The biosynthesis of the thiopeptide antibiotic thiostrepton

Thesis (Ph. D.)--University of Washington, 1999Thiostrepton is a sulfur rich, highly modified peptide antibiotic produced by the soil bacteria Streptomyces laurentii, Streptomyces azureus , and Streptomyces hawaiiensis. It is a member of the structurally similar thiopeptide antibiotic family. Thiostrepton has been found to show strong antibiotic activity towards Gram-positive bacteria by inhibition of ribosomal protein biosynthesis. Thiostrepton is used as a veterinary antibiotic, and as a selective marker in Streptomycete research. Previous studies on this system have elucidated the amino acid precursors of thiostrepton, demonstrated that the peptide likely is synthesized nonribosomally, and found that the biosynthetic genes are not clustered with the known gene conferring resistance to the antibiotic. Three approaches to finding the thiostrepton biosynthetic gene cluster in S. laurentii are presented in this study. Attempted first was reverse genetics method in which a pathway enzyme was chosen for purification in order to obtain amino acid sequence information for use in designing oligonucleotides for Southern hybridization experiments. The enzyme was not successfully purified, due to its low stability and concentration in cell-free extract. Second, specific hybridization to restriction endonuclease digested genomic DNA from three thiopeptide producers was not observed using a Southern probe sythesized from a gene encoding an enzyme catalyzing nonribosomal peptide synthesis from a related species. A novel approach developed by Marahiel and co-workers was undertaken. In this approach, PCR primers are designed based on conserved amino acid sequences found in nonribosomal peptide synthetases. These primers were used to amplify DNA fragments from nonribosomal peptide synthetases in S. laurentii . A cluster of these enzymes was found by this method, partially characterized, and proven to not be involved in thiostrepton biosynthesis by single crossover integrational mutants in that region. The experiment was repeated, and two other clusters of nonribosomal peptide synthetases were isolated and demonstrated to not be located near the known cluster, or to each other. Whether these are involved in thiostrepton biosynthesis remains an open question