Several cosmid clones from Streptomyces ambofaciens containing the spiramycin resistance gene srmB were introduced into S.fradiae PM73, a mutant defective in tylosin synthesis, resulting in tylosin synthesis. The DNA responsible for this complementation was localized to a 10.5-kilobase EcoRI fragment. A 32-kilobase DNA segment which included the srmB spiramycin resistance gene and DNA which complemented the defect in strain PM73 were mutagenie in vivo with TnlO carrying the gene for Nmr (which is expressed in Streptomyces spp.) or M vitro by insertional mutagenesis with a dnug resistance gene (Nmr) cassette. When these mutaenized DNA segments were crossed into the S. ambofaciens chromosome, three mutant classes blocked in spiramycin synthesis were obtained. One mutant accumulated two precursors of spiramycin, platenolide I and platenolide II. Two mutants, when cofermented with the platenolide-accumulating mutant, produced spiramycin. Tylactone supplementation of these two mutants resulted in the synthesis of a group of compounds exhibiting antibiotic activity. Two other mutants failed to coferment with any of the other mutants or to respond to tylactone supplementation.Spiramycin, a macrocyclic lactone antibiotic (macrolide) synthesized by Streptomyces ambofaciens (44), is used in both veterinary medicine (58) and human medicine (40). Spiramycin has a 16-member lactone ring (platenolide) to which three sugars are attached-mycaminose, mycarose, and forosamine (43). Structural similarities between spiramycin and tylosin (see Fig. 1) are mirrored by similarities in the two biosynthetic pathways, as evidenced by the ability of a cerulenin (polyketide synthase inhibitor)-inhibited culture of S. ambofaciens to synthesize chimeramycin, a tylosin-spiramycin hybrid, when supplemented with the tylosin lactone tylactone (42). We are interested in isolating spiramycin biosynthetic genes to study their regulation and to use them for the synthesis of novel antibiotics (3,24,26,41,42).Like many antibiotic-producing streptomycetes (14), S. ambofaciens is resistant to the antibiotic it produces, i.e., spiramycin. We have isolated three different spiramycin resistaince genes from S. ambofaciens in a shuttle cosmid vector (48). Since there is precedence for clustering of antibiotic biosynthetic genes in streptomycetes and for the association of resistance genes with these clusters (36, 52), we examined the possibility that DNA segments adjacent to the spiramycin resistance genes code for spiramycin biosynthetic enzymes. In this report, we present evidence for the existence of three spiramycin biosynthetic genes. These are located on both sides of the srmB spiramycin resistance gene. Culture conditions and genetic manipulations. Escherichia coli and Streptomyces strains were grown as described earlier (45, 48). E. coli transformations were done as described by Maniatis et al. (35). Streptomyces transformations were done as previously described (45). Selection for antibiotic resistance was on TY, TS, or modified R2 medium (45) sup...