| Thiostrepton is a member of a family of sulfur-rich peptides, known as thiopeptide antibiotics. These compounds are produced mostly by bacteria of the genus Streptomyces. Thiopeptide antibiotics inhibit protein synthesis in Gram-positive bacteria by forming a complex with the 23S rRNA and ribosomal protein L11. Thiostrepton is primarily used in veterinary medicine, and as a research tool in molecular biology.;The goal of this research was to determine the biosynthetic pathway for thiostrepton at the biochemical and genetic levels, so that the genetic machinery of the producing organism could eventually be used to make modified antibiotics. Nearly all antibiotic biosynthetic pathways studied share the feature that the biosynthetic genes are linked to a gene that provides self-resistance to the antibiotic produced. Based on this observation, the thiostrepton resistance (tsnR) gene from S. laurentii was cloned, sequenced, and used as a probe to clone large DNA fragments. The DNA flanking tsnR was studied by combination of, hybridization analysis, DNA sequencing, and directed mutation, and was found to contain operons for ribosomal proteins rather than biosynthetic genes.;At the biochemical level, an enzyme that activates the carboxyl group of 4-(1-hydroxyethyl)-2-quinoline-carboxylate (HEQ) to an acyl adenylate was partially purified and characterized. In S. laurentii, tryptophan is converted to HEQ and subsequently incorporated into the extra loop of thiostrepton. Purification and amino acid sequencing of this enzyme can be used as an alternative approach to cloning the biosynthetic enzymes. |
