Postradiation effect of caffeine on the producer of mycoheptin, Streptoverticillium mycoheptinicum]

The modifying effect of caffeine on irradiated spores of Streptoverticillium mycoheptinicum, producing mycoheptin was found. Postradiation treatment of the strain O883: spores with caffeine resulted in decreased survival of the spores proportionally to the radiation dose increase and postradiation caffeine treatment. An increase in the frequency of the morphologically changed colonies, as well as the low and highly active variants with respect to mycoheptin production was observed. The effect may be explained by the fact that caffeine possibly inhibited the reparation process in the irradiated spores of the strain tested. The method of postradiation treatment of the spores of the mycoheptin-producing organism with caffeine which provided selection of highly active variants by the antibiotic production with the use of definite doses may be considered promising in selection of actinomycetes.     

Lethal and mutagenic action of N-nitroso-N-methylbiuret on the producers of levorin, amphotericin and mycoheptin]

The lethal and mutagenic effect of N-nitrozo-N-methylbiuret (NMB) on the organisms producing levorin, amphotericin B and mycoheptin was studied. The mutagen effect depended on the dose, culture and physiological state of the spores. NMB had a low mutagenic effect on the levorin-producing organism characterized by high activity and genetic homogenicity with respect to the colony morphology and antibiotic production. As for the organisms producing amphotericin B and mycoheptin characterized by high genetic heterogenicity, significant variation of all the features studied was observed on their exposure to the mutagen. Inspite of diverse reaction of the organisms producing levorin, amphotericin B and mycoheptin to the effect of NMB mutants with increased antibiotic production were obtained from the three cultures. The lethal and mutagenic effect of NMB on the mycoheptin-producing organism depended on the process of the spore DNA replication. The spores during the DNA replication period were least sensitive to the lethal effect of the mutagen and most mutable with the respect to the colony morphology. For selection of highly active and stable strains exposure to NMB of the spores of the mycoheptin-producing organism during replication of DNA proved to be more effective than that of the spores during the lag-phase.     

Mutagenic effect of mitomycin C on different strains of oleandomycin producer Streptomyces antibioticus

Mitomycin C, a DNA-tropic antibiotic, was shown to have a lethal effect on spore sprouts of two strains of Streptomyces antibioticus, an organism producing oleandomycin. When the time of exposure to the antibiotic increased there was an almost equal decrease in the survival rate. The mutagen action on the morphological variation and antibiotic production of the two closely related strains were diverse due to their genetic differences. The strain isolated after the culture treatment with a chemical mutagen and subjected to a more prolonged maintaining selection showed lower variation with respect to its colony morphology. The other strain isolated after treatment of the culture with high concentrations of its own antibiotic showed lower variation with respect to its antibiotic production property. The shift in the antibiotic production in the direction of the low active variants was characteristic of the both highly productive strains.     

Pleiotropic effect of the mutation of streptomycin resistance in Micromonospora purpurea var. violacea]

Variation of different features of populations of streptomycin-sensitive and streptomycin-resistant forms of M. purpurea var. violacea, an organism producing gentamicin was studied. The population of the initial streptomycin-sensitive culture was characterized by high homogeneity with respect to the cultural, morphological and some physiological properties. The variation of the features, such as the colony size, pigment formation, auxotrophic mutations, antibiotic production significantly increased in populations grown on media with streptomycin. Mutants differing from the initial strain by a complex of cultural, morphological and physiological features and in particular the antibiotic production were isolated from populations of the streptomycin-resistant variants.     

Selective action of novobiocin on Act. levoris, the producer of levorin and levoristatin]

The effect of sodium novobiocin on Act. levoris, strain LIA 0868 producing levorin and levoristatin was studied. High lethal effect of the antibiotic on Act. levoris was found. The effect increased with a rise in the antibiotic concentration in the agar from 2 to 6 lambda/ml. The inhibitory effect of novobiocin on Act, levoris was evident from a marked increase in the number of morphologically changed colonies with a low level of levorin production. The selective effect of novobiocin on the organism producing levorin and levoristatin was evident from selection of variants with high levels of levoristatin production on media containing novobiocin.     

Selection of the optimal conditions for the procurement and regeneration of protoplasts of the industrial strain of Streptomyces rimosus, the producer of oxytetracycline, and the effect of the protoplasting process on the antibiotic activity]

Optimal conditions for protoplasting of the Streptomyces rimosus industrial strain No. 1 producing oxytetracycline were developed. Observation of the early stages of the protoplast regeneration in microchambers showed that there were two regeneration types: normal and anomalous. The latter was likely defined by the glycine effect on cell wall synthesis. It was accompanied by the stage in which the protoplasts had the form of multiplying protoplast-like cells. The protoplasting of the S. rimosus culture producing oxytetracycline resulted in an increase in the variability of an antibiotic producing property and the frequency of low active variants.     

