J. Spížek

Metabolites ofStreptomyces noursei

Production of cycloheximide and fungicidin by the producing strainStreptomyces noursei 52/152 was shown to be coupled. Cycloheximide added to the culture medium increased the production of fungicidin, addition of fungicidin caused an increase of the production of cycloheximide. Antibiotic compounds added were degraded in the course of cultivation approximately to the same amount as if produced in control cultures. Cycloheximide added to the culture medium was not transformed to actiphenol. It was suggested that the synthesis of secondary metabolites ofStreptomyces noursei is controlled by mechanisms analogous to repression or allosteric inhibition.AbstractБыло доказано, что образование циклогексимида и фунгицидина у штамма Str. noursei 52/152 взаимно связаны. Прибавление к культивационной среде циклогексимида вызывало повышение продукции фунгицидина; прибавление фунгицидина повышало продукцию циклогексимида. Прибавляемые антибиотические вещества в течение культивирования деградируются приблизительно до такого же количества, какое образуется в контрольных культурах. Циклогексимид, внесенный в культивационную среду, не превращался в актифенол. —Высказывается предположение, что синтез вторичных метаболитов у Str. noursei управляется механизмами, аналогичными подавлению или аллостерическому угнетению.

Purification of DNA-dependent RNA polymerase fromStreptomyces granaticolor

RNA nucleotidyltransferase (EC 2.7.7.6) ofStreptomyces granaticolor was purified by precipitation with polymin P and ammonium sulphate, affinity chromatography on DNA-cellulose and gell filtration on Biogel A 1.5 m. SDS-polyacrylamide gel electrophoresis revealed 8 protein bands of molar mass ranging from 37 to 130 kg/mol. Proteins of molar mass of 130 and 120 kg/mol were identified to be β and β subunits, respectively. The role of other subunits of the enzyme is discussed.

Effect of ethionine on the synthesis of β-galactosidase inEscherichia coli

Ethionine at concentrations of 10−3M, 5×10−3M and 10−2M inhibits growth, both of β-galactosidase inducible ML-30 and constitutive ML-308Escherichia coli strains. The protein synthesis (measured by the incorporation of l-leucine-14C and l-aspartic-14C acid into proteins) of these strains is inhibited to the same extent as their growth. The synthesis of inducible and constitutive β-galactosidase produced by the strains ML-30 and ML-308, respectively, is considerably inhibited by ethionine.

Some Properties of DNA-Dependent RNA Polymerase from Streptomyces granaticolor

RNA nueleotidyltransferase fromStreptomyces granaticolor was purified and some of its properties were investigated. The temperature optimum of the enzyme is 35 °C, pH optimum 7.8—8.4. Antibodies against the β subunit of the enzyme fromBacillus subtilis immunologieally cross-react with the β subunit of the enzyme fromS. granaticolor. Antibodies against the β′ subunit of the enzyme fromB. subtilis immunologically cross-react with both β and β′subunits of the enzyme fromS. granaticolor.

The synthesis of β-galactosidase inEscherichia coli in the presence of 7-azatryptophan

The presence of 7-azatryptophan an analogue of tryptophan in the growth medium ofEscherichia coli resulted in a considerable inhibition of the synthesis of active β-galactosidase. No synthesis of an immunologically cross-reacting protein was detected. In addition, the replacement of tryptophan by the analogue rendered the enzyme more susceptible to heat, urea and trypsin as compared with the normal enzyme. The inhibition of growth and enzyme synthesis by 7-azatryptophan was reversed by tryptophan. The analogue did not exhibit any effect on the synthesis and activity of β-galactoside permease.

Further observations on the effect of ethionine on the synthesis of β-galactosidase inEscherichia coli: Formation of an immunologically cross-reacting protein

It was found that ethionine partially inhibits the transport of the inducer (TMG) of β-galactosidase into the cells ofEscherichia coli ML-30. The synthesis of β-galactosidase-specific messenger RNA is not inhibited. Ethionine appears to be incorporated into proteins synthesized by the strains used. The incorporation of ethionine into the molecule of β-galactosidase results in the synthesis of an enzymically inactive, immunologically cross-reacting protein.

DNA-Dependent RNA Polymerase from Streptomyces Granaticolor

DNA-Dependent RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6) plays an important role in the transcription of genetic information. This process is assumed to be regulated by the level and specificity of RNA polymerase. Although RNA polymerases of E.coli and B.subtilis have been studied in most detail, a. number of papers concerned with the isolation and characterization of this enzyme from other microorganisms have already been published (for a review see refs.l and 2). Data concerning the isolation of the enzyme from streptomycetes are quite scant and include primarily the results obtained with S.mediterranei (3), S.antibioticus (4,5,6), S.aureofaciens (7), S.hygroscopicus (8) and S.coelicolor (Westfehling and Losick, cited after (9)).

Effect of granaticin on the activity of DNA-directed RNA polymerase fromStreptomyces granaticolor

DNA-directed RNA polymerase was isolated from wild-type and mutant (asporogenic and granaticin overproducing)Streptomyces granaticolor. The effect of granaticin on the enzyme activity was compared with that of standard transcription inhibitors, rifampicin and rifamycin SV and shown to be zero.

Catabolite repression of different inducible enzymes inEscherichia coli and the effect of cAMP

Simultaneous induction of two enzymes sensitive to catabolite repression does not lead to an additive decrease of the specific activity of the two. Exogenously added cAMP increases the specific activity of catabolically repressed enzymes, irrespective of whether the enzyme is induced separately or simultaneously with another enzyme. In the presence of 12 different substrates metabolized by inducible enzymes glucose does not bring about catabolite repression. Synthesis of cAMP is identical with that occurring under conditions when glucose brings about catabolite repression.

The specific inhibition of the expression of the lactose operon by D, L-threo-phenylserine inEscherichia coli

Phenylserine, one of the phenylalanine analogues, is incorporated into proteins ofEscherichia coli and replaces the natural amino acid. The incorporation results in the inhibition of the synthesis of both inducible and constitutive β-galactosidase. The rate of the synthesis of β-galactosidase specific m-RNA is only slightly influenced by phenylserine, the steady-state level being decreased by about 40%. The m-RNA formed in the present of the analogue functions normally and its translation after the removal of the inhibitor results in the formation of normal β-galactosidase. The character of the inhibition of the enzyme synthesis by phenylserine is similar to that caused by chloramphenicol. However, phenylserine specifically inhibits only the synthesis of β-galactosidase, whereas other cell proteins are synthesized. No protein immunologically cross-reacting with the antiserum against normal β-galactosidase is formed by inducible ánd constitutiveEscherichia coli strains. The active transport is completely inhibited as the cells induced in the presence of phenylserine do not accumulate14C-TMG. It follows from the results that phenylserine inhibits both the formation of TMG-specific permease and the synthesis of the active molecule of β-galactosidase inEscherichia coli.