Cytological and biochemical evidence for an early cell dismantling event in surface cultures of Streptomyces antibioticus.

A process of programmed cell death taking place late in the aerial mycelium was previously reported in surface cultures of Streptomyces antibioticus ATCC11891. In this study, we present evidence for the occurrence of a similar process taking place early in the vegetative mycelium of surface cultures of the same strain. Several indicators, such as cell wall and membrane disruption, DNA degradation and release of the cytoplasmic content into the exocellular medium, support the existence of active, highly regulated cell suicide involving specific enzymes. Calcium-dependent proteolytic activation of a precursor of nucleases and the nucleolytic formation of a ladder of chromosomal bands are conspicuous events associated with the initiation of the death process.

Viability staining and terminal deoxyribonucleotide transferase-mediated dUTP nick end labelling of the mycelium in submerged cultures of Streptomyces antibioticus ETH7451.

Viability stain and terminal deoxyribonucleotide transferase-mediated dUTP nick end labelling (TUNEL) have been applied to submerged cultures of Streptomyces antibioticus ETH7451, the last technique after a suitable permeabilization treatment. Areas of dead mycelium can be clearly delineated by the viability stain within the network of hyphae which forms the mycelial masses characteristic of the submerged cultures. In addition, the TUNEL reaction shows that DNA fragmentation accompanies the death processes in the mycelium. These techniques permit the investigation of the influence of the medium and nutritional conditions on the viability of the cells. This has relevant biotechnological implications for the study of these important filamentous bacteria in the industrial fermentation processes. These techniques also allow a straight forward analysis of the physical and chemical reagents which provoke damage in Streptomyces DNA.

Effects of 5-azacytidine on physiological differentiation of Streptomyces antibioticus.

We studied the specificity of the effect of 5-azacytidine, a DNA-methylase inhibitor that impairs Streptomyces differentiation. We showed that this compound did not affect global DNA, RNA or protein biosynthesis in submerged cultures of S. antibioticus ETHZ 7451. Among individual proteins, enzymes such as alkaline phosphatase and intracellular protease were produced in similar amounts in the presence and absence of this compound. However, the production of extracellular protease was significantly inhibited. Also DNA-methyltransferases were inhibited, indicating that DNA methylation might be involved in the regulation of differentiation. By contrast, elevated levels of the antibiotic rhodomycin resulted when 5-azacytidine was added to the culture medium. In order to determine whether there was a correlation between sporulation and altered enzymatic activities, these activities were analysed in S. antibioticus submerged cultures. Among them, alkaline phosphatase and intracellular protease activities did not show a clear correlation with sporulation. However, high levels of extracellular protease were produced during septation of hyphae. This association between extracellular protease and sporulation suggests a specific inhibitory effect of 5-azacytidine, not only on spore formation, but also on physiological differentiation.

A membrane-like structure envelopes substrate mycelium during colony development in Streptomyces.

Colonies of Streptomyces antibioticus were studied by transmission and scanning electron microscopy. The micrographs show that substrate mycelium growth takes place among an intercellular material and this mycelium is covered by a surface film. This structure could be a boundary between the aerial mycelium and the substrate mycelium.

Synchronous germination of Streptomyces antibioticus spores: tool for the analysis of hyphal growth in liquid cultures.

We have devised a method for obtaining synchronous and dispersed growth of Streptomyces antibioticus in liquid cultures. After ultrasonic treatment, most of the spores germinated at the same time, yielding hyphae very similar in length. Dispersed growth was achieved in media without Ca2+ and in which the levels of Fe2+ and Mg2+ were carefully controlled. Studies on the kinetics of growth carried out with synchronous cultures of young hyphae revealed a multiphasic pattern of hyphal elongation, with successive periods of linear growth and changes in growth rate at defined intervals.

Sporulation of Streptomyces antibioticus ETHZ 7451 in submerged culture.

Streptomyces antibioticus ETHZ 7451 formed spores in cultures grown in a liquid medium from either a spore or a mycelium inoculum. The spores formed were similar to those formed on surface-grown cultures, except for reduced heat resistance. Both types of spores were sensitive to lysozyme, which is unusual for Streptomyces spores. Glucose and other carbon sources, which promoted different growth rates, did not affect sporulation efficiency. Nitrogen sources, such as casamino acids, that allowed high growth rates suppressed the sporulation. A remarkable repression was also observed in media with some nitrogen sources that promoted noticeably lower growth rates. In permissive media, with nitrogen sources that permitted relatively high growth rates, sporulation was conditioned to the consumption of ammonium in the medium, but not to that of other nitrogen sources, such as asparagine. Phosphate did not show a repressive effect on sporulation in the assayed conditions.

[Cyclic adenosine-3',5-monophosphate in Streptomyces antibioticus and its possible role in regulating oleandomycin biosynthesis and the growth of the culture]. / Tsiklicheskaia adenozin-3',5-monofosfornaia kislota u Strptomyces antibioticus i ee vozmozhnaia rol' v reguliatsii biosinteza oleandomitsina i rosta kul'tury.

