Abyssomicins-A 20-Year Retrospective View.

Abyssomicins represent a new family of polycyclic macrolactones. The first described compounds of the abyssomicin family were abyssomicin B, C, atrop-C, and D, produced by the marine actinomycete strain Verrucosispora maris AB-18-032, which was isolated from a sediment collected in the Sea of Japan. Among the described abyssomicins, only abyssomicin C and atrop-abyssomicin C show a high antibiotic activity against Gram-positive bacteria, including multi-resistant and vancomycin-resistant strains. The inhibitory activity is caused by a selective inhibition of the enzyme 4-amino-4-deoxychorismate synthase, which catalyzes the transformation of chorismate to para-aminobenzoic acid, an intermediate in the folic acid pathway.

Discovery of the Streptoketides by Direct Cloning and Rapid Heterologous Expression of a Cryptic PKS II Gene Cluster from Streptomyces sp. Tü 6314.

Genome sequencing and bioinformatic analysis have identified numerous cryptic gene clusters that have the potential to produce novel natural products. Within this work, we identified a cryptic type II PKS gene cluster (skt) from Streptomyces sp. Tü 6314. Facilitated by linear plus linear homologous recombination-mediated recombineering (LLHR), we directly cloned the skt gene cluster using the Streptomyces site-specific integration vector pSET152. Direct cloning allowed for rapid heterologous expression in Streptomyces coelicolor, leading to the identification and structural characterization of six polyketides (three known compounds and new streptoketides), four of which exhibit anti-HIV activities. Our study shows that the pSET152 vector can be directly used for LLHR, expanding the Rec/ET direct cloning toolbox and providing the possibility for rapid heterologous expression of gene clusters from Streptomyces.

Structures and biological activities of cycloheptamycins A and B.

The heptadepsipeptide cycloheptamycin A was isolated from the terrestrial Streptomyces sp. Tü 6314. Its constitution was elucidated on the basis of NMR spectroscopic experiments and mass spectrometric analysis. Its stereostructure was investigated by peptide hydrolysis and derivatization and firmly established by X-ray structure analysis. In addition to the parent compound, a new cycloheptamycin analog, cycloheptamycin B, was discovered and structurally assigned using comparative MS/MS experiments and NMR. The biological profile of both compounds was investigated, revealing a selective inhibitory potential of cycloheptamycins against Propionibacterium acnes.

Streptomyces AcH 505 triggers production of a salicylic acid analogue in the fungal pathogen Heterobasidion abietinum that enhances infection of Norway spruce seedlings.

The necrotrophic fungus Heterobasidion spp. is the causal agent of 'annosum root rot' of Norway spruce. In the presence of the rhizosphere bacterium Streptomyces AcH 505, enhanced colonization of Norway spruce roots with Heterobasidion abietinum 331 has previously been observed. By analyzing dual cultures of H. abietinum 331 and Streptomyces AcH 505 with HPLC, a fungal metabolite was identified that was increased in the presence of Streptomyces AcH 505. Likewise, challenge of H. abietum 331 with common antifungals produced by soil streptomycetes rendered the same effect. The structure of the compound, 5-formylsalicylic acid (5-FSA), was elucidated by HPLC-HR-ESI-Orbitrap-mass spectrometry and NMR spectroscopy. Based on in vivo measurements of maximum photosystem II efficiency of Norway spruce seedlings, 5-FSA did not influence plant vitality. However, when challenged with H. abietinum 331, ergosterol amounts in infected roots increased significantly for 5-FSA pre-treated seedlings. The severity of the infection was comparable to that observed in the presence of Streptomyces AcH 505. 5-FSA is a structural analogue of salicylic acid, an important signalling molecule active in plant defence. Thus, the expression of two defence-response related marker genes (PR1, Hel) was analysed in 5-FSA treated Arabidopsis thaliana seedlings by Northern blot analysis. The transcription of both marker genes was altered, indicating that 5-FSA is perceived by Arabidopsis in a similar manner to salicylic acid and is able to interfere with Arabidopsis defence signalling. The role of 5-FSA as a potential virulence factor of H. abietinum 331 in the presence of Streptomyces AcH 505 is discussed.

Atmospheric Dispersal of Bioactive Streptomyces albidoflavus Strains Among Terrestrial and Marine Environments.

