Comparative structural characterization of material extractable from aerial sheath of Streptomyces roseoflavus var. roseofungini

1. Tubular-like structures were regularly revealed in the surface sheath of the aerial mycelium of the parent strain of Streptomycetes roseoflavus var. roseofungini. In their shape and dimensions these structures were highly reminiscent of those massive accumulation of which was earlier reported to occur in cultures of dedifferentiated nocardioform “fructose” mutant of the same parent strain.
2. The tubular-like structures of the aerial mycelium sheath were shown to be markedly susceptible to brief acetone washing, undergoing almost complete desintegration.
3. On addition of water to crude acetone extract of the aerial mycelium precipitation and possible selfassembly of a spectrum of various structures occurred (folded scaly, bubble-like, spout-like). Some among structures so produced were reminiscent of those found in spectra of structures observed in reconstruction experiments with tubules from the mutant as well as of structures found in the aerial sheath of the parent strain and some other actinomycetes. Similarity in subunit structure of above materials was also noticeable.

The presence, in the sheath of aerial mycelium, of material with a tendency to selforganization is discussed in relation to the possible involvement of selfassembly processes in the formation of the surface sheath.     

Self-assembly of extracellular tubular structures of non-protein nature produced by an actinomycete]

When grown on the solid synthetic medium with glucose as the only carbon source the dedifferentiated "fructose" mutant of Actinomyces roseoflavus var. roseofungini accumulated aggregates of tubular-like structures. The individual tubules had the internal diametre of 80 A and external diametre of approximately 200-220 A. These structures were isolated as a distinct fraction and their non-protein nature was demonstrated. They were easily soluble in acetone and reconstitutable in vitro. The possible significance of production of self-assembling structures by a mutant with impaired differentiation is discussed. The possibility of involvement of self-assembly processes in the formation of surface sheath of aerial mycelium in normally differentiating actinomycetes is mentioned.     

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.     

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.     

Colony development in Streptomyces carpinensis: a streptomycete with substrate mycelium spores

In this paper we present an ultrastructural study of spore formation in aerial vs. substrate mycelia of Streptomyces carpinensis. Both mycelia initiated spore formation at nearly the same time of colony development but exhibited different patterns of spatial localization of sporulation: spore formation took place throughout the aerial mycelium whereas in the substrate mycelium was confined to a narrow zone at the bottom of the colony. The ultrastructural changes leading to spore formation, however, were quite similar in both mycelia, differing only with respect to the outer components of the sporal wall. Spores formed in the aerial mycelium were covered by a thin sheath whereas the spores formed in the substrate mycelium were covered by an amorphous electron-dense material.     

Effect of inorganic phosphate on levorin biosynthesis and on the mycelial makeup of Streptomyces levoris]

The effect of inorganic phosphate on biosynthesis of the polyenic antibiotic levorin by Streptomyces levoris and composition of the culture mycelium was studied. It was found that the synthetic medium with 0.4 mM of phosphate was optimal for growth of Str. levoris. When the concentration of phosphate was higher, the biomass increased, while the synthesis of levorin appeared to be inhibited and morphological changes in the culture were observed. Phosphate had a significant effect on the mycelium composition. When its concentration was increased 10 times as compared to the optimal one, the amounts of protein, RNA, total phosphorus and polyphosphates increased 1.3--1.4, 1.6--1.7, 2--3 and 10 times respectively, while the synthesis of levorin decreased 5 times. Changes in the lipid component of the mycelium were also observed. In the absence of inorganic phosphate in the medium the acetone precipitating fraction of the lipids contained 20--40 per cent of the phosphoruless compounds. During cultivation their portion increased up to 70--77 per cent. However, in the presence of its excess the polar lipids were represented only by phospholipids during the whole life cycle. The fatty acid spectrum of the lipids did not depend on the phosphate concentration and was represented mainly by saturated fatty acids with a branched chain of a series of iso- and anteiso-structures containing 14--18 carbon atoms.     

Formation by Actinomyces malachitospinus sp. nov. of a physiologically active substance that stimulates zygote formation in Mucorales]

A new species Actinomyces malachitospinus sp. nov. is described. It has spiral catenulate spores with spines, a gray aerial mycelium, and a green colonial mycelium, and contains no soluble pigments (including melanoid pigments). The strain of Act. malachitospinus INMI 217 produces a physiologically active substance that stimulates the formation of zygotes in Phycomyces blakesleeanus.     

