Taxonomic studies of Streptomyces virginiae mutants overproducing virginiamycin M1.

By using both the traditional International Streptomycetes Project methods and chemical approaches followed by a hierarchical cluster analysis, Streptomyces virginiae mutants A-1 and B-43 (yielding higher amounts of the M1 component of virginiamycin complex), their wild ancestor ATCC 13161, and another virginiamycin producer, S. pristinaespiralis NRRL 2958, were subjected to taxonomic studies to find kinship or differences among the strains. Of the methods used, only the test of carbon utilization, investigation of spore surface and analysis of sugar constituents of cell walls proved to be reliable enough to demonstrate the species identity of S. virginiae strains and to distinguish them from S. pristinaespiralis. L,L-2,6-Diaminopimelic acid was present in all strains. Analysis of fatty acids and total proteins as well as investigations of morphology and pigmentation of agar cultures led to confusing results.

Characteristics of Streptomyces globisporus strain 0234A forming endospores in submerged cultures.

Thermosensitive submerged endospores formed by Streptomyces globisporus 0234 and its natural variant A resembled those of thermoresistant actinomycetes not only in their morphology ultrastructure, but also in the content of dipicolinic acid. The production of endospores containing this substance is unusual in Streptomyces while other features of the strain indicate relatedness to other streptomycetes. Chemotaxonomic analysis of variant A revealed the cell wall to be of chemotype I and fatty acid content typical of Streptomyces. Most characteristics of surface cultures of variant A coincided with those of the original strain 0234 and its endosporeless variant B. Both the strain 0234 and its variants A and B produced identical antibiotics and pesticidal compounds.

Macrotetrolide antibiotics produced by Streptomyces globisporus.

Macrotetrolides isolated from a new producer, Streptomyces globisporus, were identified as nonactin, monactin, dinactin and trinactin. Spectroscopic characterization of these compounds was extended by 13NMR spectra. Chemical ionization with ammonia as reactive gas was proposed for mass-spectroscopic characterization of their mixtures. Their biological activity was confirmed by using larvae of the Colorado potato beetle (Leptinotarsa decemlineata) as a new test model.

Ultrastructural effects of macrotetrolides of Streptomyces griseus LKS-1 in tissues of Culex pipiens larvae.

The isolate of macrotetrolides produced by Streptomyces griseus strain LKS-1 was tested in its effect on the ultrastructure of larvae of Culex pipiens autogenicus. Changes were mainly in mitochondria where the cristae were destroyed and the outer membrane inflated. The endoplasmic reticulum was vacuolized and subsequently the nuclear membranes were seriously affected. Microvilli of the midgut epithelial cells and the surface membrane of these cells were unaltered and there were no changes in the arrangement of cells in the tissues. The effect of macrotetrolides on insect tissues is analogous to the effect of secondary metabolites of fungi such as beauvericin, destruxin E, cyclosporin or tolypin, and differs from the effects of bacterial endotoxins of Bacillus thuringiensis or B. sphaericus.

Streptomycetes producing daunomycin and related compounds: do we know enough about them after 25 years?

The growing need of highly potent anticancer agents has stimulated the investigation of streptomycetes producing daunomycin-type anthracyclines. This review compares the features of production strains and their mutants and emphasizes the necessity of application of biochemical and biophysical analytical methods for better understanding these microorganisms and, above all, their further improving and practical usage.

Regulation of nebramycin biosynthesis by inorganic phosphate.

The effect of inorganic phosphate on the biosynthesis of nebramycin factors 2, 4 and 5' was studied in Streptomyces tenebrarius strain A (forming 2, 4 and 5' in natural ratios) and its mutants B (forming predominantly 2), C (forming 2 as the only major product) and D (forming predominantly 5'). In phosphate-supplemented complex media, the production of 2 in A, B and C was reduced by 20-70%, while the yields of 5' remained unchanged in A and decreased by 30-60% in B. The production of 4 increased by 50-90% in A and was fully suppressed in B. In D the biosynthesis of the three factors was inhibited completely.

Microbial transformation of semisynthetic derivatives of daunomycinone modified in ring A.

Semisynthetic derivatives of daunomycinone with 7,9-isopropylacetal, 7-O-methyl, 7-O-(4-penten-2-yl), and 7-O-(2-hydroxyethyl) substituents were converted by Streptomyces peucetius var. caesius (an adriamycin-blocked mutant) into 7-deoxy-13-dihydrodaunomycinone, while daunomycinone was transformed into 13-dihydrodaunomycinone (predominantly) and 7-deoxy-13-dihydrodaunomycinone. S. coeruleorubidus mutants 24-74 (accumulating aclavinone derivatives instead of daunomycin and related compounds) and 96-85 (producing no anthracycline substances), and S. aureofaciens B-96 (a tetracycline-blocked mutant) transformed the above substrates into the corresponding 13-dihydro derivatives, with the exception of 7,9-isopropylacetal daunomycinone which remained intact. 7-O-Propyn-1-yl daunomycinone was not transformed by any of the strains used under the conditions.

Enzymic degradation of bromoxynil by cell-free extracts of Streptomyces felleus.