Members of the Streptomyces albidoflavus clade, identified by 16S rRNA sequencing and phylogenetic analyses, are widespread among predominant terrestrial lichens (Flavoparmelia caperata and Xanthoria parietina) and diverse intertidal and subtidal marine macroalgae, brown red and green (Phylum Heterokontophyta, Rhodophyta, and Chlorophyta) from the Cantabrian Cornice. In addition to these terrestrial and coastal temperate habitats, similar strains were also found to colonize deep-sea ecosystems and were isolated mainly from gorgonian and solitary corals and other invertebrates (Phylum Cnidaria, Annelida, Echinodermata, Arthropoda, and Porifera) living up to 4700-m depth and at a temperature of 2-4 °C in the submarine Avilés Canyon. Similar strains have been also repeatedly isolated from atmospheric precipitations (rain drops, snow, and hailstone) collected in the same area throughout a year observation time. These ubiquitous strains were found to be halotolerant, psychrotolerant, and barotolerant. Bioactive compounds with diverse antibiotic and cytotoxic activities produced by these strains were identified by high-performance liquid chromatography (HPLC) and database comparison. These include antibacterials (paulomycins A and B), antifungals (maltophilins), antifungals displaying also cytotoxic activities (antimycins and 6-epialteramides), and the antitumor compound fredericamycin. A hypothetical dispersion model is here proposed to explain the biogeographical distribution of S. albidoflavus strains in terrestrial, marine, and atmospheric environments.

Genome Mining of Streptomyces sp. Tü 6176: Characterization of the Nataxazole Biosynthesis Pathway.

Streptomyces sp. Tü 6176 produces the cytotoxic benzoxazole nataxazole. Bioinformatic analysis of the genome of this organism predicts the presence of 38 putative secondary-metabolite biosynthesis gene clusters, including those involved in the biosynthesis of AJI9561 and its derivative nataxazole, the antibiotic hygromycin B, and ionophores enterobactin and coelibactin. The nataxazole biosynthesis gene cluster was identified and characterized: it lacks the O-methyltransferase gene required to convert AJI9561 into nataxazole. This O-methyltransferase activity might act as a resistance mechanism, as AJI9561 shows antibiotic activity whereas nataxazole is inactive. Moreover, heterologous expression of the nataxazole biosynthesis gene cluster in S. lividans JT46 resulted in the production of AJI9561. Nataxazole biosynthesis requires the shikimate pathway to generate 3-hydroxyanthranilate and an iterative type I PKS to generate 6-methylsalicylate. Production of nataxazole was improved up to fourfold by disrupting one regulatory gene in the cluster. An additional benzoxazole, 5-hydroxynataxazole is produced by Streptomyces sp. Tü 6176. 5-Hydroxynataxazole derives from nataxazole by the activity of an as yet unidentified oxygenase; this implies cross-talk between the nataxazole biosynthesis pathway and an unknown pathway.

Two Streptomyces species producing antibiotic, antitumor, and anti-inflammatory compounds are widespread among intertidal macroalgae and deep-sea coral reef invertebrates from the central Cantabrian Sea.

Streptomycetes are widely distributed in the marine environment, although only a few studies on their associations to algae and coral ecosystems have been reported. Using a culture-dependent approach, we have isolated antibiotic-active Streptomyces species associated to diverse intertidal marine macroalgae (Phyllum Heterokontophyta, Rhodophyta, and Chlorophyta), from the central Cantabrian Sea. Two strains, with diverse antibiotic and cytotoxic activities, were found to inhabit these coastal environments, being widespread and persistent over a 3-year observation time frame. Based on 16S rRNA sequence analysis, the strains were identified as Streptomyces cyaneofuscatus M-27 and Streptomyces carnosus M-40. Similar isolates to these two strains were also associated to corals and other invertebrates from deep-sea coral reef ecosystem (Phyllum Cnidaria, Echinodermata, Arthropoda, Sipuncula, and Anelida) living up to 4.700-m depth in the submarine Avilés Canyon, thus revealing their barotolerant feature. These two strains were also found to colonize terrestrial lichens and have been repeatedly isolated from precipitations from tropospheric clouds. Compounds with antibiotic and cytotoxic activities produced by these strains were identified by high-performance liquid chromatography (HPLC) and database comparison. Antitumor compounds with antibacterial activities and members of the anthracycline family (daunomycin, cosmomycin B, galtamycin B), antifungals (maltophilins), anti-inflamatory molecules also with antituberculosis properties (lobophorins) were identified in this work. Many other compounds produced by the studied strains still remain unidentified, suggesting that Streptomyces associated to algae and coral ecosystems might represent an underexplored promising source for pharmaceutical drug discovery.