Lysis of the cellular walls of Streptococcus group A by enzymes produced by actinomycetes]

More than 80 cultures of actinomycetes belonging to different taxanomic groups were studied with a purpose of screening actinomycetes actively producing enzymes lyzing the cell walls of group A streptococci. 31 strains of the actinomycetes producing enzymes which lyzed the cell walls by 20-50 and 60-80 per cent within 1 and 4 hours respectively were selected. The proteolytic activity of the enzymes produced by these strains was also studied. It was shown that 4 cultures, i.e. Actinomyces albus, strains 6 and 9, Actinomyces levoris, strain 29 and Actinomyces gibsonii, strain 42 were of interest as organisms producing enzymes which lyzed the streptococcal cell wall without impairing its antigenic components.     

A decrease in GTP content is associated with aerial mycelium formation in Streptomyces MA406-A-1

Aerial mycelium formation by Streptomyces sp. MA406-A-1, a formycin-producing strain, was suppressed by the presence of excess nutrient. In such suppressed cultures, decoyinine, which specifically inhibits GMP synthetase, initiated the formation of aerial mycelium at concentrations which only partially inhibited growth. The intracellular GTP pool of organisms growing in liquid culture markedly decreased on the addition of decoyinine. Decoyinine was also effective in initiating aerial mycelium formation of three other Streptomyces spp. examined. Regardless of the successful initiation of aerial mycelium formation, the ability of the cells to produce antibiotics (formycin or actinomycin D) did not increase, but decreased, on the addition of decoyinine. It is concluded that aerial mycelium formation by Streptomyces results from a decrease in the pool of GTP (or GDP), whereas antibiotic synthesis results from a different signal(s).     

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.     

Calcium ions and the differentiation of Streptomyces hygroscopicus 155, a producer of an antibiotic complex]

The effect of Ca2+ on differentiation of Streptomyces hygroscopicus 155 and its inactive variant 155-0 was studied. Addition of Ca2+ to the medium induced formation of the aerial mycelium in the inactive variant and accelerated formation of the aerial mycelium in the parent strain. The inhibitory effect of EGTA, verapamil, nifedipin, chlorpromazine and dilthiazeme on the aerial mycelium formation demonstrated the physiological role of Ca2+ in the process. Addition of pandavir (nigericin) and azalomycin B, the antibiotics produced by the streptomycete, induced formation of the aerial mycelium in the inactive variant. The effect was higher in the presence of Ca2+. Streptomyces hygroscopicus 155 and its inactive variant synthesized a proteolytic complex containing metalloproteases and trypsin-like proteases. The total proteolytic activity of the inactive variant was lower than that of the parent strain. Addition of Ca2+ to the medium stimulated their proteolytic activity. The inducing action of the antibiotics produced by the parent strain on differentiation of S.hygroscopicus 155-0 and the increase of their action in the presence of Ca2+ suggested that they controlled the differentiation and that such a function of the antibiotics expressed itself through the Ca2+ signal system.     

Action of the products of the vital activity of yeastlike fungi on the biosynthesis of levorin, levoristatin and fatty acids by a Streptomyces levoris culture

The data on the effect of the products of vital activity of Candida tropicalis, a yeast-like fungus, on the biosynthesis of levorin, levoristatin and fatty acids by Streptomyces levoris are presented. It was shown that the effect of the biostimulators was not specific with respect to production of levorin, since in the presence of the products of vital activity of C. tropicalis an increase in the synthesis of levoristatin and fatty acids was also observed. The qualitative and quantitative composition of the fatty acids of the mycelium of S. levoris was studied. Interrelation between the biosynthesis of levorin and synthesis of unsaturated fatty acids and branched chain fatty acids was noted.     

Autoradiographic study of hyphal growth during aerial mycelium development in Streptomyces antibioticus.

The pattern of growth of aerial mycelium in Streptomyces species was investigated by autoradiography. Colonies of Streptomyces antibiotics were labeled with N-acetyl-D-[1-3H] glucosamine to localize the sites of hyphal growth during the development of aerial mycelium. Autoradiographs obtained with sections of the colonies revealed that hyphal growth occurs not only at the top of the colony but also in the inner zones of the aerial mycelium.     

Intrinsic luminescence and differentiation in Actinomyces lucensis--producer of etruscomycin]

The luminescence of Actinomyces lucensis producing etruscomycin was found to be caused not by the antibiotic but by additional luminescent substances. No direct correlation has been established between the content of the luminescent substances and the intensity of the luminscence. The latter depends on the differentiation of the colonies, i.e. on the presence of a dark layer that screens the luminescence of the colonial mycelium and is localized under the aerial mycelium. The dark layer contains pigments of the melanin type.     

