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.     

New antibiotic, parvulomycin, produced by a culture of Actinomyces parvullus var. chromogenes var. nov]

Actinomycete LIA-O784 was isolated from a soil sample. By its morphological and cultural properties the isolate was close to Act. parvullus but differed from it in synthesis of melanoid pygment, thyrosinase, hydrogen sulphide and pronounced antifungal activity. The actinomycete was classified as a new variant and designated as Actinomyces parvullus var. chromogenes var. nov. The culture produced a new polyglycoside antibiotic named parvulomycin. The physico-chemical characteristics of the antibiotic is presented.     

Characteristics of the intrinsic luminescence of Actinomyces olivocinereus--producer of heliomycin]

Some characteristics of UV-induced luminescence were studied with Actinomyces olivocinereus producing the antibiotic heliomycin. The luminescence of the growth medium was found to be caused not by heliomycin, but by some other factors. The luminescence of heliomycin in the colonies was quenched as a result of its screening with melanin pigments located in a layer between the aerial and substrate mycelium.     

Natural variability of Actinomyces lavendulae, a producer of cholesterol oxidase

Five variants of Actinomyces lavendulae differing in the morphology of their colonies were found when natural variability of this organism was studied. A correlation was established between the colony morphology of the variants and their activity of cholesterol decomposition. The variants forming colonies of the basic and folded type had the highest activity. A variant with an elevated activity of cholesterol oxidase was selected. In order to maintain the high activity of the culture, it is necessary to examine its variability and to select variants corresponding to the basic type in their morphology.     

Control of actinomycin D biosynthesis in Streptomyces parvullus: regulation of tryptophan oxygenase activity

Tryptophan oxygenase (tryptophan 2,3-dioxygenase) activity increases immediately before the initiation of actinomycin D production by Streptomyces parvullus. We have attempted to discern whether this increase is due to a release from catabolite repression or to the synthesis of an inducer substance. The standard culture medium (glutamic acid-histidine-fructose medium) used in antibiotic production studies with S. parvullus contains l-glutamate as a major constituent. l-Glutamate is almost totally consumed before the onset of actinomycin D synthesis. The addition of 10 mM l-glutamate at this stage completely abolished actinomycin D production as well as tryptophan oxygenase synthesis. Fourteen amino acids were tested for a similar effect. Of these, l-glutamate and l-aspartate had the most dramatic effect on tryptophan oxygenase and beta-galactosidase (beta-d-galactosidase), another inducible enzyme. Standard glutamic acid-histidine-fructose medium, preincubated for 23 h to remove l-glutamate, allowed the synthesis of actinomycin D and tryptophan oxygenase by cells at a stage of growth normally considered too early for antibiotic production. A chemically defined medium lacking l-glutamate and adjusted to pH 8.0 was designed to simulate the preincubation medium. The transfer of cells to this artificial preincubation medium resulted in the appearance of tryptophan oxygenase as early as 19 h before normal synthesis occurred, eliminating the possibility that an inducer molecule is synthesized and excreted during the preincubation period. The results of these studies suggest that the increase in tryptophan oxygenase activity before the onset of actinomycin D synthesis, as well as the synthesis of actinomycin D itself, is due to a release from l-glutamate catabolite repression.     

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.     

Use of antibiotics in selecting a nystatin producer]

The lethal and mutagenic effect of streptomycin and nystatin on Act. noursei, strain 408 producing nystatin was studied. The survival of the spores of strain 408 on the medium with streptomycin decreased with an increase in the antibiotic concentration. Streptomycin had a selective effect on the nystatin-producing organism decreasing the frequency of morphologically changed and low active variants and revealing highly active and antibiotic stable variants. The survival of the spores of strain 408 on the medium with nystatin (20,000 units/ml) amounted to 35 per cent. Nystatin had an inhibitory effect on the organism producing it which was evident from delayed growth and significant modification variation of the colonies, as well as from a marked increase in the number of the variants characterized by low antibiotic production.     

