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.     

Changes in ribosomal proteins during colony development in Streptomyces.

The structure and functionality of the ribosomal subunits of the substrate and the aerial mycelium of Streptomyces antibioticus were compared. Using SDS-PAGE and HPLC, several differences between the ribosomal protein pattern from both stages of development were observed, including a clear decrease in the L7/L12 content of the aerial mycelium. The activity of the aerial mycelia ribosomes was also decreased when compared with that of the substrate mycelium. This effect was more pronounced in the 50S subunit. These results suggest that during cell differentiation in Streptomyces important changes occur at the ribosomal level, particularly in the transition from the substrate to the aerial mycelium.     

DNA-methyltransferase activities in Streptomyces antibioticus

Abstract To quantitate ammonia production by the intestinal flora, ammonia levels in arterial blood and the venous effluent of the small and large bowel of conventional, selectively decontaminated, germ-free and gnotobiotic rats were measured.
When the anaerobic flora was removed by decontamination a significant decrease in ammonia levels was observed in the effluent of both the small and large intestine. Decontamination of aerobic flora did not result in depression of ammonia production. Gnotobiotic rats colonised with an anaerobic flora or with a mixed aerobic and anaerobic flora, showed a slight increase in ammonia levels. No increase in ammonia production was observed when rats were colonised with aerobic flora. These results indicate that the Enterobacteriaceae were not responsible for ammonia generation.
The increase in ammonia levels after colonisation with anaerobic or mixed anaerobic/aerobic flora did not completely restore ammonia levels, despite reaching bacterial counts which were comparable to those in conventional rats. This may be explained by the limited number of species with which the rats were colonized. The finding that aerobic flora does not significantly contribute to ammonia production suggests that neomycin, known to be exclusively effective against aerobic flora, must have other effects to explain improvement of hepatic encephalopathy.     

Hyphal death during colony development in Streptomyces antibioticus: morphological evidence for the existence of a process of cell deletion in a multicellular prokaryote

During the life cycle of the streptomycetes, large numbers of hyphae die; the surviving ones undergo cellular differentiation and appear as chains of spores in the mature colony. Here we report that the hyphae of Streptomyces antibioticus die through an orderly process of internal cell dismantling that permits the doomed hyphae to be eliminated with minimum disruption of the colony architecture. Morphological and biochemical approaches revealed progressive disorganization of the nucleoid substructure, followed by degradation of DNA and cytoplasmic constituents with transient maintenance of plasma membrane integrity. Then the hyphae collapsed and appeared empty of cellular contents but retained an apparently intact cell wall. In addition, hyphal death occurred at specific regions and times during colony development. Analysis of DNA degradation carried out by gel electrophoresis and studies on the presence of dying hyphae within the mycelium carried out by electron microscopy revealed two rounds of hyphal death: in the substrate mycelium during emergence of the aerial hyphae, and in the aerial mycelium during formation of the spores. This suggests that hyphal death in S. antibioticus is somehow included in the developmental program of the organism.     

Restriction-modification systems in Streptomyces antibioticus

Several restriction systems were detected in different strains of Streptomyces antibioticus by using actinophages as biological indicators. Adsorption of phages to the bacteria, together with the study of the efficiency of plating gave an initial indication of restriction in three strains. The alternation of efficiency of plating values obtained from restricting and nonrestricting hosts, gave evidence for the presence of a restriction-modification system in another strain. No common modification systems were detected among the different strains tested. Two specific endonucleases with a possible role in restriction were detected in strains ATCC 11891 and ETH 7451, respectively.     

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

Abstract 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.     

Pattern of protein degradation during germination of Streptomyces antibioticus spores

The pattern of protein degradation during germination of Streptomyces antibioticus spores was studied by the pulse and chase technique. Two different protein fractions were found. First, a fraction of the proteins synthesized during the darkening process (20-30%) was quickly degraded in the 30 min following the labelling period. This rapid protein degradation was partially inhibited by protease inhibitors: p-chloromercuribenzoic acid, phenylmethylsulphonylfluoride, and o-phenanthroline. Second, the remaining 70-80% and the entire protein population formed during spore swelling and germ tube emergence were degraded with a lower and constant rate (3.3-6.0% /h). A stable mRNA fraction of the dormant spores was translated upon incubation of the spores in a minimal synthetic medium (MSM) or in distilled water. However, the degradation of these proteins did not occur unless the spores were then incubated in the MSM. A strong correlation between the degradation pattern of these proteins and that of those quickly degraded at the beginning of germination was observed. Protease activity in cell-free extracts of dormant spores was detected. Inhibition studies suggest the presence of serine, thiol, and metalloproteases. The protease activity, using casein as substrate, remained constant during the darkening process and started to increase progressively from the beginning of spore swelling.     

Fusion of protoplasts in Streptomyces antibioticus

The analysis of Streptomyces antibioticus recombinants isolated under non-selective conditions for stability of inheritance of parental properties demonstrated the existence of non-stable diploids and stable haploid recombinants. Also, genetic analysis of heteroclones isolated on selective medium after plating spores of regenerants was conducted. The results of analysis of haploid recombinant and heteroclones pointed to linkage of genetic loci and the possibility to use the technique of protoplast fusion for establishment of genetic map of S. antibioticus.     

Induction of mutations in Streptomyces antibioticus

Mutagenic action of UV-light, nitrosoguanidine and nitrosomethylbiuret was studied in Streptomyces antibioticus VNIIA 1607. Nitrosomethylbiuret appeared to be most effective inducer of auxotrophic mutations (mutation frequency reached 15%). By means of hybridological analysis, it was shown that heterokaryons predominated in the progeny of mixed cultures of multiply marked strains. The test for functional allelism using heterokaryons permitted us to divide 93 independently obtained mutations into 28 complementation groups.     