Streptomycin resistance in chloramphenicol-producing strains of Streptomyces species 3022a.

Resistance to low (5 mug/ml) concentrations of streptomycin in agar media was not inherited by all of the surviving population. Outgrowth of cultures in liquid media supplemented with the antibiotic depended upon inoculum size. Antibiotic titers in the supplemented cultures decreased during incubation, and an inactive radioactive product was detected when [14C] streptomycin was used. This low-level resistance is, therefore, attributed to enzymic inactivation of the antibiotic. Growth 10 mug/ml or higher concentrations of streptomycin on agar media was due to selection of resistant variants present in the parent strain. A range of such variants existed, decreasing in frequency as their degree of resistance increased. Examination of one that was resistant to moderate concentrations of streptomycin, (25 mug/ml) and a second that was resistant to high (100 mug/ml) concentrations of streptomycin suggested that both possed ribosomes which had lower affinity for the antibiotic than those of the parent strain, and that tolerance to high levels of streptomycin was due to a resistant ribosomal system for protein biosynthesis.     

Mutagenesis in Streptomycete cultures exposed to bleomycin

Mutagenic properties of bleomycin, an antitumor antibiotic were studied with respect to 2 species of streptomycetes producing practically important antibiotics. A multifold increase in the frequency of prototrophic revertants among the survivors of strains His- and Met- of Actinomadura carminata exposed to bleomycin was observed. Bleomycin was effective in induction of various morphological mutants, and auxotrophs at a high survival rate of the spores of Str. cremeus var. tobramycini, a tobramycin-producing organism. It was shown with the method of subsequent mutagenesis that the efficacy of induction of morphological and auxotrophic mutants in germinating spores of Actinomadura carminata, a carminomycin-producing organism by bleomycin in a concentration of 100 micrograms/ml and an exposure time of 5 minutes was much higher that in the latent spores. The mutagenic effect of bleomycin is comparable with that of ionizing radiation.     

Rsistance to streptomycin in a producing strain of Streptomyces griseus.

Streptomyces griseus S 104 was sensitive to streptomycin during exponential growth in a medium which, in the subsequent stationary phase, supported production of the antibiotic in yields above 200 mug/ml. When antibiotic production began cultures developed a tolerance toward their lethal metabolite. This was not due to an increase in pH associated with antibiotic production, since pH effects on streptomycin sensitivity in S. griseus were in the reverse direction. However, the degree of tolerance was directly related to the amount of cell material present. Streptomycin production caused no change in the proportion of resistant variants in the population, nor did it cause the severe inhibition of protein synthesis observed in non-producing cultures exposed to the antibiotic. The lack of an effect on protein synthesis is attributed to the absence of streptomycin with in the cytoplasm since soluble extracts from mycelium harvested in the production phase were inactive when bioassayed immediately after cell disruption. However, they developed antibacterial activity rapidly when heated, and more slowly when incubated at 25 degrees C. The addition of phosphatase inhibitors during incubation prevented the appearance of antibiotic activity, and it was concluded that a small amount of streptomycin phosphate is present in the mycelium during antibiotic production. Differences in (14C) streptomycin uptake suggested that the mycelium was appreciably less permeable to the antibiotic in the production phase than during exponential growth. However, a small amount was taken up and much of it was in the soluble fraction of disrupted cells. Bioassays showed that this 14C-labeled antibiotic within the cells had been partially inactivated, suggesting that conversion of streptomycin to an inactive derivative is involved in the mechanism which protects the organism from its metabolite.     

Studies on a new marine streptomycete BT-408 producing polyketide antibiotic SBR-22 effective against methicillin resistant Staphylococcus aureus

A new actinomycete strain designated as BT-408 producing polyketide antibiotic SBR-22 and showing antibacterial activity against methicillin resistant Staphylococcus aureus has been characterized and found to be a novel strain of Streptomyces psammoticus. Nutritional and cultural conditions for the production of antibiotic by this organism under shake-flask conditions have been optimized. Glucose and ammonium nitrate were found to be best carbon and nitrogen sources respectively for growth and antibiotic production. Similarly initial medium pH of 7.2, incubation temperature of 30 degrees C and incubation time of 96 h were found to be optimal. Optimization of medium and cultural conditions resulted in 1.82-fold increase in antibiotic yield.     

