Properties of chorismate mutase ofStreptomyces venezuelae

Zusammenfassung Aus Kulturen von Streptomyces collinus (Stamm Tü 365) wurde ein neues, gelbes Antibioticum isoliert und durch analytische, spektroskopische und mikrobiologische Daten charakterisiert.

---

Metabolic products of microorganisms99. Kirromycin

Summary Kirromycin, a new yellow antibiotic, has been isolated from cultures of strain Tü 365 of Streptomyces collinus. It has been characterized by analytical, spectroscopic, and microbiological data.

---

---

98. Mitt.: Bayer, E., Gugel, K. H., Hägele, K., Hagenmaier, H., Jessipow, S., König, W. A., Zähner, H.: Helv. chim. Acta 55, 224–239 (1972).     

Identification and activation of novel biosynthetic gene clusters by genome mining in the kirromycin producer <Emphasis Type="Italic">Streptomyces collinus</Emphasis> Tü 365

Streptomycetes are prolific sources of novel biologically active secondary metabolites with pharmaceutical potential. S. collinus Tü 365 is a Streptomyces strain, isolated 1972 from Kouroussa (Guinea). It is best known as producer of the antibiotic kirromycin, an inhibitor of the protein biosynthesis interacting with elongation factor EF-Tu. Genome Mining revealed 32 gene clusters encoding the biosynthesis of diverse secondary metabolites in the genome of Streptomyces collinus Tü 365, indicating an enormous biosynthetic potential of this strain. The structural diversity of secondary metabolisms predicted for S. collinus Tü 365 includes PKS, NRPS, PKS-NRPS hybrids, a lanthipeptide, terpenes and siderophores. While some of these gene clusters were found to contain genes related to known secondary metabolites, which also could be detected in HPLC–MS analyses, most of the uncharacterized gene clusters are not expressed under standard laboratory conditions. With this study we aimed to characterize the genome information of S. collinus Tü 365 to make use of gene clusters, which previously have not been described for this strain. We were able to connect the gene clusters of a lanthipeptide, a carotenoid, five terpenoid compounds, an ectoine, a siderophore and a spore pigment-associated gene cluster to their respective biosynthesis products.     

A novel tyrosine-phosphorylated protein inhibiting the growth of Streptomyces cells

Very few of the tyrosine-phosphorylated proteins in Streptomyces have been identified. Here, we identify a tyrosine-phosphorylated protein from Streptomyces coelicolor A3(2), designated as SCO5717. The protein possesses Walker motifs and a tyrosine cluster at the C-terminus. When sco5717 harboring its own promoter was introduced into the S. coelicolor cell, the growth was inhibited. An sco5717-disrupted mutant formed aerial mycelium earlier than the wild-type strain, suggesting that SCO5717 controls the cell growth of S. coelicolor. Although the recombinant SCO5717 showed an ATPase activity, it lacked self-phosphorylation ability, suggesting that SCO5717 is a novel tyrosine-phosphorylated protein, which is distinguishable from bacterial protein tyrosine kinases known so far.     

Protein synthesis elongation factor EFTu from streptomyces collinus producing kirromycin

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.     

Identification and biochemical characterization of a 5-dimethylallyl tryptophan synthase in Streptomyces coelicolor A3(2)

The genome sequencing of Streptomyces coelicolor A3(2) has lead to the identification of numerous cryptic gene clusters involved in the biosynthesis of secondary metabolites; throwing open the challenge of identifying the enzymatic functions that the gene clusters are associated with. In this work, we report the biochemical characterization of one such cryptic gene, SCO7467 from S. coelicolor A3(2), which is annotated as a prenyltransferase. Based on LC–MS and 2D-NMR studies, we show that SCO7467 acts as a 5-dimethylallyl tryptophan synthase (5-DMATS), and catalyzes the transfer of a dimethylallyl group to the C-5 position of the indole ring of l-tryptophan. The studies indicate that SCO7467 could be involved in the synthesis of C-5 prenylated indole alkaloids, which may exhibit unique pharmacological and biological properties.     

Novel Tryptophan Metabolism by a Potential Gene Cluster That Is Widely Distributed among Actinomycetes

The characterization of potential gene clusters is a promising strategy for the identification of novel natural products and the expansion of structural diversity. However, there are often difficulties in identifying potential metabolites because their biosynthetic genes are either silenced or expressed only at a low level. Here, we report the identification of a novel metabolite that is synthesized by a potential gene cluster containing an indole prenyltransferase gene (SCO7467) and a flavin-dependent monooxygenase (FMO) gene (SCO7468), which were mined from the genome of Streptomyces coelicolor A3(2). We introduced these two genes into the closely related Streptomyces lividans TK23 and analyzed the culture broths of the transformants. This process allowed us to identify a novel metabolite, 5-dimethylallylindole-3-acetonitrile (5-DMAIAN) that was overproduced in the transformant. Biochemical characterization of the recombinant SCO7467 and SCO7468 demonstrated the novel l-tryptophan metabolism leading to 5-DMAIAN. SCO7467 catalyzes the prenylation of l-tryptophan to form 5-dimethylallyl-l-tryptophan (5-DMAT). This enzyme is the first actinomycetes prenyltransferase known to catalyze the addition of a dimethylallyl group to the C-5 of tryptophan. SCO7468 then catalyzes the conversion of 5-DMAT into 5-dimethylallylindole-3-acetaldoxime (5-DMAIAOx). An aldoxime-forming reaction catalyzed by the FMO enzyme was also identified for the first time in this study. Finally, dehydration of 5-DMAIAOx presumably occurs to yield 5-DMAIAN. This study provides insight into the biosynthesis of prenylated indoles that have been purified from actinomycetes.     

