in two higher producing strains about twofold those found in the mycelium of a lower producing one suggesting that in this organism an enhanced production of acetyl-CoA should be one of the prerequisites necessary for an improved antibiotic biosynthesis. No clear interrelationship was established, however, between the biosynthesis of the secondary metabolite A 6599 on the one hand and the acetate and propionate kinase content on the other hand. In S. hygroscopicus JA 6599 the carboxylation of acetyl-CoA or propionyl-CoA seems to be the major pathway giving malonyl-CoA or methylmalonyl-CoA, respectively. Thus, the activities of acetyl-CoA and propionyl-CoA carboxylases corresponded with both the levels of antibiotic production in several strains and with variations observed in the specific antibiotic production rate during the cultivation. Some other pathways synthesizing these precursors, e.g. via oxaloacetate, are assumed to be negligible since even in the mycelium of the lower producing strain increased activities of phosphoenolpyruvate carboxylase were present.     

Cyclic adenosine-3',5-monophosphate in Streptomyces antibioticus and its possible role in regulating oleandomycin biosynthesis and the growth of the culture

When glucose is substituted for sucrose in the fermentation medium for Streptomyces antibioticus, the pH of the cultural broth becomes more acidic, the rate of protein synthesis in the mycelium rises, and the rate of oleandomycin synthesis decreases abruptly. The dynamics of cAMP (cyclic monophosphate) accumulation was studied in the process of biosynthesis by the culture in different media. Most of the synthesized cAMP (80-90%) was shown to be excreted into the medium. Glucose stimulates cAMP synthesis and excretion from the mycelium by a factor of 1.5-3. No distinct correlation was found between cAMP content in S. antibioticus cells and the level of oleandomycin biosynthesis. A correlation between changes in the concentration of exocellular cAMP and protein synthesis in the mycelium suggests that the excreted cAMP may be involved in regulating the growth of the culture producing the antibiotic.     

The effect of heat shock on the formation and composition of actinomycins]

The effect of various conditions of heat shock on production of actinomycins by Streptomyces chrysomallus 2 and their composition was studied. The actinomycin biosynthesis was shown to be the function of the growing mycelium and changed in accordance with changes in the volume of the mycelium and its morphological features after heat shock at various suboptimal temperatures. The temperature shock had a specific action on the antibiotic synthesis: the index of the actinomycin maximum quantity increased after the heat shock at 35 and 38 degrees C and lowered more sharply than that of the biomass volume after the heat shock at the temperatures of 40, 42, 45 and 50 degrees C for 1 hour. After the shock at 38 degrees C the component composition of the actinomycin complex did not significantly change while with addition of exogenic amino acids such as L-valine, L-leucine and L-isoleucine the shock effect on the component composition of the actinomycin complex was marked.     

Analysis of the neurohypophyseal components accumulating in the supraoptic nucleus of the rat after injection of colchicine

[35S]Cysteine has been injected into the supraoptic nuclei of normal rats and of animals given 7 micrograms colchicine into the cerebrospinal fluid to inhibit transport of neurosecretory granules. Analysis of extracts of the supraoptic nuclei 20 min or 6 h after isotope injections showed that (i) colchicine does not affect biosynthesis, i.e., incorporation of tracer into the common precursors of neurohypophyseal hormones and their associated neurophysins, and (ii) processing of precursors inside the arrested granules proceeds normally.     

The role of succinic acid in the biosynthesis of levorin

The influence of succinic acid as a component of media for biosynthesis of levorin, a polyenic antibiotic was studied. It was shown that with the use of the soybean-corn medium supplemented with succinic acid (0.05-0.4 per cent) the antibiotic content in the fermentation broth was higher than that in the control. The highest stimulating effect (135 per cent) was observed with addition of 0.1 per cent of succinic acid. For providing optimal antibiotic production in the synthetic medium supplemented with succinic acid (0.4 per cent) addition of acetic acid (0.05 per cent) was required. Studies with the soybean-corn medium with and without succinic acid revealed differences in the level of p-aminoacetophenone, an aromatic fragment of the levorin molecule. Under the conditions of the medium with succinic acid the content of p-aminoacetophenone in the mycelium was higher by 10 to 18 per cent as compared to that in the control and depended on the fermentation period. The role of succinic acid in biosynthesis of levorin is discussed.     