Growth limitations and biosynthesis of gramicidin in Bacillus brevis var. G.-B

Gramicidin S biosynthesis was studied in Bacillus brevis var. G.-B. during its batch and continuous cultivation when the culture growth was limited with nutrient sources (glycerol, ammonium nitrogen, phosphate), oxygen deficiency and the action of a physical factor (a low temperature). The antibiotic biosynthesis was shown to be induced by a change in the growth rate caused by the action of any factor decelerating the growth. The authors propose a mathematical model for the antibiotic synthesis, biomass accumulation and the utilization of a substrate limiting the growth. The model is based on the age separation of cells. The model is analyzed in terms of optimizing the one-stage continuous cultivation process. The model allows one to calculate optimal conditions of the antibiotic synthesis in the process of one-stage continuous cultivation.     

Polymorphism of a culture of the chromomycin producer, Actinomyces aburaviensis var. verrucosus]

Polymorphism of the chromomycin-producing orgnaism Act. aburaviensis var. verrucosus 144--3 was studied. The stable variants differed in the morphologo-cultural properties, assimilation of the carbon sources, the component composition of the luminescence substances and the quantitative property of the antibiotic production. A substance with violet luminescence was found in the culture of varient I in addition to chromomycin. Similarity of the phenotypes observed in variants 1 and 2 when grown on complex organic media is explained by intensive chromomycin production by variant 1 on such media. Active colonies may be selected according to the colour due to chromomycin. Forms with an activity almost 4 times higher than that of the initial culture were found among the colonies of variant 2.     

Spontaneous variability of antibiotic biosynthesis in relation to morphogenetic processes in Actinomyces chromogenes var. trienicus]

Available data on possible relationship between the antibiotic activity of actinomycetes and the level of their differentiation, especially with their spore-formation ability, present certain interest with respect to possible relationship between the synthesis of antibiotics and the formation of secondary structures. The study of spontaneous stable variants of Actinomyces chromogenes var. trienicus demonstrates that all sporogenous variants produce the same complex of antibiotics as does the original population. The loss of the ability to synthesize antibiotics is observed only in the phenotypically different dwarf variant (VI). The impaired differentiation (the loss of spore-formation ability) is accompanied by disturbances in the antibiotic synthesis: asporogenous variants are either inactive or produce only 1 antibiotic from the complex synthesized by the original population. Changes in the structure of spore chains do not probably correlate with qualitative and quantitative measurable changes in the antibiotic synthesis. The statistic evidence is suggestive of the fact that the variant with a more complex profile and topography of aerial mycelium displays a higher activity.     

Influence of the culture medium on the production of iturin A by Bacillus subtilis

The production of iturin A by Bacillus subtilis was studied with respect to the composition of the culture medium. Increasing phosphate concentrations did not modify the antibiotic yield. Fructose, sucrose and mannitol were better carbon sources than glucose for antibiotic production. The nature of the nitrogen source was an important factor in the production of antibiotic. Among the amino acids which are components of iturin A, L-asparagine was the best substrate for the biosynthesis of iturin A; L-glutamine and L-serine were rather poor substrates while L-proline and D-tyrosine gave no antibiotic. Ammonium salts permitted good synthesis of antibiotic but the addition of calcium ions to the culture medium inhibited the excretion of antibiotic from the cells.     

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.     

Kinetics of Streptomyces baarnensis growth and antibiotic synthesis in batch and dialysis cultures

The kinetics of biomass and antibiotic formation in batch and dialysis culture of Streptomyces baarnensis at various initial concentrations limiting the substrate growth (glucose) has been studied. The antibiotic substances were synthesized by actively growing culture, its concentration in the cultural media was maximum in the log-phase. In continuous dialysis culture on the background of biomass lianer growth in the course of time the constant antibiotic concentration in the media proportional to the glucose input concentration has been established. The inactivation (decomposition) of antibiotic was immediately initiated after discontinuation of substrate supply and followed first kinetics order. Observed features were used for construction of kinetical model of antibiotic biosynthesis. A conclusion has been made that the dialysis culture gives opportunity for more effective antibiotic synthesis as compared with the batch one.     

