Discovery of New Siderophores from a Marine Streptomycetes sp. via Combined Metabolomics and Analysis of Iron-Chelating Activity.

The marine-derived Streptomyces sp. FIMYZ-003 strain was found to produce novel siderophores with yields negatively correlated with the iron concentration in the medium. Mass spectrometry (MS)-based metabolomics coupled with metallophore assays identified two novel α-hydroxycarboxylate-type siderophores, fradiamines C and D (3 and 4), together with two related known siderophores, fradiamines A and B (1 and 2). Their chemical structures were elucidated by nuclear magnetic resonance (NMR) and MS experiments. The annotation of a putative fra biosynthetic gene cluster enabled us to propose the biosynthetic pathway of fradiamines A-D. Furthermore, the solution-phase iron-binding activity of fradiamines was evaluated using metabolomics, confirming them as general iron scavengers. Fradiamines A-D exhibited Fe(III) binding activity equivalent to that of deferoxamine B mesylate. Growth analysis of pathogenic microbes demonstrated that fradiamine C promoted the growth of Escherichia coli and Staphylococcus aureus, but fradiamines A, B, and D did not. The results indicate that fradiamine C may serve as a novel iron carrier applicable to antibiotic delivery strategies to treat and prevent foodborne pathogens.

The potent protein phosphatase 2A inhibitors aminocytostatins: new derivatives of cytostatin.

Specific inhibitors of protein phosphatase 2A (PP2A) mediate anticancer effects by augmenting the tumor-killing activity of natural killer (NK) cells. In this study, new PP2A inhibitors, aminocytostatins A-E, were isolated from Kitasatospora sp. MJ654-NF4 and structurally characterized. Aminocytostatins are derivatives of cytostatin, which is a specific PP2A inhibitor isolated from the same organism, and aminocytostatins have a characteristic amino group within the lactone moiety. Compared to cytostatin, aminocytostatin A showed a stronger inhibitory activity against PP2A in vitro and augmented the tumor-killing activity of NK cells in vivo. Furthermore, a docking model was generated to demonstrate the favorable activities of aminocytostatin A.

Complementary Genomic, Bioinformatics, and Chemical Approaches Facilitate the Absolute Structure Assignment of Ionostatin, a Linear Polyketide from a Rare Marine-Derived Actinomycete.

A new linear type-1 polyketide, ionostatin (1), has been fully defined using a combined genomic and bioinformatics approach coupled with confirmatory chemical analyses. The 41 carbon-containing polyether is the product of the 101 kbp ion biosynthetic cluster containing seven modular type-1 polyketide synthases. Ionostatin is composed of 15 chiral centers that were proposed using the stereospecificities installed by the different classes of ketoreductases and enoylreductases and confirmed by rigorous NMR analyses. Incorporated into the structure are two tetrahydrofuran rings that appear to be the product of stereospecific epoxidation, followed by stereospecific ring opening and cyclization. These transformations are proposed to be catalyzed by conserved enzymes analogous to those found in other bacterial-derived polyether biosynthetic clusters. Ionostatin shows moderate cancer cell cytotoxicity against U87 glioblastoma and SKOV3 ovarian carcinoma at 7.4 µg/mL.

Photopiperazines A-D, Photosensitive Interconverting Diketopiperazines with Significant and Selective Activity against U87 Glioblastoma Cells, from a Rare, Marine-Derived Actinomycete of the Family Streptomycetaceae.

Photopiperazines A-D (1-4), unsaturated diketopiperazine derivatives, were isolated from the culture broth of a rare, marine-derived actinomycete bacterium, strain AJS-327. This strain shows very poor 16S rRNA sequence similarity to other members of the actinomycete family Streptomycetaceae, indicating it is likely a new lineage within this group. The structures of the photopiperazines were defined by analysis of HR-ESI-TOF-MS spectra in conjunction with the interpretation of 1D and 2D NMR data. The photopiperazines are sensitive to light, causing interconversion among the four olefin geometrical isomers, which made purification of each isomer challenging. The photopiperazines are highly cytotoxic metabolites that show selective toxicity toward U87 glioblastoma and SKOV3 ovarian cancer cell lines.

