Variability of the levorin producer, Actinomyces levoris Krass]

Natural variation of the levorin-producing organism Act. levoris, strain 28 was studied with respect to the colony morphology and production of levorin and levoristatin. The population of strain 28 consisted of 3 morphological colony types, the main type amounting to 99.7 percent. The strain variation with respect to production of levorin and levoristatin ranged from 20 to 180 and from 0 to 300 percent respectively as compared to the control. Mutant M-28 differing from the initial strain by the colony morphology, moderate phage titer and preferable production of levoristatin was isolated as a result of repeated passages of strain 28 onto agarized Chapek media with starch without maintaining selection. Variants differing from the population of strains 28 and M-28 by the ratio of levorin and levoristatin in the culture fluid were selected. No correlation in production of the above antibiotics by strain 28 was noted. Preparations obtained with strain M-28 differed from those obtained with strain 28 in a significant content of levoristatin.     

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.     

Biosynthesis of the polyene macrolide antibiotic nystatin in Streptomyces noursei

The polyene macrolide antibiotic nystatin, produced commercially by the bacterium Streptomyces noursei, is an important antifungal agent used in human therapy for treatment of certain types of mycoses. Early studies on nystatin biosynthesis in S. noursei provided important information regarding the precursors utilised in nystatin biosynthesis and factors affecting antibiotic yield. New insights into the enzymology of nystatin synthesis became available after the gene cluster governing nystatin biosynthesis in S. noursei was cloned and analysed. Six large polyketide synthase proteins were implicated in the formation of the nystatin macrolactone ring, while other enzymes, such as P450 monooxygenases and glycosyltransferase, were assumed responsible for ring decoration. The latter data, supported by analysis of the polyene mixture synthesised by the nystatin producer, helped elucidate the complete nystatin biosynthetic pathway. This information has proved useful for engineered biosynthesis of novel nystatin analogues, suggesting a plausible route for the generation of potentially safer and more efficient antifungal drugs.     

Some characteristics of using polyalcohols as cryoprotectors in preserving Actinomyces noursei LIA-0471]

Freezing of Act. noursei cell suspensions at a rate of 0.5 degrees per minute in the presence of various substances showed that 5 per cent concentrations of glycerol, ethylen-, diethylen-, propylenglycol and polyethylenglycols had a cryoprotective effect. Polyethylenglycols with a molecular weight of 1500-20 000 had the highest protective effect on the actinomycetous cells from the damaging action of repeated freezing and thawing. The method of repeated freezing and thawing is recommended for studying the cryoprotective effect of various compounds.     

Spontaneous and induced variability in Actinomyces rubiginosohelvolus, a new producer of the antibiotic rubomycin]

Mutants possessing 3 times higher activity as compared to the initial culture were obtained as a result of selection of active variants of Act. rubiginosonelvolus, a new organism producing rubomycin against the background of variation induced by N-methyl-N1-nitro-N-nitrozoguinidine, diethylsulphate and their combinations. Further selection among the mutants stable to 6-mercaptopurine and riboflavine resulted in obtaining an active culture No. 3912 at least 7 times more active with respect to rubomycin production than the initial soil culture.     

Study of the carbohydrate metabolic enzyme activity of Act. noursei]

Activity of aldolase and threosophosphate dehydrogenase, transketolase and phosphogluconate dehydrogenase in Act. noursei, strain 153 and its inactive mutant 149 was studied comparatively. The enzyme activity of the inactive mutant was investigated in the absence of the antibiotic production and under conditions of reduced biosynthesis of nystatin in this strain after addition of the fermentation broth filtrate of the inactive mutant 369 to the medium. The activity of the enzymes of the hexosomonophosphate metabolic pathway in the active strain 153 of Act. noursei was 2-4 times higher than that of the inactive mutant 149. The activity of the enzymes of the hexosomonophosphate metabolic pathways increased and reached the level of the enzyme of the active mutant. The high level of the enzyme activity of the hexosomonophosphate glycolysis pathway is probably one of the necessary conditions for nystatin production.     

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.     

