Hans-Peter Fiedler

Marine actinomycetes as a source of novel secondary metabolites.

A set of 600 actinomycetes strains which were isolated from marine sediments from various sites in the Pacific and Atlantic Oceans were screened for the production of bioactive secondary metabolites. Marine streptomycete strains were found to be producers of well known chemically diverse antibiotics isolated from terrestrial streptomycetes, as in the case of marine Micromonospora strains. New marine members of the rare genus Verrucosispora seem to be a promising source for novel bioactive secondary metabolites as shown in the case of the abyssomicin producing strain AB-18-032.

A guide to successful bioprospecting: informed by actinobacterial systematics

New structurally diverse natural products are discovered when novel screening procedures are introduced or when high quality biological materials from new sources are examined in existing screens, hence it is important to foster these two aspects of novelty in drug discovery programmes. Amongst prokaryotes, actinomycetes, notably streptomycetes, remain a rich source of new natural products though it has become increasingly difficult to find such metabolites from common actinomycetes as screening ‘old friends’ leads to the costly rediscovery of known compounds. The bioprospecting strategy which is the subject of this review is based upon the premise that new secondary metabolites can be found by screening relatively small numbers of dereplicated, novel actinomycetes isolated from marine sediments. The success of the strategy is exemplified by the discovery of a range of novel bioactive compounds, notably atrop-abyssomicin C and proximicins A, B and C from Verrucosispora strains isolated from sediment samples taken from the Sea of Japan and the Raune Fjord, respectively, and the dermacozines derived from Dermacoccus strains isolated from the Challenger Deep of the Mariana Trench in the Pacific Ocean. The importance of current advances in prokaryotic systematics in work of this nature is stressed and a plea made that resources be sought to train, support and employ the next generation of actinobacterial systematists.

Biosynthetic Capacities of Actinomycetes. 1 Screening for Secondary Metabolites by HPLC and UV-Visible Absorbance Spectral Libraries

Abstract Culture filtrates, their extracts and mycelium extracts of actinomycetes strains are analyzed by HPLC and diode-array detection. Comparing retention times and UV-visible spectra with data from known antibiotics and other metabolites stored in natural substance class libraries in a data base permit an effective screening for new secondary metabolites.

Auxofuran, a Novel Metabolite That Stimulates the Growth of Fly Agaric, Is Produced by the Mycorrhiza Helper Bacterium Streptomyces Strain AcH 505

ABSTRACT The mycorrhiza helper bacterium Streptomyces strain AcH 505 improves mycelial growth of ectomycorrhizal fungi and formation of ectomycorrhizas between Amanita muscaria and spruce but suppresses the growth of plant-pathogenic fungi, suggesting that it produces both fungal growth-stimulating and -suppressing compounds. The dominant fungal-growth-promoting substance produced by strain AcH 505, auxofuran, was isolated, and its effect on the levels of gene expression of A. muscaria was investigated. Auxofuran and its synthetic analogue 7-dehydroxy-auxofuran were most effective at a concentration of 15 μM, and application of these compounds led to increased lipid metabolism-related gene expression. Cocultivation of strain AcH 505 and A. muscaria stimulated auxofuran production by the streptomycete. The antifungal substances produced by strain AcH 505 were identified as the antibiotics WS-5995 B and C. WS-5995 B completely blocked mycelial growth at a concentration of 60 μM and caused a cell stress-related gene expression response in A. muscaria. Characterization of these compounds provides the foundation for molecular analysis of the fungus-bacterium interaction in the ectomycorrhizal symbiosis between fly agaric and spruce.

Caboxamycin, a new antibiotic of the benzoxazole family produced by the deep-sea strain Streptomyces sp. NTK 937*

Caboxamycin, a new benzoxazole antibiotic, was detected by HPLC-diode array screening in extracts of the marine strain Streptomyces sp. NTK 937, which was isolated from deep-sea sediment collected in the Canary Basin. The structure of caboxamycin was determined by mass spectrometry, NMR experiments and X-ray analysis. It showed inhibitory activity against Gram-positive bacteria, selected human tumor cell lines and the enzyme phosphodiesterase.

Proximicin A,B and C, Novel Aminofuran Antibiotic and Anticancer Compounds Isolated from Marine Strains of the Actinomycete Verrucosispora

A family of three novel aminofuran antibiotics named as proximicins was isolated from the marine Verrucosispora strain MG-37. Proximicin A was detected in parallel in the marine abyssomicin producer “Verrucosispora maris” AB-18-032. The characteristic structural element of proximicins is 4-amino-furan-2-carboxylic acid, a hitherto unknown γ-amino acid. Proximicins show a weak antibacterial activity but a strong cytostatic effect to various human tumor cell lines.

