Macrocin-O-methyltransferase (MacOMeTmse) catalyzes the final enzymatic step in the biosynthesis of tylosin in Streptomycesfradiae. A 44-base mixed oligonucleotide probe containing only guanosine and cytidine in the third position of degenerate codons was synthesized based on the amino acid sequence of the amino terminus of MacOMeTase. Plaque blot hybridization to a bacteriophage A library and colony blot hybridization to a cosmid library of S. fradiae DNA identified recombinants that contained overlapping fragments of chromosomal DNA. The nucleotide sequence of the cloned DNA verified that the DNA contained the coding sequence for MacOMeTase. Recombinant plasmids transformed mutants blocked in tylosin biosynthesis and complemented tyIF (the structural gene for MacOMeTase) and tyl mutations of eight other classes.

A gene conferring high-level resistance to tylosin in Streptomyces lividans and Streptomyces griseofuscus was cloned from a tylosin-producing strain of Streptomyces fradiae. The tylosin-resistance (Tylr) gene (tlrA) was isolated on five overlapping DNA fragments which contained a common 2.6 Kb KpnI fragment. The KpnI fragment contained all of the information required for the expression of the Tylr phenotype in S. lividans and S. griseofuscus. Southern hybridization indicated that the sequence conferring tylosin resistance was present on the same 5 kb SalI fragment in genomic DNA from S. fradiae and several tylosin-sensitive (Tyls) mutants. The cloned tlrA gene failed to restore tylosin resistance in two Tyls mutants derived by protoplast formation and regeneration, and it restored partial resistance in a Tyls mutant obtained by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The tlrA gene conferred resistance to tylosin, carbomycin, niddamycin, vernamycin-B and, to some degree, lincomycin in S. griseofuscus, but it had no effect on sensitivity to streptomycin or spectinomycin, suggesting that the cloned gene is an MLS (macrolide, lincosamide, streptogramin-B)-resistance gene. Twenty-eight kb of S. fradiae DNA surrounding the tlrA gene was isolated from a genomic library in bacteriophage lambda Charon 4. Introduction of these DNA sequence into S. fradiae mutants blocked at different steps in tylosin biosynthesis failed to restore tylosin production, suggesting that the cloned Tylr gene is not closely linked to tylosin biosynthetic genes.

A spontaneous mutant of Streptomyces fradiae contained an amplifiable unit of DNA with a sequence length of approximately 10.5 kilobases that was amplified to approximately 500 copies per chromosome. The amplified DNA appears to be cryptic. SalI fragments of the amplified DNA were cloned into Escherichia coli to construct a restriction map and characterize the amplified DNA. The amplified DNA contained tandem repeats of the amplifiable unit of DNA. The unit had an average base composition of 71% guanine plus cytosine, similar to the chromosomal DNA of Streptomyces species. At least a portion of the amplifiable unit of DNA was present at a low copy number in the wild-type strain. The phenotype of amplified DNA was designated Ads1SF for amplified DNA sequence 1 in S. fradiae.

A substantial amount of information on the biosynthesis of tylosin has been obtained over the past ten years. Physiological studies and experiments with tylosin-blocked (tyl) mutants have suggested the probable pathway by which tylactone is converted to tylosin. The development of recombinant DNA methodology for streptomycetes in general, and for Streptomyces fradiae in particular, has allowed us to apply gene cloning techniques in further studies of tylosin biosynthesis in S. fradiae. The macrocin O-methyltransferase (MOMT), which catalyzes the last step in tylosin biosynthesis, was purified, and the sequence of the 35 amino acids at its amino-terminus was determined. A synthetic 44 base oligonucleotide probe was constructed on the basis of the amino acid sequence. The probe was used to identify sequences containing the MOMT structural gene in bacteriophage and cosmid libraries of S. fradiae DNA. Complementation of tyl mutants with the cloned DNA sequences identified nine tyl biosynthetic genes (tylC, D, E, F, H, J, K, L, and M) in a 42 kb stretch of DNA. Genes complementing four mutant classes, tylA, B, G, and I were not found. A tylosin-resistance gene, tlrB, was located just left of the tyl gene cluster. Tylosin-sensitive mutants of S. fradiae, which were isolated from regenerated protoplasts and which have pleiotropic deficiencies in tylosin biosynthesis, contained deletions which included at least some of the identified tyl loci and one or both of two tylosin-resistance genes, tlrB and tlrC. Possible schemes for the functional organization of the tyl region of the S. fradiae genome are discussed.

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