New species, Actinomadura fulvescens sp. nov. and Actinomadura turkmeniaca sp. nov. and their antagonistic properties

Two new species of Actinomadura isolated from soil samples of the Turkmen SSR, i.e. Actinomadura fulvescens sp. nov. and Actinomadura turkmeniaca sp. nov. are described. The first species is characterized by formation of short (1-2 turns) spiral spore chains, smooth spores, scanty white aerial mycelium, colourless or yellowish substrate mycelium on synthetic media and brownish-yellow substrate mycelium and soluble pigment of the same colour on organic media. No melanoid pigment is secreted. The type culture is designated as INA 3321. The cultures of A. fulvescens show antibiotic activity. A. turkmeniaca is characterized by formation of short straight or spiral spore chains, smooth spores, scanty white aerial mycelium, substrate mycelium and soluble pigment of pinkish-violet colour, absence of melanoid pigment. The type culture is designated as INA 3344. The strains of this species have low antibiotic activity. The study on the use of carbon sources by the representatives of 7 species (9 strains) of Actinomadura showed that the majority of the cultures (5 species, 7 strains) produced no growth on the Pridham and Gottlieb medium (ISP-9) with various carbon sources, including glucose. Possibly this medium cannot be used as the main medium for investigation of the spectrum of carbohydrate consumption in Actinomadura.     

硝酸盐、镁盐对林可霉素生物合成的促进作用

在林肯链霉菌生物合成林可霉素代谢调节的研究中,发现硝酸盐可明显促进林可霉素的生物合成．加入硝酸钾０．８％,林肯链霉菌合成林可霉素的产量可增加３７％．在发酵９６ｈ之前加入硝酸盐均能促进林可霉毒的合成,但产量的增加随加入时间的延迟而降低．硝酸钾在促进产量的同时,使菌体生长减少,看来硝酸盐对林可霉素的合成与菌体生长之间起着调节作用．洗涤菌体试验指出,硝酸盐的加入诱导了林可霉素合成所需要的酶系,这可能是加入硝酸盐后,产生进一步氮代谢的结果;蛋白胨不能代替硝酸盐,进一步说明硝酸钾的作用并不是作为氮源利用．在蛋白质合成抑制剂氯霉素存在下,硝酸盐不再能促进林可霉素的合成,说明氯霉素抑制了硝酸盐或其代谢中间物所诱导的酶系的合成．同时还报导了镁盐促进林可霉素生物合成现象的初步观察结果．硫酸镁在促进林可霉素产量提高的同时,使菌体生长延迟．硫酸镁的这种作用机制可能是通过磷酸镁铵沉淀,降低了培养基中游离氨和可溶性磷酸盐浓度,解除了铵盐和磷酸盐对林可霉素合成的抑制．     

Effect of inorganic phosphate on levorin biosynthesis and on the mycelial makeup of Streptomyces levoris]

The effect of inorganic phosphate on biosynthesis of the polyenic antibiotic levorin by Streptomyces levoris and composition of the culture mycelium was studied. It was found that the synthetic medium with 0.4 mM of phosphate was optimal for growth of Str. levoris. When the concentration of phosphate was higher, the biomass increased, while the synthesis of levorin appeared to be inhibited and morphological changes in the culture were observed. Phosphate had a significant effect on the mycelium composition. When its concentration was increased 10 times as compared to the optimal one, the amounts of protein, RNA, total phosphorus and polyphosphates increased 1.3--1.4, 1.6--1.7, 2--3 and 10 times respectively, while the synthesis of levorin decreased 5 times. Changes in the lipid component of the mycelium were also observed. In the absence of inorganic phosphate in the medium the acetone precipitating fraction of the lipids contained 20--40 per cent of the phosphoruless compounds. During cultivation their portion increased up to 70--77 per cent. However, in the presence of its excess the polar lipids were represented only by phospholipids during the whole life cycle. The fatty acid spectrum of the lipids did not depend on the phosphate concentration and was represented mainly by saturated fatty acids with a branched chain of a series of iso- and anteiso-structures containing 14--18 carbon atoms.     

Study of the chemical makeup of the mycelium from an active strain of Act. rimosus and from an inactive mutant in relaiton to oxytetracycline biosynthesis]

Chemical composition of the mycelium of the active and inactive mutants of Act. rimosus grown under conditions favourable for oxytetracycline biosynthesis on the starch or maltose medium and under favourable conditions on the glucose medium was studied. It was shown that according to its chemical composition the above strains did not practically differ. When grown on the starch medium the mycelium of both strains contained great amounts of carbohydrates and comparatively small amounts of nucleic acids and nitrogen. Replacement of starch in the medium by glucose or maltose induced significant changes in the mycelium composition: the synthesis of intracellular polysaccharides was markedly suppressed and the synthesis of nucleic acids and nitrogen containing compounds increased. RNA was the main nucleic acid in both strains on starch and glucose media. The content of DNA was low and did not practically change. The mycelium of both strains contained small amounts of lipids which did not significantly change during the process of cultivation and did not correlate with the antibiotic activity.     

