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.

Novel polyketide metabolites from a species of marine fungi.

Fermentation of a marine fungal species obtained from a tissue sample of a marine sponge collected in Indonesia in October 1996, yielded the novel hexaketide compounds iso-cladospolide B (1); seco-patulolide C (2); the 12-membered macrolides, pandangolide 1 (3) and pandangolide 2 (4); and the known terrestrial fungal metabolite, cladospolide B (5).

Marine microorganisms as a source of new natural products.

Over the past decade, marine microorganisms have become recognized as an important and untapped resource for novel bioactive compounds. The oceans cover greater than 70% of the earth's surface and, taking this into account by volume, represent better than 95% of the biosphere. Given this fact, the oceans present themselves as an unexplored area of opportunity for the discovery of pharmacologically active compounds. In this review, data have been presented to illustrate the diversity of microorganisms living in the sea and the plethora of chemical compounds that have been discovered from them. However, it is important to pursue basic research on the marine environment in order to permit the continued isolation of unique microorganisms. There is still limited knowledge of the physiological requirements of most marine microorganisms, and a greater understanding of their conditions for growth will offer new insights into the complex world of marine microbiology. Clearly, a greater investment in the development of marine biotechnology will produce novel compounds that may contribute significantly toward drug development over the next decade.

Gamma-indomycinone, a new pluramycin metabolite from a deep-sea derived actinomycete.

A new member of the pluramycin class of antibiotics, gamma-indomycinone [1], has been isolated along with the known compounds rubiflavinone C-1 [2] and beta-indomycinone [3] from the culture broth of a Streptomyces sp. obtained from a deep-sea sediment core. Each compound is composed of an anthraquinone-gamma-pyrone nucleus, but bears a different side-chain. Compounds 2 and 3 were identified by comparison of their spectral data with published data, while gamma-indomycinone [1] was characterized using 1H-nmr and mass spectrometry.

Bioxalomycins, new antibiotics produced by the marine Streptomyces sp. LL-31F508: taxonomy and fermentation.

An actinomycete strain designated LL-31F508 was isolated from an intertidal sediment sample collected in Key West, Florida. Culture LL-31F508 was assigned to the Streptomyces genus based on the presence of LL-diaminopimelic acid (DAP) in the cell wall and observations of spiny spores using scanning electron microscopy (SEM). Excellent antimicrobial activity against Staphylococcus and Enterococcus spp. were detected in both the supernatant and cell extract samples from fermentations of culture LL-31F508. Production of antibiotic activity peaked at 48-50 hours and closely paralleled cell growth, during which time glucose was more rapidly assimilated than dextrin. A series of new antibiotics called the bioxalomycins was identified as the antibacterial products from fermentations of this culture. Fermentation conditions for production of bioxalomycin alpha differed substantially from those required for production of a related compound, naphthyridinomycin, by the reference culture Streptomyces lusitanus NRRL 8034.

Martinomycin, a new polyether antibiotic produced by Streptomyces salvialis. I. Taxonomy, fermentation and biological activity.

Actinomycete culture LL-D37187 has been found to produce the new polyether antibiotic martinomycin. Taxonomic studies, including morphological, physiological, and cell wall chemistry analyses, revealed that culture LL-D37187 is a novel streptomycete species, and the proposed name is Streptomyces salvialis. Martinomycin exhibits activity against the Southern Army Worm (Spodoptera eridania) and Gram-positive bacteria.

Glycothiohexides, novel antibiotics produced by "Sebekia" sp. LL-14E605. I. Taxonomy, fermentation and biological evaluation.

The new glycothiohexide antibiotics, which are related to nosiheptide, were identified in fermentations of an actinomycete belonging to the genus "Sebekia". Strain LL-14E605 was classified as a "Sebekia" based on the presence of both mesodiaminopimelic acid and madurose in the cell wall and the presence of pseudosporangia encasing the spores. Culture LL-14E605 was successfully fermented in 10 to 3,000 liters of a complex medium. Antibiotic activity closely followed cell mass accumulation and usually peaked after 4 to 5 days of incubation. Glycothiohexide alpha demonstrated excellent in vitro activity against Gram-positive bacteria with MICs of 0.03 to 0.06 microgram/ml against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. However, glycothiohexide alpha failed to protect mice against a lethal challenge with Staphylococcus aureus Smith unless it was administered prior to challenge.

A study on the mechanism of action of sceptrin, an antimicrobial agent isolated from the South Pacific sponge Agelas mauritiana.

The mechanism of action of sceptrin, an antimicrobial agent isolated from the sponge Agelas mauritiana, was investigated. Sceptrin has been reported to exhibit antibacterial and antifungal activities. In our studies, sceptrin demonstrated a bacteriostatic rather than bactericidal effect on exponentially growing Escherichia coli cells at the MIC. Under these conditions, the culture produced chains of cells, and incorporation of radio-labelled precursors into DNA, protein, and cell wall was unaffected, whereas incorporation of 3H-uridine into RNA was slightly inhibited. At concentrations higher than the MIC, sceptrin was bactericidal, inhibited the incorporation of all radiolabelled precursors, and induced the formation of unusual spheroplasts. Peptidoglycan turnover in E. coli appeared to be stimulated by sceptrin as demonstrated by a release of diaminopimelic acid-containing high molecular weight material. Subsequent studies of the release of potassium ions from E. coli and the lysis of red blood cells suggested that sceptrin disrupts the cell membranes of both prokaryotic and eukaryotic cells. It is proposed that spheroplasts formation may reflect a cell wall effect that occurs subsequent to membrane damage.