Karel Mikulík

Microbial growth and production of antibiotics.

Antibiotic compounds do not constitute a homogeneous group. From the chemical point of view they are highly heterogeneous and it is difficult to formulate a general hypothesis that would explain the causes of production of these compounds on the basis of data obtained so far (Donovick and Brown, 1965). This difficulty is also reflected by contradictory assumptions concerning the importance of these compounds for production microorganisms.

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.

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).

Role of polyamines in the binding of initiator tRNA to the 70S ribosomes of extreme thermophilic bacterium Calderobacterium hydrogenophilum

Slowly cooled cells of an extreme thermophilic eubacterium Calderobacterium hydrogenophilum possess ribosomes with weakly associated subunits. These ribosomal subunits are capable of association to 70S ribosomes either at higher Mg2+ concentrations (30–40 mM) or at 4–10 mM Mg2+ and in the presence of polyamines. The contribution of 30S and 50S subunits to the hydrodynamic stability of ribosomes was examined by forming hybrid 30S–50S couples from C. hydrogenophilum and Escherichia coli. At lower Mg2+ (4–10 mM) heterogeneous subunits containing 30S E. coli and 50S C. hydrogenophilum and homogeneous subunits of the thermophilic bacterium associated only in the presence of polyamines. Ribosomal subunits associated at 30 mM Mg2+ lose thermal stability and activity concerning poly(AUG)-dependent binding of f[3H]Met-tRNA to the P-site on 70S ribosomes or translation of poly(UG). Poly(AUG), deacylated-tRNA or initiator-tRNA have no valuable effect on association of 30S and 50S subunits. Protein synthesis initiation factor IF3 of C. hydrogenophilum prevents association of ribosomal subunits to 70S ribosomes at physiological temperature (70°C). The factor also stimulates dissociation of 70S ribosomes of E. coli at 37°C. The codon-specific binding of f[3H]Met-tRNA to homogeneous 70S ribosomes of C. hydrogenophilum at 70°C is dependent on the presence of initiation factors and concentrations of tri-pentaamines. However, excess of polyamines inhibited the reaction. Our results indicate that tri-pentaamines enhance conformational stability of 70S initiation complex at elevated temperatures.

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.

Polypeptide synthesis on ribosomes of an extreme thermophilic hydrogen bacterium Calderobacterium hydrogenophilum

Ribosomes of the extreme thermophilic hydrogen-oxidizing bacterium Calderobacterium hydrogenophilum interact with a broad spectrum of polyamines. In the absence of polyamines and at 70°C close to the growth optimum (75°C), high salt washed ribosomes lost their activity in the poly(UG)-directed polypeptide synthesis. At 70°C the in vitro system synthesized the polypeptide in the presence of spermidine, spermine or natural polyamines (tri-pentaamines) isolated from ribosomal extracts but not in the presence of putrescine. The activity of ribosomes was affected by a number of antibiotics interfering with functions of typical eubacterial 70S, such as tetracyclines, lincomycin, chloramphenicol and erythromycin. However, the ribosomes were relatively resistant to streptomycin and insensitive to 80S inhibitors, such as ricin and cycloheximide. The 30S and 50S subunits have structural features typical of eubacterial ribosomes.

Protein synthesis elongation factor EFTu from Streptomyces collinus producing kirromycin.

Abstract Elongation factor EFTu of Streptomyces collinus has been purified. The molecular weight of the factor determined by SDS-polyacrylamide gel electrophoresis is 52,500 (±1,500). The factor shares common immunochemical determinants with EFTu of Streptomyces aureofaciens and Escherichia coli . The factor is not involved in the mechanism of resistance to the drug produced, since translation of poly(U) in the presence of EFTu and ribosomes of S. collinus is sensitive to the kirromycin effect.

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.