Study of the structure of colonies of active and inactive Actinomyces parvullus variants using luminescence and scanning microscopy]

The colony structure of the active and inactive proactinomycete-like variants of Actinomyces parvullus producing actinomycin D was studied with luminescent and scanning microscopy. Clear differentiation of the colony profile was shown by the structure and functions of the mycelium layers. A zone of active synthesis and accumulation of the antibiotic was observed in the colonies of the active variant in the upper part of the substrate mycelium with reddish-yellow self luminescence in UV light and characteristic close hyphae "cemented" by the intracellular substance. Formations of the granule type were often noted on the hyphae of that layer. The layer of the aerial mycelium was loosely connected with the substrate mycelium and consisted of sporophores and spore chains partially broken into single spores. The colonies of the inactive proactinomycete-like variant had a slightly differentiated profile with a sponge-like structure, no zones of the antibiotic synthesis being found. The presence of the intracellular substance was observed in the upper part of the colony supersubstrate mycelium.     

Regulation of Nigeran Accumulation by Aspergillus aculeatus

Nigeran (mycodextran), an unbranched glucan consisting of alternating alpha,1-4 and alpha,1-3 linkages, is accumulated intracellularly by Aspergillus aculeatus, under conditions of nitrogen limitation and low pH (optimum pH 5.0). Both conditions are necessary. Nigeran is synthesized de novo from exogenous glucose and accounts for about 20% of the glucose transported by the mycelium. The polymer is diluted out but not degraded when the mycelium is transferred to fresh medium containing NH(4) (+). Cycloheximide prevents development of the nigeran biosynthetic system. The addition of substrate levels of NH(4) (+) to nigeran-accumulating mycelium immediately suppresses further nigeran buildup, presumably by diverting glucose to the biosynthesis of amino acids, nucleotides, etc.     

Effect of labuctril 25 and three other herbicides on wild soil isolates and mutant strains of streptomycetes

Labuctril 25 (3,5-dibromo-4-hydroxybenzonitrile, LAB) at concentrations up to 100μg/mL inhibits effectively growth, morphology, and pigmentation of most soil streptomycete isolates grown under laboratory conditions. Oxytril CM (OXT), Basagran (BAS) and Faneron 50 WP (FAN) applied at the same concentrations had no detectable effect on growth of substrate mycelium but suppressed both aerial mycelium and pigment formation, the effectivity decreasing in the order OXT—BAS—FAN. The LAB-sensitivity of mutant strains was markedly higher as compared with that of the soil isolates. A wild strain resistant to 100–400μg of LAB per mL (depending on the medium composition) was isolated. It was capable of supporting the growth and development of sensitive strains on the LAB-containing medium. A stimulatory effect of low doses of LAB (10–20μg/mL) on the antibiotic activity of streptomycetes was observed.     

Mitotic activity in cells of the wool follicle bulb

Mitotic activity in the cells of the germinative region of wool follicle bulbs was quantified by using small (0.1-0.5 ml) intradermal doses of colchicine and selective staining of the metaphase-blocked nuclei using either crystal violet, iodine and eosin or haematoxylin and eosin. The number of metaphase nuclei present 3 h after colchicine administration increased with colchicine dose from 0 to 1 microgram and thereafter remained relatively constant up to 200 micrograms colchicine. The accumulation of metaphase nuclei was linear for up to 6 h after intradermal colchicine. The metaphase-blocking effect of intradermal colchicine was confined to a radius of less than 5 cm from the injection site, allowing a number of estimates of mitotic rates to be made over a small area of skin. Such estimates revealed little variation in mitotic activity over the midside region of the sheep, although there were substantial differences in follicle activity at different sites over the body. The technique is simple, allows serial or concurrent estimates of mitotic activity to be made in the same animal, and eliminates problems associated with intravenous colchicine administration. It was used to derive the relationship between follicle activity and fibre production after nutritional changes, and to define the time course of mitotic events after administration of the antimitotic defleecing agent cyclophosphamide.     

Morphological and biosynthetic activity of Trichothecium roseum in relation to the polynuclear conidia of the fungus]

The effect of the number of the nuclei on the morphological and synthetic variability was studied with the conidia of Trichothecium roseum. Treatment of suspensions of the conidia with 0.5--1.0 percent solutions of colchicine during two hours increased the number of the nuclei in the conidia, induced the formation of giant colonies, and augmented the ability to produce antibiotics. No reliable differences have been found in the levels of proteolytic activity between the control strains and the strains treated with colchicine.     

Media for cultivation of indoor streptomycetes

The growth of 10 indoor Streptomyces spp. isolates on nutritionally complex and selective 26 media revealed that the mycelium production had a tendency to increase in order: starch-casein < glycerol-arginine < glucose-tryptone, and NH(4)NO(3) < Na-caseinate-asparagine. Yeast extract increased mycelium biosynthesis, but not always the growth rate. The strains belonging to streptomycetes most common environmental isolates produced visible mycelium in 5 days on all media.