Site-specific mutagenesis and domain substitutions in the loading module of the nystatin polyketide synthase,and their effects on nystatin biosynthesis in Streptomyces noursei

The loading module for the nystatin polyketide synthase (PKS) in Streptomyces noursei is represented by the NysA protein composed of a ketosynthase (KS(S)), acyltransferase, dehydratase, and an acyl carrier protein. The absolute requirement of this protein for initiation of nystatin biosynthesis was demonstrated by the in-frame deletion of the nysA gene in S. noursei. The role of the NysA KS(S) domain, however, remained unclear, since no data on the significance of the "active site" serine (Ser-170) residue in the loading modules of type I PKSs were available. Site-specific mutagenesis of Ser-170 both in the wild-type NysA and in the hybrid loading module containing malonyl-specific acyltransferase domain from the extender module had no effect on nystatin biosynthesis. A second mutation (S413N) of the NysA KS(S) domain was discovered that completely abolished the ability of the hybrids to restore nystatin biosynthesis, presumably by affecting the ability of the resulting proteins to catalyze the required substrate decarboxylation. In contrast, NysA and its Ser-170 mutants bearing the same S413N mutation were able to restore nystatin production to significant levels, probably by using acetyl-CoA as a starter unit. Together, these data suggest that the KS(S) domain of NysA differs from the KS(Q) domains found in the loading modules of several PKS type I systems in that the active site residue is not significant for its activity.     

Stability of quaternary structure and mode of dissociation of fructosediphosphate aldolase isoenzymes

Using a highly sensitive "subunit exchange" assay, we have studied the relative strengths of interactions between different subunit types (A and C) of fructosediphosphate aldolase and have determined the mode of dissociation of aldolase tetramers in vitro. Interactions between C subunits within C4 tetramers were found to be considerably more resistant to disruption than were interactions between A subunits in A4 tetramers with regard to increasing concentrations of H+, OH-, or urea. Slight dissociation of A4 was also observed in 1.2 M magnesium chloride. These observations suggest that the quaternary structure of aldolase C4 is inherently more stable than that of aldolase A4. Also, the symmetrical heterotetramer A2C2 was found to be more resistant to urea-mediated dissociation than was the aldolase A4 homotetramer; this observation suggests that, even when in heteromeric combination, C subunits have a stabilizing influence on the quaternary structure of aldolase tetramers. In no case did we find evidence for a stable dimeric intermediate in the dissociation of aldolase tetramers to monomers. These observations are considered in terms of the tetrahedral arrangement of subunits in the aldolase tetramer. The general applicability of the subunit exchange assay described here for studying the subunit structure and mode of dissociation of oligomeric enzymes is discussed.     

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.     

Auxotrophic mutants in the selection of the rubomycin producer, Actinomyces coeruleorubidus

A total of 351 auxotrophic mutants with different antibiotic activity, including several mutants with activity higher than that of the parent prototrophic strains were obtained under the effect of gamma-rays from 3 prototrophic strains of Act. coeruleorubidus. It was shown that most of the auxotrophic mutants did not preserve the property of biochemical insufficiency on passages on complete media. A mutant strain 1059-32 with activity 2 times higher than that of the prototrophic strain 2-39 and the parent auxotrophic culture was obtained from the revertants. Requirements in 29 growth factors including 17 amino acids, 4 nitrous bases, 8 vitamins and coenzymes were determined in 46 stable auxotrophic mutants isolated. The effect of the specific and non-specific growth factors on the culture antibiotic production was studied.     

L-valine transport by the actinomycete Actinomyces species 26-115, the producer of actinomycin C]

Transport of L-valine by Actinomyces species 26-115, an organism producing actinomycin C depended on L-valine concentration in the medium and temperature and required a source of intrinsic energy. Km for L-valine transport was 3.5.10(-6)--6.0.10(-6) M. It somewhat differed from experiment to experiment. The above system transported also other neutral amino acids. L-isoleucine was a competing inhibitor of L-valine transport. The transport of L-valine was stereospecific. The activity of the transport system was regulated by the intracellular content of L-valine. Probably because of this the amino acid transport depended on the culture age, so far as the level of free valine in the mycelium at various stages of development was different.     

Isolation of the membranes of Actinomyces sp. 26-115, a producer of actinomycin C]

A summation fraction of the membranes of Actinomyces sp. 26-115 was obtained as a result of lysis of its protoplasts in a hypotonic medium. The qualitative content of protein, lipids, phospholipids, nucleic acids, glucosamine and muramic acid was determined in the membranes at various stages of the organism development. Phosphatidylcholine is the main component of phospholipids in this organism. Intracellular actinomycin was found inside the protoplasts. Electrophoregrams of the microprotoplasts and membranes are presented. Actinomycin was also detected in the membranes. Still, it is not clear whether it is a component of the membrane or it is adsorbed on the membrane during the process of its isolation. The final conclusion on the relationship between the membrane and localization of actinomycin in the cell requires further investigation.