Draft Genome Sequence of Marine Actinomycete Streptomyces sp. Strain NTK 937, Producer of the Benzoxazole Antibiotic Caboxamycin.

Streptomyces sp. strain NTK 937 is the producer of the benzoxazole antibiotic caboxamycin, which has been shown to exert inhibitory activity against Gram-positive bacteria, cytotoxic activity against several human tumor cell lines, and inhibition of the enzyme phosphodiesterase. In this genome announcement, we present a draft genome sequence of Streptomyces sp. NTK 937 in which we identified at least 35 putative secondary metabolite biosynthetic gene clusters.

Geodermatophilus brasiliensis sp. nov., isolated from Brazilian soil.

A Gram-reaction-positive bacterial isolate, designated Tü 6233(T), with rudimentary, coral-pink vegetative mycelium that formed neither aerial mycelium nor spores, was isolated from a Brazilian soil sample. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Geodermatophilus. Cell-wall hydrolysates contained meso-diaminopimelic acid as the diagnostic diamino acid and galactose as the diagnostic sugar. The major fatty acids were iso-C(16 : 0), iso-C(15 : 0) and C(17 : 1)ω8c and the predominant menaquinone was MK-9(H4). The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylinositol, an unknown glycophospholipid and an unknown phospholipid. The DNA G+C content of the strain was 75.4 mol%. The 16S rRNA gene sequence identity with members of the genus Geodermatophilus was 94.2-98.7%. Based on phenotypic, chemotaxonomic and phylogenetic data, strain Tü 6233(T) is proposed to represent a novel species, Geodermatophilus brasiliensis sp. nov., with the type strain Tü 6233(T) ( = DSM 44526(T) = CECT 8402(T)).

Stereochemistry and conformation of skyllamycin, a non-ribosomally synthesized peptide from Streptomyces sp. Acta 2897.

Skyllamycin is a non-ribosomally synthesized cyclic depsipeptide from Streptomyces sp. Acta 2897 that inhibits PDGF-signaling. The peptide scaffold contains an N-terminal cinnamoyl moiety, a ß-methylation of aspartic acid, three ß-hydroxylated amino acids and one rarely occurring α-hydroxy glycine. With the exception of α-hydroxy glycine, the stereochemistry of the amino acids was assigned by comparison to synthetic reference amino acids applying chiral GC-MS and Marfey-HPLC analysis. The stereochemistry of α-hydroxy glycine, which is unstable under basic and acidic conditions, was determined by conformational analysis, employing a combination of data from NOESY-NMR spectroscopy, simulated annealing and free MD simulations. The simulation procedures were applied for both R- and S-configured α-hydroxy glycine of the skyllamycin structure and compared to the NOESY data. Both methods, simulated annealing and free MD simulations independently support S-configured α-hydroxy glycine thus enabling the assignment of all stereocenters in the structure of skyllamycin and devising the role of two-component flavin dependent monooxygenase (Sky39) as S-selective.

Dermacozines H-J isolated from a deep-sea strain of Dermacoccus abyssi from Mariana Trench sediments.

Dermacoccus abyssi sp. nov. strains MT1.1 and MT1.2 are actinomycetes isolated from a Mariana Trench sediment at a depth of 10 898 m. The fermentation process using complex media led to the production of three new pigmented heteroaromatic (oxidized and reduced) phenazine compounds, dermacozines H-J (1-3). Extensive use was made of 1D and 2D NMR experiments and high-resolution MS to determine the structures of the compounds. The new dermacozines showed radical scavenging activity, and the highest activity was observed for dermacozine H (1), with an IC50 value of 18.8 µM.

New bhimamycins from Streptomyces sp. AK 671.

