Three distinct classes of mutations affecting the biosynthesis of actinomycin have been established in Streptomyces chyrsomallus by crossing various actinomycin-nonproducing mutants with each other by protoplast fusion. In crosses between members of different classes of mutations, actinomycin-producing recombinant progeny arose, whereas in crosses between members of the same class, no actinomycin-producing recombinants were seen. Biochemical examination of a number of mutants revealed that the expression of all actinomycin synthetases was reduced by about 1 order of magnitude in mutants belonging to class II. In mutants of class I, the specific activities of the actinomycin synthetases were comparable with those measured in their actinomycin-producing parents. Feeding experiments with 4-methyl-3-hydroxyanthranilic acid (4-MHA), the biosynthetic precursor of the chromophore moiety of actinomycin, with representative mutants of the three genetic classes revealed formation of actinomycin in minute amounts by mutants of class I. It is suggested that mutants belonging to class I are mutated at a genetic locus involved in the biosynthesis of 4-MHA. Mutants belonging to class II appear to carry mutations at a locus involved in the regulation of the expression of the actinomycin synthetases. The role of the locus in class III mutations could not be assigned. Mapping studies in S. chrysomallus based on conjugal matings revealed the chromosomal linkage of all three loci. Mutations belonging to classes I and III were closely linked. Their genetic loci could be localized in a map interval of the chromosomal linkage group which is significantly distant from the gene locus represented by mutations belonging to class II.
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