Molecular characterization of SCO0765 as a cellotriose releasing endo-<Emphasis Type="Italic">β</Emphasis>-1,4-cellulase from <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A(3)

The sco0765 gene was annotated as a glycosyl hydrolase family 5 endoglucanase from the genomic sequence of Streptomyces coelicolor A3(2) and consisted of 2,241 bp encoding a polypeptide of 747 amino acids (molecular weight of 80.5 kDa) with a 29-amino acid signal peptide for secretion. The SCO0765 recombinant protein was heterogeneously over-expressed in Streptomyces lividans TK24 under the control of a strong ermE* promoter. The purified SCO0765 protein showed the expected molecular weight of the mature form (718 aa, 77.6 kDa) on sodium dodecyl sulfate-polyacryl amide gel electrophoresis. SCO0765 showed high activity toward β-glucan and carboxymethyl cellulose (CMC) and negligible activity to Avicel, xylan, and xyloglucan. The SCO0765 cellulase had a maximum activity at pH 6.0 and 40°C toward CMC and at pH 9.0 and 50–60°C toward β-glucan. Thin layer chromatography of the hydrolyzed products of CMC and β-glucan by SCO0765 gave cellotriose as the major product and cellotetraose, cellopentaose, and longer oligosaccharides as the minor products. These results clearly demonstrate that SCO0765 is an endo-β-1,4-cellulase, hydrolyzing the β-1,4 glycosidic bond of cellulose into cellotriose.     

A possible extended family of regulators of sigma factor activity in Streptomyces coelicolor

SCO4677 is one of a large number of similar genes in Streptomyces coelicolor that encode proteins with an HATPase_c domain resembling that of anti-sigma factors such as SpoIIAB of Bacillus subtilis. However, SCO4677 is not located close to genes likely to encode a cognate sigma or anti-anti-sigma factor. SCO4677 was found to regulate antibiotic production and morphological differentiation, both of which were significantly enhanced by the deletion of SCO4677. Through protein-protein interaction screening of candidate sigma factor partners using the yeast two-hybrid system, SCO4677 protein was found to interact with the developmentally specific σ^F, suggesting that it is an antagonistic regulator of σ^F. Two other proteins, encoded by SCO0781 and SCO0869, were found to interact with the SCO4677 anti-σ^F during a subsequent global yeast two-hybrid screen, and the SCO0869-SCO4677 protein-protein interaction was confirmed by coimmunoprecipitation. The SCO0781 and SCO0869 proteins resemble well-known anti-anti-sigma factors such as SpoIIAA of B. subtilis. It appears that streptomycetes may possess an extraordinary abundance of anti-sigma factors, some of which may influence diverse processes through interactions with multiple partners: a novel feature for such regulatory proteins.     

Analysis of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> M145 genes <Emphasis Type="Italic">SCO4164</Emphasis> and <Emphasis Type="Italic">SCO5854</Emphasis> encoding putative rhodaneses

Streptomyces coelicolor genome carries two apparently paralogous genes, SCO4164 and SCO5854, that encode putative thiosulfate sulfurtransferases (rhodaneses). These genes (and their presumed translation products) are highly conserved and widely distributed across actinobacterial genomes. The SCO4164 knockout strain was unable to grow on minimal media with either sulfate or sulfite as the sole sulfur source. The SCO5854 mutant had no growth defects in the presence of various sulfur sources; however, it produced significantly less amounts of actinorhodin. Furthermore, we discuss possible links between basic interconversions of inorganic sulfur species and secondary metabolism in S. coelicolor.     

Stoffwechselprodukte von Mikroorganismen

Zusammenfassung Der Streptomycet Tü 901, Streptomyces tendae, bildet ein antifungisch wirkendes Nukleosid-Antibioticum, Nikkomycin. Als Angriffsort kommt die Chitinsynthese in Frage.Mit Hilfe der Massenspektrometrie und des chemischen Abbaus konnten Uracil, eine Aminohexuronsäure und eine neue, einen Pyridinring enthaltende Aminosäure nachgewiesen werden.

---

Metabolic products of microorganisms154. Nikkomycin, a new inhibitor of fungal chitin synthesis

From the fermentation broth of Streptomyces tendae Tü 901 a substance was isolated, which inhibits the growth of several fungi. The new antibiotic affects the chitchin biosynthesis. Its structure was identified by mass spectrometry of the products obtained after chemical degradation. Nikkomycin is a nucleoside-peptide antibiotic consisting of uracil, an amino hexuronic acid and a new amino acid containing a pyridin ring.