A method is described for spectrophotometric monitoring the degradation of the herbicide bromoxynil by cell-free extracts of Streptomyces felleus. The method involves a decrease in absorbance at 286 nm (absorption maximum of bromoxynil) that can be ascribed most probably to the cleavage of the aromatic ring of the bromoxynil molecule. Conditions necessary for measuring this degradation together with physico-chemical features of the degradation indicate that the reaction(s) is seemingly catalyzed by an Fe2+-dependent dioxygenase whose activity was not, however, detected in cell-free extracts of a bromoxynil-sensitive mutant of S. felleus as well as other bromoxynil-sensitive streptomycete strains.

Production of quinomycin A in Streptomyces lasaliensis.

In addition to lasalocid, an oligoether coccidiostatic compound, other compounds are synthesized by Streptomyces lasaliensis. Mutants producing either of two antibiotics, lasalocid A or quinomycin A (an antibiotic of quinoxaline character), were obtained by natural selection and by mutagenesis. Methods of isolation, purification and estimation of both compounds were established.

An antibiotic-overproducing mutant of Streptomyces granaticolor with impaired differentiation.

A thy- tetracycline-resistant mutant of Streptomyces granaticolor was prepared by mutagenesis of the parental strain ETH 7437. The mutant exhibits a different morphology and an overproduction of granaticins. The ability to form an aerial mycelium and spores has been lost. The mutant cells have a round or an atypical shape and a thick cell wall, the membraneous system is enlarged by numerous mesosomes. Division septa are formed rarely. The mutant is more sensitive to both low and high temperature than the parental strain. The altered features are stably maintained for many generations. Ribosomal proteins of the mutant do not differ substantially from those of the original strain indicating that the mutant phenotype is not due to an alteration at the translational level.

Production of rhodomycins in Streptomyces griseoruber 4620.

The strain Streptomyces griseoruber 4620 produces, besides the anthracycline antibiotics beromycins, some other anthracyclines of the rhodomycin type. Twelve isolates exhibiting a higher antibiotic activity (up to 2.5X), as compared to the parent strain, were obtained after a spontaneous selection. The following species were isolated from the hydrolysate of mycelial extract: beta-rhodomycinone, beta-isorhodomycinone, alpha 2-rhodomycinone, epsilon-rhodomycinone and 10-deoxy-beta-rhodomycinone, which has not yet been described.

Spontaneous variability of Streptomyces glomeratus, a producer of the amthracycline antibiotics beromycins.

Spontaneous variants of the beromycin-producing strain Streptomyces glomeratus 3980 were divided into five groups (A-E) according to increasing antibiotic activity. The most active variants (group E) differed from the other types and the wild strain by a suppressed ability to produce aerial mycelium and melanoid piogment and by an increased production of propionic acid. Strains with a 12-fold higher antibiotic production capacity (with respect to strain 3980) were obtained by selection of superior segregants from submerged cultures of the E type.

Effect of cultivation conditions on the activity of the beromycin producer Streptomyces glomeratus 3980 and its spontaneous variants.

Optimal conditions for the submerged cultivation of Streptomyces glomeratus 3980, producer of the anthracycline antibiotics beromycins, and its variants were sought in media with glucose, soybean meal and salts differing in the content of ammonium sulphate. As compared with the original activity of the strain the antibiotic titre of some variants increased about 12 times on increasing the glucose concenration from 3 to 5%, or on omitting CaCO3 from the medium (i.e. under conditions leading to an increased production of propionic acid and suppression of production of the melanin-like pigment). In melanin-less variants accumulating propionate even under standard conditions the activity increased about 18-40 times in the medium with 3% glucose and 0.2% CaCO3 under conditions of more intensive aeration (i.e. under conditions when no propionic acid accumulated). Individual strains also differed in the requirement for (NH4)2SO4 in the medium, their response to changes of volume of the vegetative inoculum and semsitivity to MgSO4 x 7H2O. The biosynthetic activity of all strains was inhibited by the addition of ZnSO4 x 7H2O or CaCl2 and substitution of glucose with starch, lactose or sucrose.

Partial purification and properties of glucosyltransferase from Streptomyces aureofaciens.

Differential centrifugation, precipitation with ammonium sulphate and chromatography on DEAE-cellulose led to a twenty-fold purification of glucosyltransferase from Streptomyces aureofaciens B 96. The Michaelis constants for glucosyluridyl diphosphate (UDP-glucose) was 10.8 microM for 1,2-dihydroxy-9,10-anthraquinone (alizarin) 110 microM; the maximum rate of glucosylation reaction was 5.32 mumol per s per mg protein. The pH optimum was at 7.1; the flat temperature optimum was at 30 degrees C. Using some hydroxy derivatives of 9,10-anthraquinone it was found that the production of glucosides from aglycones with alpha-hydroxyl groups was about 1/8 of the values obtained with beta-hydroxyl substrates. In both types of aglycones the presence of another hydroxyl group led to a higher glucoside production.

Intra- and interspecific cosynthetic activity of mutants of Streptomyces coeruleorubidus and Streptomyces galilaeus impaired in the biosynthesis of anthracyclines.