[ANTINEMATICIDAL ACTIVITY OF METABOLITES PRODUCED BY SOIL STREPTOMYCETE].

GOAL: To study in vitro the antinematicide activity of soil streptomycetes metabolite and bioformulations based on them against root-knot, cyst and leaf nematodes of species. METHODS: Action of streptomycetes metabolites and their bioformulations were determined in vitro against larvae at two age of root-knot nematode M incognita (Kofoid & White, 1919) Chitwood 1949 and sugar beet cyst nematode H. schachtii A. Schmidt 1871, and leaf nematodes ofAphlenchoides genus by cultivation them in solutions of biomass ethanol extracts separate individual components metabolites and bioformulations during 24 hours. Nematicide and nematistatic effects of the substances were evaluated by the change in the motility activity of the nematodes. RESULTS: Soil streptomycetes S. violaceus IMV Ac-5027, S. averinitilis IMV Ac-5015 and metabolite bioformulations based on them (Violar, Aver- com and Avercom-nova, respectively) showed significant nematicidal activity against plant pathogenic nematodes M incognita and H. schachtii. The biomass extracts of S. avermiti- lis IMV Ac-5015 and S. violaceus IMV Ac-5027 caused 100 % death of nematodes, and S. netropsis IMV Ac-5025 caused only 75 % mortality during 24 hours of action. Using an antibiotic complex from S. violaceus IMV Ac-5027 was divided on six fractions that were different from the avermectin complex. Purified fractions of individual antibiotic complex of S. violaceus IMV Ac-5 027 have a negative impact against the root-knot nematode M incognita. The highest nematicidal activity showed fractions #4 and #5 since their efficiency was over 90 % after a 0.5 hours of action. Nematicidal efficiency of fractions ## 1, 2, 3 and 6 remained between 72.6-86.2 % during 24 hours of action. Fractions 4 and 5 showed high nematicidal effectiveness against leaf nematodes of Aphelenchoides genus where 90-95 % level of helminthes death was observed after 4 hours of action. Among created complex metabolite bioformulations Avercom-nova was the most effective which caused 100 % mortality of nematodes M incognita and H. schachtii after 24 hours of action. Bioformu- lation Violar caused 96.6-96.8 and 95.2-97.0 % of death of nematodes M incognita and H. schachtii, after 24 hours of action. Phytovit based on S. netropsis IMV Ac-5025 showed low nematicide activity against of nematodes, which did not exceed 15.2-18.4 % of their death after 24 hour of action. Bioformulations based on streptomycetes metabolites did not inferior to chemical insecticide Konfidor Maxi, which caused the death of 93.3-94.6 % nematodes at doses recommended by the manufacturer. CONCLUSION: Soil streptomycetes S. violaceus IV Ac-5027 and S. avermitilis IMV Ac-5015 are promising producers for creation of bioformulations with nematicidal activity against plant-parasitic nematode of M incoenita and H. schachtii.

Streptacidiphilus gen. nov., acidophilic actinomycetes with wall chemotype I and emendation of the family Streptomycetaceae (Waksman and Henrici (1943)AL) emend. Rainey et al. 1997.