153. Mitteilung: G. Wolf, J. Wörth, H. Achenbach: Untersuchungen der Pigmente aus Streptomyces michiganesis. Arch. Microbiol. 106, 245–249 (1975)     

Polyhydroxyalkanoate accumulation in Streptomyces coelicolor affected by SCO7613 gene region

Polyhydroxyalkanoate (PHA) is stored as an important carbon and energy source in bacterial cells. For biomedical applications, gram-positive bacteria can be better sources of PHAs, since they lack outer membrane lipopolysaccharide. Although gram-positive Streptomyces coelicolor A3(2) has been indicated as a high potential PHA producer, pha C gene that encodes the key enzyme PHA synthase in the metabolic pathway is not determined in its genome. BLAST search results of the GenBank database argued that SCO7613 could specify a putative polyhydroxyalkanoate synthase (PhaC) responsible for PHA biosynthesis. Deduced amino acid sequence of SCO7613 showed the presence of conserved lipase box like sequence, ⁵⁵⁵GASAG⁵⁵⁹, in which serine residue was present as the active nucleophile. Present study describes deletion of putative S. coelicolor pha C gene via PCR dependent method. We showed that SCO7613 is not an essential gene in S. coelicolor and its deletion affected PHA accumulation negatively although it is not ceased. Transcomplementation abolished the mutant phenotype, demonstrating that the decrease in PHA resulted from the deletion of SCO7613.     

Zur Entwicklung des Subcommissuralorgans der Ratte Morphologische und histochemische Untersuchungen

Zusammenfassung Die Entwicklung des Subcommissuralorgans (SCO) der Ratte wurde morphologisch und histochemisch untersucht. Zwischen dem 15. und 16. Embryonaltag steigt mit Ausbildung der hochprismatischen Ependymzellen die Enzymaktivität des Energiestoff-wechsels (GAP-DH, LDH, SDH, CyO) deutlich an, wobei die anaerobe Glykolyse überwiegt. Die gleichzeitige Aktivitätszunahme der G6P-DH, NADPH₂-Reduktase, Thiaminpyrophos-phatase und sauren Phosphatase führt zu dem für das Ependym der adulten Ratte charakteristischen Enzymmuster. Dieser bis zum 18. Tag anhaltende Anstieg wird mit der beginnenden sekretorischen Tätigkeit in Beziehung gebracht. Der Glykogengehalt nimmt ebenfalls allmählich zu. Nach der Geburt fällt die Enzymaktivität des Energiestoffwechsels allmählich ab. Der Glykogengehalt und die Zahl apikaler Protrusionen nehmen anfänglich noch zu, dann aber ebenfalls langsam ab. Es wird auf einen Rückgang der sekretorischen Aktivität geschlossen.An dem nur spärlich ausgebildeten Hypendym treten erst in der 3. Lebenswoche ähnliche Änderungen der Enzymaktivitäten und des Sekretgehalts auf. Mit dieser verzögerten Entwicklung des Hypendyms schließt die Differenzierung des SCO in der 4. Lebenswoche ab.Die Anlage eines besonderen Ependymbereichs, der okzipital an das SCO anschließt, wird beschrieben. Vor der Geburt weist er dem SCO vergleichbare morphologische und histochemische Eigenschaften auf, fehlt jedoch bei der erwachsenen Ratte. Die mögliche Bedeutung dieser Kückbildung wird diskutiert.

---

The development of the subcommissural organ of the rat morphological and histochemical investigations

Summary The development of the Subcommissural organ (SCO) of the rat has been investigated morphologically and histochemically. Between the 15th and 16th prenatal day elongated ependymal cells appear. The activities of enzymes of the energy metabolism (GAP-DH, LDH, SDH, CyO), particularly of the anaerobic glycolysis, increase. With the synchronous increase of activities of G6P-DH, NADPH₂-reductase, thiamine pyrophosphatase and acid phosphatase, an enzyme pattern appears which is characteristic for the ependyma of the adult rat. This increase which is observed to the 18th day may correspond to the onset of the secretory activity. The content of glycogen increases gradually. After birth the enzyme activities of the energy metabolism decrease gradually. After an initial increase, the glycogen content and the amount of apical protrusions decrease, too. The amount of secretory material increases till the 10th day, and remains unchanged thereafter. This may reflect a decrease in secretory function.The hypendyma is poorly developed. Similar changes in enzyme activities and secretory content appear in the 3rd postnatal week. In the 4th week the differentiation of the SCO is completed.The anlage of a specialized ependyma adjoning the SCO caudally is described. Prenatally it has some of the morphological and histochemical characteristics of the SCO. It has not been observed in the adult rat. The possible significance of this involution is discussed.

---

---

Frau I. Köhl danken wir für ihre sorgfältige technische Hilfe, Frau H. Asam für die Durch-führung der Photoarbeiten.

---

Die vorliegende Arbeit ist ein wesentlicher Teil der Inauguraldissertation, die von Thomas Linderer der Medizinischen Fakultät der Universität München vorgelegt wird.