Cosynthesis of anthracycline compounds was followed in five phenotypic groups of mutants of Streptomyces coeruleorubidus (A--E), blocked in the biosynthesis of the daunomycine complex, and in two mutant types of Streptomyces galilaeus (F, G) blocked in the biosynthesis of glycosides of epsilon-pyrromycinone and aklavinone. Glycosides of daunomycinone and 13-dihydrodaunomycinone were produced in combinations A+B, A+C, A+D, A+E and A+F, epsilon-rhodomycinone was synthesized in combinations A+E, A+F, B+E and B+F. During the cultivation of types B--E with type G or F non-anthracycline compounds, typical of S. galilaeus, were cosynthesized. No cosynthesis could be observed in other combinations of the mutant types. Negative results were also obtained with combinations of mutants of the same group and during cultivation of all mutant types with streptomycetes not producing anthracyclines. A scheme illustrating metabolic pathways leading to the biosynthesis of daunomycinone, aklavinone, epsilon-rhodomycinone in S. coeruleorubidus and S. galilaeus was constructed.

Biotransformations of anthracyclinones in Streptomyces coeruleorubidus and Streptomyces galilaeus.

The ability to transorm biologically exogenous daunomycinone, 13-dihydrodaunomycinone, aklavinone, 7-deoxyaklavinone, epsilon-rhodomycinone, epsilon-isorhodomycinone and epsilon-pyrromycinone was studied in submerged cultures of the following strains: wild Streptomyces coeruleorubidus JA 10092 (W1) and its improved variants 39-146 and 84-17 (type P1) producing glycosides of daunomycinone and of 13-dihydrodaunomycinone, together with epsilon-rhodomycinone, 13-dihydrodaunomycinone and 7-deoxy-13-dihydrodaunomycinone; in five mutant types of S. coeruleorubidus (A, B, C, D, E) blocked in the biosynthesis of glycosides and differing in the production of free anthracyclinones; in the wild Streptomyces galilaeus JA 3043 (W2) and its improved variant G-167 (P2) producing glycosides of epsilon-pyrromycinone and of aklavinone together with 7-deoxy and bisanhydro derivatives of both aglycones; in two mutant types S. galilaeus (F and G) blocked in biosynthesis of glycosides and differing in the occurrence of anthracyclinones. The following bioconversions were observed: daunomycinone leads to 13-dihydrodaunomycinone and 7-deoxy-13-dihydrodaunomycinone (all strains); 13-dihydrodaunomycinone leads to 7-deoxy-13-dihydrodaunomycinone (all strains); daunomycinone or 13-dihydrodaunomycinone leads to glycosides of daunomycinone and of 13-dihydrodaunomycinone, identical with metabolites W1 and P1 (type A), or only a single glycoside of daunomycinone (type E); aklavinone leads to epsilon-rhodomycinone (types A and B); aklaviinone leads to 7-deoxyaklavinone and bisanhydroaklavinone (type C); epsilon-rhodomycinone leads to zeta-rhodomycinone (types C, E); epsilon-rhodomycinone leads to glycosides of epsilon-rhodomycinone (types W2, P2); epsilon-isorhodomycinone leads to glycosides of epsilon-isorhodomycinone (types W2, P2); epsilon-pyrromycinone leads to a glycoside of epsilon-pyrromycinone (types W1, P1). 7-Deoxyaklavinone remained intact in all tests. Exogenous daunomycinone suppressed the biosynthesis of its own glycosides in W1 and P1; it simultaneously increased the production of epsilon-rhodomycinone in P1.

Isolation of ekatetrone, a new metabolite of producing variants of Streptomyces aureofaciens.

From a mixture of substances formed by producing strains of Streptomyces aureofaciens under conditions of submerged fermentation a new metabolite, ekatetrone, was isolated. Its isolation and basic physical and chemical data are described. Ekatetrone is a quinone derivative with a carboxamide group. In tests in vitro with cells of Ehrlich's ascites tumour evidence was provided that ekatetrone inhibits proteo- and nucleosynthesis.

Development mutants of Streptomyces coeruleorubidus, a producer of anthracyclines: isolation and preliminary characterization.

The wild strain Streptomyces coeruleorubidus JA 10092 was found to segregate into two spontaneous morphological variants (spo-1 and bld-1) with a different ability to form aerial mycelium in media with glucose as the main carbon source. Six new types of developmental mutants were obtained from the bald variant bld-1 after treatment with mutagens (UV light, gamma radiation, nitrous acid) and after natural selection. Formation of the aerial mycelium was fully suppressed in the bld-2 type growing on media both with glucose and with starch. The other types were bald only on starch media, forming the aerial mycelium on media with glucose; types spo-2, spo-3, spo-4 and spo-5 differed in size, shape and surface structure of spores, the type whi formed asporogenous aerial hyphae.

7-Deoxy-13-dihydrodaunomycinone in cultures of Streptomyces coeruleorubidus.

7-Deoxy-13-dihydrodaunomycinone was isolated from three strains of Streptomyces coeruleorubidus. Its production was found to rise at the end of cultivation and to be stimulated by lowered aeration intensity.