Synergistic effect of cobalt on the induction by A-factor of the formation of aerial mycelium and anthracyclines by a blocked mutant of Streptomyces griseus.

Cobalt ions synergistically supported the inducing effect of A-factor 1a on the formation of aerial mycelium and anthracyclines by the blocked S. griseus mutant 86. This suggested an involvement of effector 1a in the transport or metabolism of this trace element. In accordance with the proposed role of 1a as an autoregulatory secondary metabolite, the parent strain S. griseus JA 3933 secreted 1a into the medium after a decrease of the specific growth rate and before the onset of antibiotic production.

Pleiotropic effects of a butyrolactone-type autoregulator on mutants of Streptomyces griseus blocked in cytodifferentiation.

Mutants of Streptomyces griseus blocked in cytodifferentiation regained their capacity to form differentiated mycelia and/or anthracycline pigments in the presence of butyrolactone-type autoregulatory effectors such as trans-2-(6'-methylheptanol-1'-yl)-3-hydroxymethyl-4-butanolide+ ++. In the pertinent indicator strains, the effect has been correlated with the increase of lipid synthesis, with changes in the composition of lipid fraction and with the restoration of the production of neutral proteinases. The results suggest that autoregulatory butyrolactones from streptomycetes stimulate cytodifferentiation of their producers at an early stage of development.

Modification by genetic changes of the pleiotropic interference of butyrolactone-type autoregulators with differentiation of Streptomyces griseus.

Two series of aerial-mycelium-negative (Amy-), anthracycline-nonproducing (Ant-) mutants were obtained from ancestral Amy+Ant+ strains of S. griseus: a) derivatives represented by the met- strain 39 which could not differentiate although they were still producing both the butyrolactone-type autoregulator 1 and NADP-glycohydrolase, and b) mutants whose incapability to form spores and anthracycline pigments was apparently caused by the loss of autoregulator production. These latter mutants responded to the addition of 1 or the naturally occurring dihydro derivative 2 with complete or at least partial reconstitution of differentiation-associated functions. All of the b)-type mutant strains exhibited similar biochemical alterations in the presence of 1 or 2 regardless of the presence of additional genetic changes in the primary metabolism. Two mutants, however, displayed an altered pattern of secondary product formation. In submerged cultures the major biochemical changes observed in presence of 1 (or 2) were an increase of the lipid level in the mycelium, an alteration of the lipid composition, and a stimulation of neutral proteinase production. All of the blocked autoregulator-negative mutants were discernible from the ancestral strains and strain 39 by their lack of NADP-glycohydrolase production. This suggested the existance of a common genetic locus or a common pleiotropic regulator gene controling both gene functions. Present ideas concerning the role of butyrolactone-type autoregulator 1 as a pleiotropic effector molecule interacting with development of S. griseus are summarized in a hypothetical scheme.

[Leukemomycin-blocked mutants of Streptomyces griseus and their pigments. II. New 7-hydroxy-bisnahydro-rhodomycinones from the mutant ZIMET 41707/1P]. / Leukaemomycin-geblockte Mutanten des Streptomyces griseus und ihre Pigmente. II. Neue 7-Hydroxy-bisanhydro-rhodomycinone aus der Mutante ZIMET 41707/1P.

Various blocked mutants were isolated from three leukaemomycin-(daunomycin-)producing strains IMET JA 3933, IMET JA 5142 and IMET JA 5570 of Streptomyces griseus by NTG and UV treatments. Among them, one class of four mutants ZIMET 43707/1P, IMET JA 5570/3P, IMET JA 5570/10P and IMET JA 5142/01P1 produced new blue and red pigments. Two red compounds designated 1PI and 1PII are the main components of the pigment complex produced by culture of the blocked mutant ZIMET 43707/1P. This paper describes the isolation of 1PI and IPII; furthermore, the spectral and physicochemical properties of these anthracyclinones and the elucidation of their structures are reported.

Inducers of both cytodifferentiation and anthracycline biosynthesis of Streptomyces griseus and their occurrence in actinomycetes and other microorganisms.

Many taxonomically different strains of actinomycetes as well as several other procaryotic and eucaryotic microorganisms were shown to produce inducers of cytodifferentiation and anthracycline biosynthesis being active towards blocked mutant ZIMET 43682 of Streptomyces griseus. 37 out of 40 strains of the species of S. griseus (92.5%) yielding different kinds of antibiotics were able to induce both the formation of aerial mycelium in the indicator strain ZIMET 43682 concomitant with the biosynthesis of anthracycline antibiotics. 80 (26.3%) out of 304 strains belonging to 94 different Streptomyces species were also producers of inducing agents. Among 77 strains of actinomycetes belonging to 28 different genera being apart from Streptomyces, 12 strains displayed inducing activity (15.6%). The results obtained so far support the conclusion that the formation of inducing agents was neither associated with a particular species of Streptomyces nor its capacity to produce a given antibiotic. The occurrence of inducers has been observed even with members of other genera. Few strains were capable of inducing only one of the differentiation-associated functions. Some strains of Streptomyces species produced at least two different kinds of inducers discernible by their Rf values on TLC.

