Ivan Janda

Macromolecular synthesis accompanying the transition from spores to vegetative forms ofStreptomyces granaticolor

The rates of RNA, protein and DNA synthesis were estimated in synchronously germinating spores ofStreptomyces granaticolor. Rapid uptake of labelled precursors of RNA and proteins was observed after 20 s. The germination process took place through a sequence of time + ordered events. RNA synthesis started after 3 min of germination, protein synthesis began at 4 min and net DNA synthesis at 60–70 min of germination. A characteristic feature of germination was the biphasic pattern in the rate of RNA and protein synthesis. Spores ofStreptomyces granaticolor were sensitive to actinomycin D, rifampicin and chloramphenicol even at the start of germination. Protein synthesis during germination was dependent on new mRNA synthesis and was independent during the first 60–70 min on replication of the spore genome.

Production, long term preservation and synchronous germination of aerial spores of Streptomyces

Abstract Vigorous vegetative growth of various Streptomyces species (S. auroefaciens, S. collinus and S. granaticolor) was achieved in a new semisynthetic liquid medium. Unlike the media commonly used for the cultivation of the submerged mycelia of different streptomycetes, this one does not contain insoluble material which enables direct and reliable measurement of net production of biomass. The medium was formulated to meet the nutritional requirements of all the three species. Is also supported production of antibiotic in each of the strains. A method for bulk preparation of Streptomyces aerial spores, involving cultivation on agar plates covered with cellophane, was developed. Advantage of this method lies in higher yields of spores, their higher purity and easier harvesting. The spores were activated by amild treatment with an Ultra-Turrax homogenizer resulting in the breakage of fibrous sheath, suspended in 20% glycerol, and stored at −60°C. Thus, treated spores germinated synchronously even after several months of the storage. Hence, such spore material may be used for precise inoculation in a large series of experiments implying synchronous germination, and the inoculations can be carried out from the same batch over a long period.

Susceptibility of ribosomes of the tetracycline-producing strain of Streptomyces aureofaciens to tetracyclines.

Ribosomes from cells of Streptomyces aureofaciens producing tetracycline antibiotics (Tc‐ribosomes) differ in electrophoretic mobility of ribosomal proteins S2, S10 and L19 from those of the same strain, where the production of tetracyclines was suppressed by changed cultivation conditions (C‐ribosomes). Purified tight vacant couples C‐ and Tc‐ribosomes are equally active in the translation of poly(U). Both types of S. aureofaciens ribosomes are more sensitive to tetracycline and chlortetracycline than ribosomes of Escherichia coli in the Phe‐tRNA binding and the translation of poly(U).

Ribosomal proteins of Streptomyces aureofaciens producing tetracycline

Three different two-dimensional polyacrylamide gel electrophoretic systems were employed for identification of individual ribosomal proteins of Streptomyces aureofaciens. Proteins of small subunits were resolved into 21 spots. Larger ribosomal subunits contained 35 proteins. The separated ribosomal proteins from 50 S subunits were transferred on nitrocellulose membranes for immunochemical estimations. Antibodies developed against 50 S proteins of S. aureofaciens and Escherichia coli were used for identification of structural homologies between 50 S proteins of the two species. Results of the experiments indicate that about one half of the 50 S proteins of S. aureofaciens share common immunochemical determinants with corresponding proteins of 50 S subunits of E. coli. Evidence is presented that acidic ribosomal protein SL5 of large ribosomal subunits of S. aureofaciens can be assembled to E. coli P0 cores lacking proteins L7/L12. Reconstitution of the P0 cores with proteins SL5 or L7/L12 led to restoration of 78% activity in polyphenylalanine synthesis.

Ribosomal proteins of Streptomyces granaticolor

Abstract A method for large-scale isolation of streptomycete ribosomal subunits involving centrifugation in hyperbolic sucrose density gradients in a zonal rotor was developed. Ribosomal proteins were extracted from 30 S and 50 S subunits of Escherichia coli A19 and primary mycelium of Streptomyces granaticolor . Their two-dimensional electropherograms differed considerably. Purified 30 S and 50 S subunits from S. granaticolor mycelium contained 21 and 36 ribosomal proteins, respectively. Only 8 proteins in the mycelial ribosomes possessed identical electrophoretic mobilities as corresponding E. coli ribosomal proteins, viz., S4, S12, S16, L1, L2, L14, L16 and L19. Despite the differences in physico-chemical properties, functional correspondence is likely to exist between certain ribosomal proteins from the two bacteria. The range of molecular weights of vegetative S. granaticolor ribosomal proteins was similar to that in other prokaryotes. Ribosomal proteins were further isolated from 70 S ribosomes of S. granaticolor dormant spores. The spore ribosomal proteins differed markedly from those of the primary mycelium and their total number was lower. The ribosomal protein alterations are presumed to take part in the regulation of the streptomycete cell differentiation.