Use of acridine yperite in the selection of Act. nodosus, a producer of amphotericin B]

The effect of acridine-yperite (AY) on Act. nodosus LIA 0861 at various physiological stages of development was studied. It was shown that both the spores and the germs were sensitive to AY. The level of the mutagen effect depended on the exposure time and the physiological state of the Act. nodosus cells during the treatment. The treatment of the spore suspension with AY had an insignificant effect on the strain morphological variation, while the number of the morphologically changed colonies markedly decreased when 5-hour germs were subjected to the mutagen effect. A decrease in the average antibiotic activity of Act. nodosus was observed, when its spores were subjected to the treatment with AY, which was associated with a decrease in the number of the plus variants. The treatment of 5-hour germs increased the average antibiotic activity of the culture which correlated with an increase in the number of the plus variants.     

Use of nystatin for random spore selection in the yeast Saccharomycopsis lipolytica

Nystatin was used to develop a new method to select spores of the yeast Saccharomycopsis lipolytica. At low concentrations nystatin killed preferently growing cells of this yeast. At high concentrations nongrowing cells were affected as well. In contrast, spores were not sensitive to nystatin action. This differential response to the antibiotic suggested its use to select spores from sporulated yeast cultures.     

Action of mitomycin C on the variability of Actinomyces hygroscopicus in forming a proteolytic complex of hygrolytin enzymes and the antibiotic, hygromycin B]

The lethal and mutagenic effect of mitomycin C in doses of 10 and 15 micrograms/ml on the spores and 24-hour culture of Act. hygroscopicus, strain O878 producing hygrolytin, a proteolytic enzyme and hygromycin B, an antibiotic was studied. It was found that mitomycin C had a high lethal effect on the organism. The lethal effect of the antibiotic depended on the stage of the culture development, mitomycin C dose and exposure time. The 24-hour culture was most sensitive to the effect of mitomycin in a dose of 50 micrograms/ml. Exposure to mitomycin increased the actinomycete variation with respect to the colony morphology and induction of new morphological mutations. Exposure of strain O878 to mitomycin C significantly increased the culture variation with respect to the quantitative features of production of the hygrolytin proteolytic enzyme complex and hygromycin B. The character of the strain induced variation with respect to the features studied was different which indicated the absence of correlation between them. The use of mitomycin C proved to be promising in selection of Act. hygroscopicus with a purpose of increasing the culture proteolytic and antibiotic activity.     

Effect of gramicidin C on the formation and germination of Bacillus brevis var. GB (P+-variant) spores]

The effect of gramicidin C added to the medium at various periods of cultivation in concentrations of 20, 40 and 100 gamma/ml on sporulation of P+-variant of Bac. brevis var. GB was studied. The most effective increase in the sporulation rate and percentage of the cells germinating into the spores was observed on addition of the antibiotic to the medium in amounts of 20 and 40 gamma/ml in 13 hours of the culture development. The amount of gramicidin C during sporulation decreased and partially passed into the spores which did not differ after germination from those of P+-variant grown on the synthetic medium with glucose and without preliminary addition of the antibiotic. Addition of gramicidin C in an amount of 100 gamma/ml at the end of the lag phase, i.e. 4 hours after the culture inoculation suppressed sporulation and had no effect on growth of the cells of its own producing organism.     

Developmental cycle and the cytomorphological characteristics of the oleandomycin producer Streptomyces antibioticus

The developmental cycle and cytomorphological features of the industrial strain OL-1 and its variant 0968 of the oleandomycin-producing organism were studied. Variant 0968 was obtained as a result of exposure of the spores of strain OL-1 to UV light. When grown under submerged conditions in flasks with the rich medium, the strains were characterized by a complete developmental cycle consisting of three generations of the hyphae. Every generation had a tendency for formation of submerged spores. The UV-induced variant differed from the industrial strain by higher levels of the antibiotic accumulation which correlated with higher rates of the spore germination. The strains were characterized by polymorphism of the mycelium and formation of submerged spores during their cultivation which is likely to prolong the antibiotic synthesis from 120 to 216 hours from the inoculation moment. The long-term selection of the oleandomycin-producing organism on the rich medium markedly changed the culture genotype and resulted in significant changes in the developmental cycle under submerged cultivation conditions. The data may be used for the microscopic control of the process of oleandomycin production.     

Actinomyces streptomycini mutants blocked in the biosynthesis of the streptobiosamine portion of the streptomycin molecule

Four low active mutants of Act. streptomycini were obtained under the effect of nitrozo-methylbiuret. The mutants increased their antibiotic production level, when streptobiosamine was present in the formentation medium. It was shown that in 3 out of the 4 mutants the highest antibiotic yield was in the medium containing streptobiosamine in an amount of 1 mg/ml and in 1 strain the highest yield was in the medium with 0.5 mg/ml of streptobiosamine. When the mutants were grown in a medium containing N-methyl-L-glucosamine no increase in streptomycin production was registered.     