Simocyclinone D8, an Inhibitor of DNA Gyrase with a Novel Mode of Action

ABSTRACT We have characterized the interaction of a new class of antibiotics, simocyclinones, with bacterial DNA gyrase. Even though their structures include an aminocoumarin moiety, a key feature of novobiocin, coumermycin A1, and clorobiocin, which also target gyrase, simocyclinones behave strikingly differently from these compounds. Simocyclinone D8 is a potent inhibitor of gyrase supercoiling, with a 50% inhibitory concentration lower than that of novobiocin. However, it does not competitively inhibit the DNA-independent ATPase reaction of GyrB, which is characteristic of other aminocoumarins. Simocyclinone D8 also inhibits DNA relaxation by gyrase but does not stimulate cleavage complex formation, unlike quinolones, the other major class of gyrase inhibitors; instead, it abrogates both Ca2+- and quinolone-induced cleavage complex formation. Binding studies suggest that simocyclinone D8 interacts with the N-terminal domain of GyrA. Taken together, our results demonstrate that simocyclinones inhibit an early step of the gyrase catalytic cycle by preventing binding of the enzyme to DNA. This is a novel mechanism for a gyrase inhibitor and presents new possibilities for antibacterial drug development.

Cloning and analysis of the simocyclinone biosynthetic gene cluster of Streptomyces antibioticus Tü 6040

Abstract. The biosynthetic gene cluster of the aminocoumarin antibiotic simocyclinone D8 was cloned by screening a cosmid library of Streptomyces antibioticus Tü 6040 with a heterologous probe from a gene encoding a cytochrome P450 enzyme involved in the biosynthesis of the aminocoumarin antibiotic novobiocin. Sequence analysis of a 39.4-kb region revealed the presence of 38 ORFs. Six of the identified ORFs showed striking similarity to genes from the biosynthetic gene clusters of the aminocoumarin antibiotics novobiocin and coumermycin A1. Simocyclinone also contains an angucyclinone moiety, and 12 of the ORFs showed high sequence similarity to biosynthetic genes of other angucyclinone antibiotics. Possible functions within the biosynthesis of simocyclinone D8 could be assigned to 23 ORFs by comparison with sequences in GenBank. Experimental proof for the function of the identified gene cluster was provided by a gene inactivation experiment, which resulted in the abolishment of the formation of the aminocoumarin moiety of simocyclinone. Feeding of the mutant with the aminocoumarin moiety of novobiocin led to a new, artificial simocyclinone derivative.

A crystal structure of the bifunctional antibiotic simocyclinone D8, bound to DNA gyrase.

Targeting DNA Gyrase DNA gyrase, an enzyme that unwinds double-stranded DNA, is essential in bacteria, but missing in humans, and is thus an important antibiotic target. DNA gyrase is inhibited by the well-known fluoroquinolines and aminocoumarins antibiotics, as well as by symocyclinones—bifunctional antibiotics comprising an aminocoumarin and a polyketide group. Surprisingly, symocyclinones, unlike aminocoumarin inhibitors, do not inhibit DNA gyrase GTPase activity, but instead inhibit binding to DNA. Now Edwards et al. (p. 1415) use biochemical and structural studies to show that the two functional groups of the antibiotic bind in separate pockets on the gyrase. Each group is a relatively weak inhibitor that together potently inhibit DNA binding. The molecular mechanism is revealed by which an antibiotic prevents DNA binding by a bacterial DNA gyrase. Simocyclinones are bifunctional antibiotics that inhibit bacterial DNA gyrase by preventing DNA binding to the enzyme. We report the crystal structure of the complex formed between the N-terminal domain of the Escherichia coli gyrase A subunit and simocyclinone D8, revealing two binding pockets that separately accommodate the aminocoumarin and polyketide moieties of the antibiotic. These are close to, but distinct from, the quinolone-binding site, consistent with our observations that several mutations in this region confer resistance to both agents. Biochemical studies show that the individual moieties of simocyclinone D8 are comparatively weak inhibitors of gyrase relative to the parent compound, but their combination generates a more potent inhibitor. Our results should facilitate the design of drug molecules that target these unexploited binding pockets.

Abyssomicins G and H and atrop-Abyssomicin C from the Marine Verrucosispora Strain AB-18-032 †

Abyssomicin C is a complex polyketide-type antibiotic and the first natural inhibitor of the p-aminobenzoate biosynthesis produced by the marine Verrucosispora strain AB-18-032. We have now isolated three novel naturally produced abyssomicins, among them the even more active atrop-abyssomicin C. The chemical structures were elucidated by mass spectrometry and NMR spectroscopy.