Translation of poly(U) on ribosomes from Streptomyces aureofaciens

Abstract Slowly cooled cells of Streptomyces aureofaciens contained mainly tight-couple ribosomes. Maximum rate of polyphenylalanine synthesis on ribosomes of S. aureofaciens was observed at 40°C, while cultures grew optimally at 28°C. Ribosomes of S. aureofaciens differed from those of E. coli in the amount of poly(U) required for maximum synthetic activity. The polyphenylalanine-synthesizing activity of E. coli ribosomes was about 3-times higher than that of S. aureofaciens ribosomes. The addition of protein S1 of E. coli or the homologous protein from S. aureofaciens had no stimulatory effect on the translation of poly(U). In order to localize alteration(s) of S. aureofaciens ribosomes in the elongation step of polypeptide synthesis we developed an in vitro system derived from purified elongation factors and ribosomal subunits. The enzymatic binding of Phe-tRNA to ribosomes of S. aureofaciens was significantly lower than the binding to ribosomes of E. coli. This alteration was mainly connected with the function of S. aureofaciens 50 S subunits. These subunits were not deficient in their ability to associate with 30 S subunits or with protein SL5 which is homologous to L7/L12 of E. coli.

Interaction of granaticin B with the transcription system ofBacillus subtilis

The interaction of granaticin B, a quinone antibiotic produced byStreptomyces granaticolor, with some biologically important bivalent metal ions, DNA and ATP was demonstrated speetrophotometrically. The activity of isolated RNA polymerase was higher when the DNA of phage SP 50 served as template than with DNA isolated fromBacillus subtilis. Granaticin B inhibitedin vitro RNA synthesis, similarly to certain other antibiotics (the inhibition was three times lower than that caused by actinomycin D or streptolydigin and slightly higher than that by ε-pyrromycinone). The inhibitory effect was higher when the Mg2+ concentration in the reaction mixture was decreased. The inhibition was then proportional to the concentration of the DNA template. DNA-dependent RNA synthesis is thus inhibitedin vitro by granaticin B but this does not appear to be the only site of action of this antibioticin vivo.

Translational system of the hydrogen-oxidizing bacterium Alcaligenes eutrophus

Some structural and functional properties of ribosomes from the hydrogen-oxidizing bacterium Alcaligenes eutrophus were studied in order to investigate the background of expression of genetic information at the translational level. Ribosomal proteins from 30S subunits of A. eutrophus H16 were separated by two-dimensional gel electrophoresis into 21 spots, those from 50S subunits into 32 spots. While electrophoretic mobilities of several ribosomal proteins differed markedly from those of Escherichia coli, proteins sharing common immunological determinants with E. coli ribosomal proteins S1 and L7/L12 were found in A. eutrophus. Shifting from heterotrophic to autotrophic conditions of growth had no influence on the ribosomal protein pattern. Ribosomes of A. eutrophus had similar requirements for Mg2+ and poly(U) concentrations for optimum polyphenylalanine synthesis as those of E. coli. Protein synthesis elongation factors Tu from A. eutrophus and E. coli were immunologically similar. Efficiency of the A. eutrophus polyphenylalanine-synthesizing system was comparable to that of an analogous system derived from E. coli. This suggests that A. eutrophus could be employed for efficient expression of recombinant DNA.

Initiation of Translation of Genetic Information in Streptomyces Aureofaciens

It is now well established that small ribosomal subunits from different prokaryotic species differ in their ability to initiate translation on specific mRNAs (1–3). Ribosomes from Gram-negative bacteria translate mRNA from both Gram-positive and Gram-negative cells while ribosomes from Gram-positive cells efficiently translate only mRNA of Gram-positive microorganisms (4,5).