Effect of the autoregulator from Streptomyces griseus JA 5142 on surface cultures of blocked mutant ZIMET 43682.

Zero time addition of the autoregulator (L-factor) from S. griseus (Lkm+Amy+) to surface cultures of its bald mutant ZIMET 43 682 (Amy-Lkm-) restored the capacity to form both anthracycline-type antibiotic leukaemomycin and aerial mycelium. The pertinent mycelia displayed the same growth rate and cellular levels of nucleic acids as the asporogeneous phenotype but the composition of fatty acids and phospholipids as well as the ratio of cytochromes b and c were altered. These differences indicate alterations in the cellular architecture of substrate and aerial hyphae. The results suggest that the autoregulator triggers the onset of a complex programme of differentiation at a very early stage.

Genetic segregation in a high-yielding streptomycin-producing strain of Streptomyces griseus.

The streptomycin-producing Streptomyces griseus HP spontaneously segregated non-reverting derivatives with altered phenotypes. Clones characterized by increased spore formation and decreased streptomycin production were found. Two other types of derivatives were defective in aerial mycelium and streptomycin formation as well, but differed in the capacity to synthesize a yellow pigment. These derivatives were examined with respect to further properties. The stability of S. griseus HP was investigated in relation to conditions of continuous culture. Both at 26 and 30 degrees C, under glycerol and NH4Cl limitation a rapid segregation and enrichment of streptomycin-non-producing derivatives occurred. At 34 degrees C and glycerol limitation segregation began only after about 35 generations of continuous culture. In NH4Cl-limited chemostats the original strain was stable during 80 generations. In the course of the continuous culture experiments it was shown that the onset of genetic segregation within mycelia can be detected before it becomes obvious in colonies grown from the mycelia. This was achieved by fractionation of the mycelia by protoplast formation and subsequent plating on regeneration medium allowing colony growth and differentiation.

A screening method for autoregulators of anthracycline-producing streptomycetes.

A novel method was proposed for screening of autoregulators by anthracycline-producing strains of Streptomyces griseus. By means of a modified cosynthesis procedure, an indicator strain was detected capable of producing both leukaemomycin and aerial mycelium only in the presence of specific inducing factors (autoregulators) which were produced not only by anthracycline-yielding organisms and their mutants, but also by a streptomycin-producing strain of Streptomyces griseus as well as by its non-differentiating derivative.

[Leukaemomycin-blocked mutants of Streptomyces griseus and their pigments]. / Leukaemomycin-geblockte Mutanten des Streptomyces griseus und ihre Pigmente.

In a continued search for leukaemomycin-blocked mutants of three leukaemomycin-producing strains IMET JA 3933, IMET JA 5142 and IMET JA 5570 of Streptomyces griseus, 32 mutants producing aerial mycelium and spores were detected. Furthermore, in all mutants cosynthetic capability has been observed. This report describes characterization of leukaemomycin-blocked mutants obtained by mutagenic treatment experiments using NTG and combined UV-/X-rays. According to the biosynthetic capability for anthracyclinones or other pigments the mutants could be divided into six classes. The first class contains 14 leukaemomycin-blocked mutants unable to synthesize anthracyclinones. Besides two classes of mutants (12)synthesizing well-known anthracyclinones as epsilon-rhodomycinone, 7-deoxy-epsilon-rhodomycinone, 11-deoxy-derivatives of daunomycinone, three new classes of mutants (6) synthesizing reddish-brown, brown and blue-violet pigments on solid media with structures not elucidated as yet, will be described.

[Fertility mutants of the turimycin-producing Streptomyces hygroscopicus IMET JA 6599]. / Fertilitätsmutanten des Turimycin-Bildners Streptomyces hygroscopicus IMET JA 6599.

In a continuing search for fertility mutants of the turimycin-producer, Streptomyces hygroscopicus, strain IMET JA 6599, from more than 683 pairs of auxotrophic mutants 19 pairs were found to develop colonies after replica plating on selective media. The frequency of recombinant production in mixed cultures on complex medium varies from 10(-6) to 10(-2) in different combinations of S. hygroscopicus mutants. Typical frequencies representing the proportion of recombinant colonies (from spores and/or fragments of substrate mycelium) and a visual indication of the different fertility levels using the familial fertility test method are given in Tables and Figures. Additionally, reversion rates of markers and different types of colonies of fertility mutants used in some S. hygroscopicus crosses will be shown.