Dereplication of Streptomyces soil isolates and detection of specific biosynthetic genes using an automated ribotyping instrument.

The discrimination of distinct cultures among morphologically similar Streptomyces soil isolates (dereplication) and the detection of specific biosynthetic pathways in these strains are important steps in the selection of microorganisms to include in a natural products library. We have developed methods for analysis of actinomycetes using the RiboPrinter microbial characterization system, an automated instrument that performs ribotyping on bacterial samples. To evaluate our dereplication method, 26 Streptomyces isolates, obtained from soil samples collected in Maui, Hawaii, were ribotyped and compared with each other, using the RiboPrinter. The strains were also compared by 16S rDNA sequence analysis, MIDI fatty acid analysis, and LC-MS profiling of fermentation extracts. The RiboPrinter was able to identify closely related isolates and to discriminate between morphologically similar isolates with unique genetic, fatty acid and fermentation profiles. For the detection of biosynthetic genes, a 1,006-bp probe containing a portion of an adenylation domain of a non-ribosomal peptide synthetase (NRPS) was employed. Using this alternate probe in place of the standard ribosomal probe, the RiboPrinter was able to detect NRPS genes in several strains of Streptomyces. These results demonstrate that the RiboPrinter has multiple applications in a natural products research program.

Mannopeptimycins, new cyclic glycopeptide antibiotics produced by Streptomyces hygroscopicus LL-AC98: antibacterial and mechanistic activities.

Mannopeptimycins alpha, beta, gamma, delta, and epsilon are new cyclic glycopeptide antibiotics produced by Streptomyces hygroscopicus LL-AC98. Mannopeptimycins gamma, delta, and epsilon, which have an isovaleryl substitution at various positions on the terminal mannose of the disaccharide moiety, demonstrated moderate to good antibacterial activities. Mannopeptimycin epsilon was the most active component against methicillin-resistant staphylococci and vancomycin-resistant enterococci (MICs, 2 to 4 micro g/ml for staphylococci and streptococci and 4 to 32 micro g/ml for enterococci), while mannopeptimycins gamma and delta were two- to fourfold less active. Mannopeptimycins alpha and beta, which lack the isovaleryl substitution and the disaccharide moiety, respectively, had poor antibacterial activities. The in vivo efficacies of the mannopeptimycins in Staphylococcus aureus mouse protection studies paralleled their in vitro activities. The median effective doses of mannopeptimycins gamma, delta, and epsilon were 3.8, 2.6, and 0.59 mg/kg of body weight, respectively. The mannopeptimycins were inactive against cell wall-deficient S. aureus and caused spheroplasting of Escherichia coli imp similar to that observed with penicillin G in an osmotically protective medium. Mannopeptimycin delta rapidly inhibited [(3)H]N-acetylglucosamine incorporation into peptidoglycan in Bacillus subtilis and had no effect on DNA, RNA, or protein biosynthesis. On the basis of the observations presented above, an effect on cell wall biosynthesis was suggested as the primary mode of action for mannopeptimycin delta. The mannopeptimycins were inactive against Candida albicans, did not initiate hemolysis of human erythrocytes, and did not promote potassium ion leakage from E. coli imp, suggesting a lack of membrane damage to prokaryotic or eukaryotic cells.

Biological and mechanistic activities of xanthorrizol and 4-(1',5'-dimethylhex-4'-enyl)-2-methylphenol isolated from Iostephane heterophylla.

Xanthorrizol (1) and 4-(1',5'-dimethylhex-4'-enyl)-2-methylphenol (2) were identified as the principal antimicrobial components of a CH(2)Cl(2)-MeOH (1:1) extract derived from Iostephane heterophylla. Compound 2 is a new natural product, but has been synthesized. Both compounds exhibited low level activity (MICs of 16-32 microg/mL) against methicillin-resistant staphylococci and vancomycin-resistant enterococci. They were either inactive or poorly active against Gram-negative bacteria and yeast. Mechanistic studies performed in Escherichia coli imp suggested nonspecific inhibition of DNA, RNA, and protein synthesis by both of these compounds. Compound 1 was tested in an in vivo model; it did not provide protection to mice infected with Staphylococcus aureus.

The cytosporones, new octaketide antibiotics isolated from an endophytic fungus.

[structure] Organic extracts from cultures of endophytic fungi collected in the Guanacaste Conservation Area of Costa Rica were screened for antibiotic activity. Two endophytes CR200 (Cytospora sp.) and CR146 (Diaporthe sp.) were found to have potent antibiotic activity. Bioassay-guided fractionation of the extracts from these fungi led to the identification of cytosporones D and E, antibacterial active trihydroxybenzene lactones, and three related but inactive metabolites. The five new octaketides were characterized using X-ray crystallography and NMR.

Cytoskyrins A and B, new BIA active bisanthraquinones isolated from an endophytic fungus.

[structure] The biochemical induction assay (BIA) is a rapid (colorimetric) bacterial assay used to identify compounds that damage DNA or inhibit DNA synthesis and thereby identify potential natural product anticancer agents. Bisanthraquinones based on a 1,3,6, 8-tetrahydroxyanthraquinone-type carbon skeleton were isolated from an endophytic fungus and characterized by NMR and X-ray crystallography. Cytoskyrin A (1) is highly active in the biochemical induction assay, while the closely related cytoskyrin B (2) has no detectable activity in this assay.

Biological activity of guanacastepene, a novel diterpenoid antibiotic produced by an unidentified fungus CR115.

Fermentation extracts of culture CR115, an unknown plant endophyte originally isolated from Costa Rica, were found to be active against antibiotic-resistant bacteria. The metabolite responsible for activity was identified as a novel diterpenoid antibiotic guanacastepene (mol. wt. 374.47 and mol. formula C22H30O5). Mechanistic studies done in an E. coli imp strain suggested membrane damage as the primary mode of bactericidal action. This compound also lysed human RBCs and caused leakage of intracellular potassium from E. coli imp.