When glucose is substituted for sucrose in the fermentation medium for Streptomyces antibioticus, the pH of the cultural broth becomes more acidic, the rate of protein synthesis in the mycelium rises, and the rate of oleandomycin synthesis decreases abruptly. The dynamics of cAMP (cyclic monophosphate) accumulation was studied in the process of biosynthesis by the culture in different media. Most of the synthesized cAMP (80-90%) was shown to be excreted into the medium. Glucose stimulates cAMP synthesis and excretion from the mycelium by a factor of 1.5-3. No distinct correlation was found between cAMP content in S. antibioticus cells and the level of oleandomycin biosynthesis. A correlation between changes in the concentration of exocellular cAMP and protein synthesis in the mycelium suggests that the excreted cAMP may be involved in regulating the growth of the culture producing the antibiotic.

Isolation and properties of Streptomyces spore membranes.

A simple procedure for the isolation of membranes from Streptomyces spores is described which produces about 12 mg of membrane protein per g of dry weight. The membrane fractions were contaminated by low levels of DNA, RNA, and hexosamines. The functional integrity of the membrane is conserved through the isolation procedure, as evaluated by the presence of several activities of the membrane-bound electron transport chain. This isolation procedure allowed the determination of the biosynthesis of proteins and phospholipids of the membrane. Both biosynthetic processes started in the first 5 min of germination and increased progressively during spore germination. A stable mRNA fraction of the dormant spore encoded 44% of the membrane proteins synthesized early in germination, but most of the phospholipid biosynthesis was not dependent on this fraction.

High calcium content in Streptomyces spores and its release as an early event during spore germination.

The metal ion content of spores of five Streptomyces species was studied. A general feature of this study was the finding of a very high calcium content (1.1 to 2.1% of the dry weight). Accumulation of calcium occurred preferentially during the sporulation process. Spore calcium was located in the integument fraction, and more than 95% of the calcium was removed from intact spores by ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid. Several divalent cations (Mg2+, Mn2+, Zn2+, and Fe2+) which induced darkening of spores and loss of heat resistance also caused the release of calcium from spores. In addition, darkening of spores was blocked by metabolic inhibitors, whereas calcium excretion was not affected. Two different categories of events in the initiation of germination may be differentiated; first, calcium release from spores which is not energy dependent and is a consequence of triggering of germination by some divalent cations, and second, some other events including loss of heat resistance, loss of spore refractility, and a decrease in absorbance, with at least one energy-dependent step.

Fine structure, physiology and biochemistry of arthrospore germination in Streptomyces antibioticus.

During germination, Streptomyces antibioticus arthrospores passed through stages: darkening, swelling and germ tube emergence. The first stage, darkening, whose main features were a decrease in absorbance and a loss of refractility, only required exogenous divalent cations (Ca2+, Mg2+ or Fe2+) and energy that can be obtained from the spore reserves. This stage was blocked by agents that inhibit ATP formation but not by antibiotics that inhibit macromolecular synthesis. The second stage, swelling, needed an exogenous carbon source and was not blocked by mitomycin C. In this stage, the spores exhibited the highest cytochrome oxidase and catalase activities and respiratory quotient. The last stage, germ tube emergence, required additional carbon and nitrogen sources. Ammonium compounds were superior to nitrate. Dry weight remained constant during the stages of darkening and swelling, with a rapid increase from the moment of germ tube emergence. Optimum pH and temperature for germination were 8.0 and 45 degrees C, respectively. Heat treatment (55 degrees C for 10 min) had no effect on germination. The fine structure of the spore underwent important changes during germination. The wall of the swollen spore became stratified and the inner layer was continuous with the germ tube wall. Macromolecular synthesis occurred in the sequence RNA, protein and then DNA. Rifampicin, streptomycin and mitomycin C prevented synthesis when added at the start of incubation. The same effect was obtained if the addition was made during germination, except with mitomycin C which inhibited DNA, but not RNA and protein synthesis.

Ultrastructural studies of sporulation in Streptomyces.

This is the first study of sporogenesis in Streptomyces carried out on a relatively high number of species (seven) which allows us, using also previously published results, to establish a general picture of this process. In the sporogenesis of Streptomyces two basic stages can be considered: the sporulation septum synthesis and the arthrospore maturation. Our ultrastructural study of the sporulation septum formation suggests the existance within this genus of three basic types. Type I is distinguished because the septum is formed from the beginning by two separate cross walls. Within this type we include Streptomyces erythraeus, Streptomyces albus, and Streptomyces aureofaciens and also include Streptomyces venezuelae, Streptomyces griseus, and Streptomyces osteogriseus. Type II is distinguished because there is a deposit of material previous to the synthesis of the double annulus which completes the septum. This type can be divided into two subtypes. In the first the deposits are wedge-shaped and the double annulus is clearly visible, and to this group belong Streptomyces flaveolus, Streptomyces ambofaciens, and Streptomyces coelicolor. In the second the deposits, which have a different shape and are very well developed, constitute almost entirely the sporulation septum in which the double annulus is barely visible; Streptomyces antibioticus and also Streptomyces viridochromogenes belong to this group. Type III, represented by Streptomyces cinnamonensis, is distinguished because the septum is formed by a single cross wall.