The taxonomic position of acidophilic actinomycetes selectively isolated from acidic soils and litter was examined using a polyphasic approach. The distinct 16S rDNA phyletic branch formed by representative strains was equated with related monophyletic clades that corresponded to the genera Kitasatospora and Streptomyces. The acidophilic isolates also exhibited a distinctive pH profile, a unique 16S rDNA signature, and contained major amounts of LL-diaminopimelic acid, galactose and rhamnose in whole-organism hydrolysates. It is proposed that these acidophilic actinomycetes be assigned to a new genus, Streptacidiphilus gen. nov., on the basis of genotypic and phenotypic differences. Three species were defined on the basis of DNA:DNA pairing and phenotypic data, namely, Streptacidiphilus albus sp. nov., the type species, Streptacidiphilus neutrinimicus sp. nov. and Streptacidiphilus carbonis sp. nov. Members of the genera Kitasatospora, Streptacidiphilus and Streptomyces share a number of key characteristics and form a stable monophyletic branch in the 16S rDNA tree. It is, therefore, proposed that the description of the family Streptomycetaceae be emended to account for properties shown by Kitasatospora and Streptacidiphilus species.

A novel peptide probe for studying the transbilayer movement of phosphatidylethanolamine.

Ro09-0198 is a cyclic peptide isolated from Streptoverticillium griseoverticillatum which recognizes strictly the structure of phosphatidylethanolamine (PE) and forms an equimolar complex with the phospholipid on biological membranes. To use the peptide as a probe for analyzing the transbilayer movement of PE, we labeled the amino-terminal amino acid of the peptide with biotin without changing either the reactivity or specificity of the peptide. The amount of the peptide bound to the membrane was measured by enzyme linked immunosorbent assay (ELISA) after extraction of the peptide from the membrane. The peptide showed a strict temperature-dependent binding to human erythrocytes and the binding increased with increasing temperature. Since the peptide bound to PE in a temperature-independent manner and the binding to membrane PE is not affected by membrane proteins, the present temperature-dependent binding of the peptide to the cell membranes was likely to reflect temperature-dependent translocation of PE. The binding of the peptide to erythrocytes differed greatly among animal species. The peptide also showed temperature-dependent binding to a human histocytic lymphoma cell line, U937, suggesting that the peptide will provide a novel and convenient probe for analyzing the transbilayer movement of PE in eukaryotic cells.

Primary structure and reactive site of Streptoverticillium anticoagulant (SAC), a novel protein inhibitor of blood coagulation produced by Streptoverticillium cinnamoneum subsp. cinnamoneum.

The complete amino acid sequence of SAC I, a novel protein inhibitor of blood coagulation produced by Streptoverticillium cinnamoneum subsp. cinnamoneum IFO 12852, was determined. Automated Edman degradation was employed for its fragment peptides, which were obtained by specific cleavage procedures including tryptic, chymotryptic, and peptic digestions, and cyanogen bromide treatment. SAC I is composed of 110 amino acid residues with a molecular weight of 11,642. It has two intramolecular disulfide bonds, Cys31-Cys46 and Cys68-Cys98, but no cysteine residues. The overall sequence homology of SAC I is 58% to plasminostreptin, a protein protease inhibitor produced by Streptomyces antifibrinolyticus, and 52% to S-SI, Streptomyces subtilisin inhibitor, produced by Streptomyces albogriseolus. Subtilisin [EC 3.4.21.14]-modified SAC I, which was prepared by incubating SAC I with subtilisin, had a newly-generated amino terminal sequence, Glu71-Trp72-Asn73-, in addition to the original amino terminal sequence, and a newly-generated carboxyl terminal arginine in addition to the original phenylalanine. These results clearly show that the Arg70-Glu71 bond was specifically cleaved on the limited proteolysis with subtilisin and that the bond is the relative site for subtilisin.

New teleocidin-related metabolites, (-)-7-geranylindolactam V and blastmycetin F, from Streptoverticillium blastmyceticum.

Two new teleocidin-related metabolites, (-)-7-geranylindolactam-V [2] and blastmycetin F [3], were isolated from fermentation broths of the actinomycete Streptoverticillium blastmyceticum NA34-17, and their structures were determined by spectroscopic methods. Compound 2 bound strongly to phorbol ester receptors in a mouse epidermal particulate fraction, suggesting that it is a potent in vivo tumor promoter comparable to teleocidins A-1 [4] and B-4 [5].