From the wild-type strain Steptomyces sp. AK 671, three nitrogen-containing octaketides were isolated, bhimamycins F, H and I, besides the known azaanthraquinone utahmycin A and polyketide shunt products SEK 4, SEK 4b, mutactin, dehydromutactin and EM18. The structures were characterized by MS and NMR experiments. The hitherto unknown absolute configuration of the two enantiomers of EM18 was determined by online-CD spectroscopy and quantum-chemical CD calculations. Bhimamycins H and I show weak antibacterial activities, whereas the enzyme phosphodiesterase 4 is strongly inhibited by bhimamycins H and I, which has never been reported for nitrogen-containing octaketides. In addition, bhimamycin H inhibits the enzyme glycogen synthase kinase-3ß.

Langkocyclines: novel angucycline antibiotics from Streptomyces sp. Acta 3034(*).

Langkocyclines A1-A3 and B1 and B2, five new angucycline antibiotics produced by Streptomyces sp. Acta 3034, were detected in the course of our HPLC-diode array screening. The producing strain was isolated from the rhizospheric soil of a Clitorea sp. collected from Burau Bay, Langkawi, Malaysia, and was characterized by morphological, physiological and chemotaxonomic features in addition to 16S ribosomal RNA gene sequence information. Strain Acta 3034 is closely related to Streptomyces psammoticus NBRC 13971(T) and Streptomyces lanatus NBRC 12787(T). Langkocyclines consist of an angular tetracyclic benz[a]anthracene skeleton and hydrolyzable O-glycosidic sugar moieties. The yellow-colored A-type langkocyclines differ in their aglycon from the blue-lilac-colored B-type langkocyclines. The A-type langkocycline aglycon is identical to that of aquayamycin and urdamycin A. The chemical structures of the langkocyclines were elucidated by HR-MS, 1D and 2D NMR experiments. They are biologically active against Gram-positive bacteria and exhibit a moderate antiproliferative activity against various human tumor cell lines.

Warkmycin, a novel angucycline antibiotic produced by Streptomyces sp. Acta 2930*.

A novel angucycline-type antibiotic, warkmycin, was isolated from the culture filtrate of Streptomyces strain Acta 2930. Its chemical structure was elucidated by HR-MS, one-dimensional and 2D NMR experiments. The compound inhibits the growth of Gram-positive bacteria and shows a strong antiproliferative activity against mouse fibroblast cell line NIH-3T3 and human cancer cell lines HepG2 and HT29.

Growth inhibition of an Araucaria angustifolia (Coniferopsida) fungal seed pathogen, Neofusicoccum parvum, by soil streptomycetes.

BACKGROUND: Araucariaceae are important forest trees of the southern hemisphere. Life expectancy of their seedlings can largely be reduced by fungal infections. In this study we have isolated and characterized such a fungus and investigated the potential of Streptomyces Actinobacteria from the respective rhizosphere to act as antagonists. RESULTS: The pathogenic fungus from Araucaria angustifolia seeds was identified by morphological markers (pore-associated Woronin-bodies) as belonging to the Pezizomycotina. Molecular data identified the fungus as Neofusicoccum parvum (Botryosphaeriaceae). Co-cultures on agar of this fungus with certain streptomycete isolates from the rhizosphere, and from the surface of Araucaria roots significantly reduced the growth of the fungus. HPLC analysis of the agar yielded streptomycete-specific exudate compounds which were partly identified. There were differences in compounds between single (bacteria, fungus) and dual cultures (bacteria + fungus). CONCLUSION: Streptomycetes from the rhizosphere of Araucariaceae produce exudates which can suppress the development of pathogenic fungi in their seeds.

Biotransformation of the fungal phytotoxin fomannoxin by soil streptomycetes.

