ISOLATION OF STREPTOMYCES TENDAE MUTANTS WITH AN ALTERED NIKKOMYCIN SPECTRUM

Abstract

To isolate Streptomyces tendae mutants blocked in the biosynthesis of the nikkomycin nucleoside base 4-formyl-4-imidazoline-2-one, an assay was developed to detect the formation of nikkomycins containing this base during growth on solid medium. The assay is based on the reaction of the 4-formylimidazolone structure of nikkomycins with the aldehyde reagent barbituric acid leading to red-colored products. Among 18, 000 N-methyl-N'-nitro-N-nitrosoguanidine treated clones tested in the barbituric acid assay, we isolated one mutant which was incapable of forming any nikkomycins containing the 4-formylimidazolone base (nikkomycins C_x, X and I) but instead produced nikkomycins containing uracil (nikkomycins C, Z and J). In addition, we isolated strains with mutations affecting the biosynthesis of 2-amino-4-hydroxy-4-(5-hydroxy-2-pyridyl)-3-methylbutyric acid, the unusual amino acid of nikkomycins Z, X, J and I. By analyzing colonies derived from single spores or protoplasts of S. tendae Tü901/395, a mutant producing besides nikkomycins Z, X, J and I, also nikkomycins K_z/K_x and O_z/O_x, we obtained strains which only formed nikkomycins K_z/K_x and O_z/O_x with 2-amino-4-hydroxy-4-(2-pyridyl)butyric acid and 2-amino-4-hydroxy-4-(5-hydroxy-2-pyridyl)-butyric acid as amino acids. Mutation of such a strain (Tü901/395-11) by UV_365nm in the presence of 8-methoxypsoralen and selection of S-2-aminoethyl-L-cysteine-resistant clones led to the isolation of Tü901/AECl and AEC2 which produced exclusively nikkomycins K_z and K_x. According to their nikkomycin spectrum, these strains were blocked at the hydroxylation step occuring at the pyridyl residue during biosynthesis of the nikkomycin amino acid.