Lyophilization of the spores and vegetative cells of Bacillus brevis var. G-B.--producer of gramicidin S

Viability, antibiotic properties and variation of 4 variants of Bac. brevis var. G.-B. were studied after lyophilization and storage for a year in the lyophilized state. It was shown that the spores and vegetative cells of S and P- variants not synthesizing gramicidin S were somewhat more stable than the spores and cells of R and P+ variants producing the antibiotic. The latter dissociated by 10 per cent towards the cells producing and not producing gramicidin. The developmental rate of the lyophilized vegetative cells was higher than that of the lyophilized spores. Under analogous cultivation conditions they produced higher amounts of the biomass and antibiotic. The lyophilization method described may be recommended for the maintenance of viability and stability of the spores and vegetative cells of Bacillus brevis var. G.-B. producing gramicidin S.     

Obtaining a productive strain of Streptoverticillium griseocarneum var. bleomycini that yields the new antineoplastic antibiotic, bleomycetin

Selection of the organism producing bleomycin, a multicomponent antitumor antibiotic, was performed. The aim was the selection of a more active strain with preferable synthesis of component A5 (bleomycetin) of the bleomycin complex. Optimal variants of the conditions for mutagensis with UV light and gamma-rays in step-wise selection of the active strain were determined and a possibility of selecting analog-resistant mutants with the use of structural analogs of the metabolites participating in synthesis of the bleomycin molecule was studied. A mutant analog-resistant strain capable of supersynthesis of bleomycin A5 (bleomycetin) was selected, the biosynthetic capacity of Streptoverticillum griseocarneum var. bleomycini being increased at least 19 times.     

Biosynthesis of Streptomycin

Investigation was carried out on demonstration of two substances constructing a precursor system located at a late stage of streptomycin biosynthesis by Streptomyces griseus. One of them is thought to be a natural precursor of Streptomycin(L) and the other is suggested as an enzymatic substance(H) transforming L to streptomycin. Both substances had no antibiotic potency and H was inactivated at low pH. L was obtained from a cell-free supernatant (active supernatant) prepared from suspension of young mycelium of Streptomyces griseus in glucose solution. H was obtained not only from active supernatant but also from cell-free extract of the organism.

Two ways of isolation were established for L. Active supernatant was adsorbed on a CM-cellulose column equilibrated with 0.05 m Tris-maleate buffer (pH 8.0). Elution of this column with the same buffer as was used for equilibration gave L-containing fraction separated from streptomycin which was eluted with the buffer including 1% of sodium chloride. L was adsorbed also on active carbon in aqueous solution at neutral pH and liberated from it at acidic pH with 95% methyl alcohol. The former method was useful to separate L from streptomycin, and the latter one was so to concentrate L.

H was isolated by using a column chromatography on DEAE-cellulose. After adsorbing active supernatant or cell-free extract of organism on a column equilibrated previously with the same buffer as above, H was eluted with the buffer including 1% of sodium chloride. Cell-free extract of S. griseus was a better source of H supply than the active supernatant.     

Polymorphism of a culture of Actinomyces chromogenes var. trienicus, the producer of chromotrienine]

Variation of Actinomyces chromogenes var. trienicus 141-18 MSU, an organism producing trienin was studied under laboratory conditions. Nine stable spontaneous variants were isolated from the population of the initial culture when grown on Gause medium No. 1. The variants varied in differentiation and biosynthetic capacity, including such characteristics as size and form of the colonies, ability for formation of the aerial mycelium and its colour, capacity for sporulation, form of the spore chains and antibiotic production property. In the secondary structures the spores formed only in 6 variants out of 9 isolates. The spore form and spore membrane surface were close in all sporogenic variants, while there were significant differences in the structure of the sporophores. The variants forming the aerial mycelium of the same colour as that of the initial culture did not differ from it also by the nature of the spore chains (spirals with 3--8 turns). The variants with lighter aerial mycelium than that of the initial population formed straight sporophores or spirals with a small number of the turns (1--3). The comparative study of the antimicrobial spectrum of the variants and the component composition of the synthesized antibiotic complex showed that the asporogenic variants and dwarf variant signifcantly differed with respect to their phenotypes from the other cultures and had no antagonistic action. One of the assporogenic variants had only insignificant activity. All the spore forming variants did not differ from the initial culture in the complex of the antibiotics synthesized.