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-formylimidazolonestructure of nikkomycinswith the aldehyde reagent barbituric acid leading to red-colored products. Among18,000 AT-methyl-JV'-nitro-TV-nitrosoguanidine treated clones tested in the barbituric acid assay, weisolated one mutant which was incapable of forming any nikkomycins containing the 4-formylimidazolone base (nikkomycins Cx, X and I) but instead produced nikkomycins containing uracil (nikkomycins C, Z and J). led to the isolation of Tu901/AECl and AEC2which produced exclusively nikkomycins Kz and Kx. 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.
913Nikkomycins belong to the nucleoside peptide antibiotics and act as potent inhibitors of chitin synthetases from fungi and insects1~4). Streptomyces tendae Tu901 produces a spectrum of various nikkomycins with 4-formyl-4-imidazoline-2-one and uracil as variable bases5>6). The nucleoside moiety of biologically active nikkomycins produced by the wild type as major components is peptidically linked to the unusual amino acid 2-amino-4-hydroxy-4-(5-hydroxy-2-pyridyl)-3-methylbutyric acid (nikkomycin D, Fig. 1). Studies on the biosynthesis of nikkomycins revealed that the imidazolone base is derived from L-histidine7) and uracil from pyrimidine metabolism8). Furthermore, L-lysine was shown to be the precursor of the pyridyl residue and the attached hydroxymethylene carbon in nikkomycin D9). There is little knownabout enzymatic reactions and intermediate structures of nikkomycin biosynthesis. Therefore we decided to screen for mutants blocked in nikkomycin biosynthesis which could be employed as hosts for cloning experiments to isolate nikkomycin biosynthetic genes. We describe the isolation of strains of S. tendae with mutations affecting the biosynthesis of the imidazolone base and nikkomycin D.
Streptomyces tendae Tü 901 produces the nucleoside peptide antibiotic nikkomycin. In shot-gun cloning experiments using pIJ699 as vector we isolated a 9.4-kb DNA fragment from S. tendae which complemented the nikkomycin nonproducing mutant NP9 to the formation of nikkomycin C/Cx and Kx. Nikkomycins were identified by HPLC analyses and their characteristic UV spectra. In Southern hybridization experiments the cloned DNA exclusively reacted with S. tendae DNA sequences. As shown by Northern dot blotting, transcripts of the isolated DNA fragment were only detected during stationary growth and correlated with the extent of nikkomycin production. When the recombinant plasmid pNP113 containing the 9.4-kb DNA fragment was transferred into the over-producing mutant Tü901/S2566, transformants exhibited a significantly decreased capacity for forming nikkomycin. Southern analysis of genomic DNA of these transformants revealed that severe rearrangements occurred in DNA sequences homologous to the 9.4-kb insert of pNP113.
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