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By cleavage with restriction endonucleases and cloning the resultant fragments, the A-factor determinant cloned from streptomycin-producing Streptomyces bikiniensis (Horinouchi et al., J. Bacteriol. 158: 481 approximately 487, 1984) was narrowed down to a 1.1-kilobase (kb) fragment. A hybrid multicopy plasmid (pAFB15) carrying the 1.1-kb fragment conferred A-factor production in a large quantity to A-factor-deficient mutants of S. bikiniensis and Streptomyces griseus as well as afsA mutants of Streptomyces coelicolor A3(2). A transcriptional control signal in the 1.1-kb fragment was identified by using a promoter-probe vector pARC 1. Plasmid pAFB15 also caused A-factor production with a marked gene dosage effect in four different Streptomyces strains which originally had no ability to produce A-factor and no DNA sequence homologous to the S. bikiniensis A-factor determinant, suggesting that precursors of A-factor are common metabolites in streptomycetes and that acquisition of only a single key enzyme encoded by the 1.1-kb fragment is sufficient for Streptomyces strains to synthesize A-factor.

The cloned Streptomyces bikiniensis A-factor determinant.