Induced tolerance of Sclerotium cepivorum to antibiotics in the presence of onion exudates

The onion white rot pathogen Sclerotium cepivorum was cultured on agar media containing 2% malt extract and one of the antifungal antibiotics, endomycin, griseofulvin, venturicidin and cycloheximide at concentrations that reduced but did not prevent growth of mycelium. When onion seeds or agar discs impregnated with diffusates from onion bulbs were placed on the antibiotic media, radial growth of the fungus was greatly increased, and there was a profuse development of aerial mycelium. Gaseous diffusates from onion tissue and from impregnated agar discs were also effective. On the antibiotic media, tomato, cabbage and radish seeds did not stimulate the growth of S. cepivorum and the onion exudates did not stimulate the growth of four other fungi. This and other evidence is considered to show that the stimulation of growth of S. cepivorum was not caused by any direct effect on the antibiotics but by a tolerance of the fungus to them, which was specifically induced by an exudate from its host plant. The phenomenon may be related to the reported reversal by onion extracts of the inhibitory effects of soil mycostasis on germination of sclerotia of the fungus.     

The physicochemical and biochemical characteristics of the cell walls in M+ and M- variants of Streptococcus group A type 29

The amino acid composition of cell walls and surface proteins, isolated from virulent (M+) and avirulent (M-) streptococcal strains (group A, type 29) has been determined by the method of E. H. Beachey et al. The kinetics of the lysis and proteolysis of streptococcal cell walls with muramidase and protease obtained from Actinomyces levoris and streptolysin has been studied. The constants describing the progress rates of these processes has been determined; their values in case of both lysis and proteolysis are higher in virulent strains than in avirulent ones.     

THE FINE STRUCTURE OF STREPTOMYCES VIOLACEORUBER (S. COELICOLOR) : III. The Walls of the Mycelium and Spores

A study of thin sections of hyphae of Streptomyces violaceoruber in the electron microscope showed that the structure of the walls and the mode of formation of cross-walls are similar to those of Gram-positive bacteria. A beaded structure was seen in some regions of the wall, and the significance of this observation is discussed in relation to previous studies of the fine structure of bacterial cell walls. Elements of the intracytoplasmic membrane system appear to be involved in the process of cross-wall formation. The walls of the hyphae of the aerial mycelium divide into two layers before the spores are formed, and only the inner component of the wall grows inwards to form the cross-walls and so delimit the spores. The outer component remains intact for a time and acts as a sheath around the developing spores. Finally the sheath breaks and the spores are liberated. This process is contrasted with the formation of endospores in eubacteria. When the spores germinate, the walls of the germ tubes are continuous with those of the spores.     

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.     

Motile tubular vacuoles in extramatrical mycelium and sheath hyphae of ectomycorrhizal systems

Summary Extramatrical mycelium and outer hyphae of the sheath ofEucalyptus pilularis-Pisolithus tinctorius mycorrhizas contain abundant motile tubular vacuoles which accumulate the carboxyfluorescein analogue Oregon Green 488 carboxylic acid. The fluorochrome accumulates in a system of small vacuoles, tubules, and larger vacuoles, which are interlinked, motile, and pleiomorphic, in external hyphae, cords, and hyphae of the outer sheath. There is often a difference in fluorescence between two neighbouring cells, indicating that the dolipore septum exercises control on the movement of material between cells. Generally the motile tubular vacuole system in mycorrhizas resembles that previously found in isolated mycelium. The majority of fungal cells in the sheath contain no fluorochrome even after long exposure of the mycorrhiza to the solution, but with differential interference optics the cells are clearly seen to be alive and to contain vacuoles resembling those in the outer hyphae. In translocation experiments, long-distance transport of the fluorochrome is slow and slight, or even nonexistent in some cases.Abbreviations carboxy-DFF Oregon Green 488 carboxylic acid - carboxy-DFFDA Oregon Green 488 carboxylic acid diacetate - DIC differential interference contrast Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Neo-clerodane diterpenoids from Ajuga genevensis

Three new neo-clerodane diterpenoids, ajugavensins A–C, have been isolated from the acetone extract of the aerial parts of Ajuga genevensis and their structures established by spectroscopic means and by comparison with closely related compounds.