Rhizosphere-associated Streptomyces sp. AcH 505 (AcH 505) promotes infection of Norway spruce (Picea abies) with the pathogenic fungus Heterobasidion abietinum 331, while Streptomyces sp. GB 4-2 (GB 4-2) enhances spruce defense against the fungus. To identify whether these bacteria influence the availability of the fungal phytotoxin fomannoxin and hence spruce infection, we analyzed the fomannoxin yield in H. abietinum 331-AcH 505 dual cultures. Further, the fate of fomannoxin was studied by adding the compound to cultures of AcH 505, GB 4-2 and nine other soil streptomycetes. Culture filtrates were extracted with ethyl acetate and analyzed by HPLC. Structures of novel compounds were elucidated by HPLC-HR-ESI-Orbitrap-MS and NMR spectroscopy. Phytotoxicity of the compounds was determined by in vivo measurement of maximum photosystem II efficiency of Arabidopsis thaliana seedlings. The amount of fomannoxin in H. abietinum 331-AcH 505 dual cultures was reduced compared to axenic fungus cultures and fungus-plant dual cultures. Following addition of fomannoxin to AcH 505 cultures, the compound disappeared and three novel fomannoxin derivatives without phytotoxic activity were detected. Another novel compound, fomannoxin amide, was discovered following fomannoxin addition to GB 4-2 cultures. Nine other streptomycetes converted fomannoxin into fomannoxin acid or fomannoxin amide. Both compounds exhibit the same phytotoxicity as fomannoxin. We, thus, conclude that the streptomycete-mediated modulation of spruce infection with H. abietinum 331 does not depend on the availability of fomannoxin. We further add evidence to the observation that the lipophilic side chain of fomannoxin is an important structural element for its phytotoxicity.

Elaiomycins K and L, new azoxy antibiotics from Streptomyces sp. Tü 6399*.

Elaiomycins K and L, two new azoxy-type antibiotics, were detected by HPLC-diode array screening in the culture filtrate extract of Streptomyces sp. Tü 6399. The structures were determined by high-resolution MS and 2-dimensional (1)H and (13)C correlated NMR spectroscopy including (15)N-NMR experiments and established these compounds as new members of the elaiomycin family. Both metabolites show a weak antibacterial activity against Bacillus subtilis and Staphylococcus lentus as well as against the phytophathogenic strain Xanthomonas campestris.

Production of fungal and bacterial growth modulating secondary metabolites is widespread among mycorrhiza-associated streptomycetes.

BACKGROUND: Studies on mycorrhiza associated bacteria suggest that bacterial-fungal interactions play important roles during mycorrhiza formation and affect plant health. We surveyed Streptomyces Actinobacteria, known as antibiotic producers and antagonists of fungi, from Norway spruce mycorrhizas with predominantly Piloderma species as the fungal partner. RESULTS: Fifteen Streptomyces isolates exhibited substantial variation in inhibition of tested mycorrhizal and plant pathogenic fungi (Amanita muscaria, Fusarium oxysporum, Hebeloma cylindrosporum, Heterobasidion abietinum, Heterobasidion annosum, Laccaria bicolor, Piloderma croceum). The growth of the mycorrhiza-forming fungus Laccaria bicolor was stimulated by some of the streptomycetes, and Piloderma croceum was only moderately affected. Bacteria responded to the streptomycetes differently than the fungi. For instance the strain Streptomyces sp. AcM11, which inhibited most tested fungi, was less inhibitory to bacteria than other tested streptomycetes. The determined patterns of Streptomyces-microbe interactions were associated with distinct patterns of secondary metabolite production. Notably, potentially novel metabolites were produced by strains that were less antagonistic to fungi. Most of the identified metabolites were antibiotics (e.g. cycloheximide, actiphenol) and siderophores (e.g. ferulic acid, desferroxiamines). Plant disease resistance was activated by a single streptomycete strain only. CONCLUSIONS: Mycorrhiza associated streptomycetes appear to have an important role in inhibiting the growth of fungi and bacteria. Additionally, our study indicates that the Streptomyces strains, which are not general antagonists of fungi, may produce still un-described metabolites.

Langkolide, a 32-membered macrolactone antibiotic produced by Streptomyces sp. Acta 3062.

A new 32-membered macrolactone antibiotic, named langkolide, was isolated from the mycelium of Streptomyces sp. Acta 3062. The langkolide structure was determined by HR-MS and 1D and 2D NMR as a 32-membered macrolactone connected from an overhanging polyketide tail to a naphthoquinone unit mediated by two carbohydrate moieties. The producing strain was isolated from a rhizosphere soil of Clitorea sp. collected at Burau Bay, Langkawi, Malaysia, and was characterized by its morphological and chemotaxonomic features in addition to its 16S rRNA gene sequence. It was identified as a member of the Streptomyces galbus clade. Langkolide exhibited various bioactivities including antimicrobial and antiproliferative activities. Furthermore, langkolide inhibited human recombinant phosphodiesterase 4 with an IC(50) value of 0.48 µM.