A time-correlated expression of eukaryotic-like protein Ser/Thr kinase Pkg2 of Streptomyces granaticolor was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) and by transcriptional fusion experiments. In a complex medium the activity of pkg2 promoter was constant during the life cycle. Direct RNA analysis proved the presence of corresponding pkg2 transcript. S1 nuclease protection analysis of the transcription initiation site showed that pkg2 gene is expressed as a leaderless mRNA. Under phosphate starvation the promoter activity was detectable merely in the early exponential phase. Under these conditions turning off of pkg2 promoter and cessation of pkg2 transcript level coincided with the start of granaticin production.

• MeSH
• Phosphates metabolism   MeSH
• Transcription, Genetic MeSH
• Catechol 2,3-Dioxygenase metabolism   MeSH
• Culture Media MeSH
• Promoter Regions, Genetic MeSH
• Protein Serine-Threonine Kinases genetics   metabolism   MeSH
• Gene Expression Regulation, Bacterial * MeSH
• Streptomyces enzymology   genetics   growth & development   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phosphates MeSH
• Catechol 2,3-Dioxygenase MeSH
• Culture Media MeSH
• Protein Serine-Threonine Kinases MeSH

A 4.2-kb SphI-BamHI fragment of chromosomal DNA from Streptomyces granaticolor was cloned and shown to encode a protein with significant sequence similarity to the eukaryotic protein serine/threonine kinases. It consists of 701 amino acids and in the N-terminal part contains all conserved catalytic domains of protein kinases. The C-terminal domain of Pkg2 contains seven tandem repeats of 11 or 12 amino acids with similarity to the tryptophan-docking motif known to stabilize a symmetrical three-dimensional structure called a propeller structure. The pkg2 gene was overexpressed in Escherichia coli, and the gene product (Pkg2) has been found to be autophosphorylated at serine and threonine residues. The N- and C-terminal parts of Pkg2 are separated with a hydrophobic stretch of 21 amino acids which translocated a PhoA fusion protein into the periplasm. Thus, Pkg2 is the first transmembrane protein serine/threonine kinase described for streptomycetes. Replacement of the pkg2 gene by the spectinomycin resistance gene resulted in changes in the morphology of aerial hyphae.

• MeSH
• Genes, Bacterial MeSH
• DNA, Bacterial genetics   MeSH
• DNA Primers genetics   MeSH
• Escherichia coli genetics   MeSH
• Gene Expression MeSH
• Phenotype MeSH
• Phosphorylation MeSH
• Cloning, Molecular MeSH
• Microscopy, Electron, Scanning MeSH
• Molecular Sequence Data MeSH
• Mutation MeSH
• Protein Serine-Threonine Kinases chemistry   genetics   metabolism   MeSH
• Recombinant Fusion Proteins chemistry   genetics   metabolism   MeSH
• Restriction Mapping MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Streptomyces enzymology   genetics   ultrastructure   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Bacterial MeSH
• DNA Primers MeSH
• Protein Serine-Threonine Kinases MeSH
• Recombinant Fusion Proteins MeSH

Protoplasts of Streptomyces granaticolor were found to be transformable by the broad-host-range plasmid pIJ350 but no transformants were detected when the narrow-host-range plasmid pIJ2 or the shuttle vector pPM66 (pIJ350--pBR322) isolated from E. coli cells were used. The onset of blue colour granaticin production by S. granaticolor cells was used as a marker to prepare protoplasts with a high transformation capacity. The presence of a restriction system is discussed.

• MeSH
• Transformation, Bacterial * MeSH
• Plasmids * MeSH
• Recombination, Genetic * MeSH
• DNA Restriction Enzymes MeSH
• Spores, Bacterial MeSH
• Streptomyces genetics   growth & development   physiology   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA Restriction Enzymes MeSH

beta-D-glucosidase in Streptomyces granaticolor is an inducible enzyme. Methyl-beta-D-glucoside or cellobiose, added to a glycerol-containing medium, are most suitable inducers. The activity of beta-D-glucosidase in a culture fully induced by cellobiose is 50 times higher than the basal level of the enzyme. beta-D-glucosidase is an intracellular enzyme, whose inducibility differ with culture age and reaches its maximum in a 10-h-old mycelium. The enzyme synthesis begins 2 h after the addition of the induced and reaches its maximum after a 10-h-induction.

• MeSH
• beta-Glucosidase biosynthesis   MeSH
• Cell-Free System MeSH
• Cellobiose metabolism   MeSH
• Enzyme Induction MeSH
• Glucosidases biosynthesis   MeSH
• Hydrogen-Ion Concentration MeSH
• Streptomyces enzymology   growth & development   metabolism   MeSH
• Temperature MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• beta-Glucosidase MeSH
• Cellobiose MeSH
• Glucosidases MeSH

Nonfilamentous forms of Streptomyces granaticolor are formed in a medium with amino acids and glucose. They form filaments again after transfer to a medium with glucose and peptone. The nonfilamentous forms do not produce granaticin. Formation of nonfilamentous forms depends on the concentration of the inoculum, on the cultivation temperature and on the presence of simple sugars. Ultrathin sections revealed atypical septation in the nonmycelial forms and non-uniform accumulation of the wall material.

• MeSH
• Culture Media MeSH
• Streptomyces growth & development   ultrastructure   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Culture Media MeSH

The rates of RNA, protein and DNA synthesis were estimated in synchronously germinating spores of Streptomyces 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 of Streptomyces 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.

• MeSH
• RNA, Bacterial biosynthesis   MeSH
• DNA, Bacterial biosynthesis   MeSH
• Macromolecular Substances MeSH
• Carbon Radioisotopes MeSH
• Spores, Bacterial * MeSH
• Streptomyces metabolism   physiology   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• RNA, Bacterial MeSH
• DNA, Bacterial MeSH
• Macromolecular Substances MeSH
• Carbon Radioisotopes MeSH

The interaction of granaticin B, a quinone antibiotic produced by Streptomyces granaticolor, with some biologically important bivalent metal ions, DNA and ATP was demonstrated spectrophotometrically. The activity of isolated RNA polymerase was higher when the DNA of phage SP 50 served as template than with DNA isolated from Bacillus subtilis. Granaticin B inhibited in 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 epsilon-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 inhibited in vitro by granaticin B but this does not appear to be the only site of action of this antibiotic in vivo.

• MeSH
• Adenosine Triphosphate MeSH
• Bacillus subtilis growth & development   metabolism   MeSH
• RNA, Bacterial biosynthesis   MeSH
• Bacteriophages MeSH
• Benzofurans pharmacology   MeSH
• DNA-Directed RNA Polymerases metabolism   MeSH
• DNA MeSH
• Transcription, Genetic drug effects   MeSH
• Templates, Genetic MeSH
• Magnesium MeSH
• Spectrophotometry MeSH
• Terpenes pharmacology   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• RNA, Bacterial MeSH
• Benzofurans MeSH
• DNA-Directed RNA Polymerases MeSH
• DNA MeSH
• Magnesium MeSH
• Terpenes MeSH