Oleandomycin content of mycelium and fermentation solution during cultivation of Streptomyces antibioticus

The time-course of the oleandomycin content in the mycelium and fermentation broth-filtrate was studied by the microbiological assay at different periods of cultivation of strains 471 and 961 in fermenters and flasks containing a rich soybean-corn medium. It was shown that centrifugation of the mycelium over the sucrose density gradient induced a 25-80 per cent decrease in its moist weight at the expense of removal of the admixture components of the rich medium. Addition of glucose (2 per cent) to the culture-grown in a lactose medium by the 72nd hour of fermentation had no effect on further increase of the cell biomass. However, it lowered the content of the mycelium-fixed and excreted antibiotic at all the subsequent fermentation periods. The content of oleandomycin in the untreated mycelium was only 0.36 per cent of its content in the fermentation broth filtrate. After centrifugation of the mycelium over the sucrose density gradient and its intensive washing with distilled water the content of the mycelium-fixed antibiotic decreased still more. The time-course of the content of the mycelium-fixed and excreted oleandomycin was characterized by the presence of two activity peaks; by the 80-110th and by the 140-170th hour of cultivation.     

tRNA accumulation and suppression of the bldA phenotype during development in Streptomyces coelicolor

Streptomyces coelicolor undergoes distinct morphological changes as it grows on solid media where spores differentiate into vegetative and aerial mycelium that is followed by the production of spores. Deletion of bldA, encoding the rare tRNA(Leu) UAA, blocks development at the stage of vegetative mycelium formation. From previous data it appears that tRNA(Leu) UAA accumulates relatively late during growth while two other tRNAs do not. Here, we studied the expression of 17 different tRNAs including bldA tRNA, and the RNA subunit of the tRNA processing endoribonuclease RNase P. Our results showed that all selected tRNAs and RNase P RNA increased with time during development. However, accumulation of bldA tRNA and another rare tRNA(Leu) isoacceptor started at an earlier stage compared with the other tRNAs. We also introduced the bldA tRNA anticodon (UAA) into other tRNAs and introduced these into a bldA deletion strain. In particular, one such mutant tRNA derived from the tRNA(Leu) CAA isoacceptor suppressed the bldA phenotype. Thus, the bldA tRNA scaffold is not critical for function as a regulator of S. coelicolor cell differentiation. Further substitution experiments, in which the 5'- and 3'-flanking regions of the suppressor tRNA were changed, indicated that these regions were important for the suppression.     

A simple method to study colony development in Streptomyces

Abstract When sodium azide was added to cultures of Myxococcus coralloides D a rapid loss in turbidity was observed. The lysis occurred irrespective of the culture age. If the azide was added to cultures which had been division-inhibited with puromycin, lysis was also induced. Other uncoupling agents (2,4-dinitrophenol, methyltriphenylphosphonium bromide and N , N '-dicyclohexylcarbodiimide) were effective to induce lysis, but not the ionophores gramicidin D or valinomycin. Energizing the membrane by the addition of glycerol, glucose or ascorbate to prelytic cultures was a means of preventing the lytic events.     

Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans

In two separate studies a BclI-generated DNA fragment coding for the enzyme tyrosinase, responsible for melanin synthesis, was cloned from Streptomyces antibioticus DNA into two SLP1.2-based plasmid vectors (pIJ37 and pIJ41) to generate the hybrid plasmids, designated pIJ700 and pIJ701, using S. lividans 66 as the host. The fragment (1.55 kb) was subcloned into the multicopy plasmid pIJ350 (which carries thiostrepton resistance and has two non-essential BclI sites) to generate four new plasmids (pIJ702-pIJ705) with the tyrosinase insert located in either orientation at each site. All six plasmids conferred melanin production (the Mel+ phenotype) on their host. As in the S. antibioticus parent, strains of S. lividans carrying the gene specifying tyrosinase synthesis possessed an enzyme activity which was inducible. Most of the tyrosinase activity was secreted during growth of S. antibioticus; in contrast, the majority remained intracellular in the S. lividans clones. The specific activity of the induced tyrosinase activity (intracellular) was higher (up to 36-fold) when the gene was present on the multicopy vector in comparison with its location on the low copy plasmids, pIJ700 or pIJ701, or in S. antibioticus. Restriction mapping of the tyrosinase fragment in pIJ702 revealed endonuclease cleavage sites for several enzymes, including single sites for BglII, SphI and SstI that are absent from the parent vector (pIJ350). Insertion of DNA fragments at any one of these sites abolished the Mel+ phenotype. The results indicate that pIJ702 is a useful cloning vector with insertional inactivation of the Mel+ character as the basis of clone recognition.     

Purification and Properties of Phospholipase D from Streptomyces antibioticus

Phospholipase D was purified from Streptomyces antibioticus by column chromatography and chromatofocusing. The enzyme preparation was electrophoretically homogeneous and the molecular weight of the enzyme was estimated to be 64,000. Its isoelectric point was around pH 6.5. The enzyme was most active at pH 5.5 and at around 60°C. It was stable between pH 4 and 8, and below 50°C.     

MsiK-dependent trehalose uptake in Streptomyces reticuli

During cultivation in the presence of trehalose Streptomyces reticuli expresses an inducible, highly specific trehalose uptake system that is absent in Streptomyces lividans. A palmitated trehalose-binding protein was identified in the cytoplasmic membrane of mycelia, extracted with the detergent Triton X-100 and purified using a trehalose affinity matrix. Immunological studies showed that within S. reticuli the synthesis of the ATP-binding protein MsiK is induced by trehalose. The data suggest that MsiK assists the trehalose ABC transporter, like the previously described ABC transport systems for maltose and cellobiose/cellotriose, respectively.