Actinomyces rimosus resistance to oxytetracycline

High frequency of spontaneous and UV-and acridine dye-induced variants susceptible to oxytetracycline (OTC) and deprived of the capacity for synthesizing this antibiotic was observed in strain LST-118 of Actinomyces rimosus. The cells of strain LST-118 of Act. rimosus contained extrachromosomal DNA not found in its OTC susceptible variant BS87, which provides evidence in favour of participation of the extrachromosomal genetic elements in control of OTC resistance of the cells of Act. rimosus, LST-118. The OTC resistance in strain LST-118 is of inducable character. The resistance level is increasing from the beginning of the antibiotic synthesis and initially the subinhibitory concentrations of OTC in the medium were the inductors triggering cellular mechanisms ensuring resistance of the cell to the increasing concentrations of OTC in the medium. The capacity for absorption of OTC in Act rimosus is 2--3 times lower than that in E. coli. The experiments with labeled tetracycline showed that the cells of the actinomycete absorbed OTC when it was present in the medium. The absorption of the main amount of the antibiotic was registered during the first 5 minutes. The difference in absorption of OTC by the cells of the antibiotic resistant and sensitive strains was insignificant.     

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.     

Influence of cultivation conditions on the synthesis of trypsin inhibitor in the submerged culture of Actinomyces janthinus 118

The influence of mass exchange and cultivation temperature on the synthesis of trypsin inhibitor in the submerged culture of Actinomyces janthinus was studied. Mass exchange parameters of the fermenter varied from 0.72 to 3.7 g O2 l/hr without oxygen limitations, and cultivation temperature ranged from 25 to 34 degrees C. The growth pattern, dynamics of substrate consumption and synthesis of trypsin inhibitor by the culture of Act. janthinus were shown to depend on mass exchange and cultivation temperature. With an increase in mass exchange the inhibitory activity reached maximum earlier but did not rise in its absolute value. With a temperature increase the inhibitory activity grew by 65%.     

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.     

The induction of antibiotic synthesis in Saccharopolyspora erythraea and Streptomyces hygroscopicus by growth rate decrease is accompanied by a down-regulation of protein synthesis rate

Abstract The relationship between antibiotic production and culture growth rate in Saccharopolyspora erythraea and Streptomyces hygroscopicus was manipulated by changing the growth-limiting substrate. Carbon- and nitrogen-limited cultures were studied and antibiotic synthesis was obtained in both cases in Saccharopolyspora erythraea cultures and in nitrogen-limited Streptomyces hygroscopicus cultures. In all cultures where antibiotic was detected, onset of antibiotic production coincided with the minimal protein synthesis rate. Further investigation in Saccharopolyspora erythraea cultures indicated that this corresponded to minimum ratio of charged to uncharged tRNA, i.e. when uncharged tRNA accumulated. This latter phenomenon was investigated in the presence of a protein synthesis inhibitor.     

Lysogeny in heat-tolerant hydrocarbon-oxidizing actinomycetes of species Actinomyces griseomycini]

Six strains of thermophilic hydrocarbon-oxidizing Actinomyces griseomycini were tested for lysogeny. The lysogenic state was detected in the four strains and their phages were isolated. The phages isolated from the three strains were virulent and cause lysis of the host culture. All isolated phages were specific and did not cause lysis of other actinomycetes species. However, the phages had different activity towards the six studied strains of Actinomyces griseomycini. One phage induced lysis only of its indicator culture, other phages caused lysis of four strains, including those two from which no phage was isolated. All phages produced negative colonies of identical morphology. The morphology of their particles was also the same. The phages induced cross-resistance and were characterized by thermoresistance.     

Preparation and characteristics of protoplasts from various strains of Streptomyces roseoflavus var. roseofungini]

It was shown that the variants of the roseofungin-producing organism, which differ in their differentiation, antibiotic activity, structure and cell wall composition had different sensitivity to the protoplasting factors. The protoplasting increased the population heterogeneity: in strain 1128 it was evident from an increased frequency of the secondary colonies, in variant 1-68, folding of the colonies increased and their consistency become milder, sectorial colonies and colonies with coremia formed. It was in principle possible to transform the protoplasts of S. roseoflavus var. roseofungini by plasmid DNA, which suggests that the roseofungin-producing culture may be useful in genetic engineering.