CLONING AND NUCLEOTIDE-SEQUENCE OF THE GENE RESPONSIBLE FOR CHLORINATION OF TETRACYCLINE

Two cosmid clones containing distinct types of self-defense gene of a 6-demethylchlortetracycline producer, Streptomyces aureofaciens NRRL32 03, were isolated. The gene responsible for chlorination of tetracycli ne (chl gene) was subcloned from one of the cosmid clones by complemen tation of a chlorination-deficient mutant, using a gene cloning system for strain NRRL3203 developed in this study. The nucleotide sequence analysis of a 4.4-kb SacI-BamHI fragment containing the chi gene showe d that the predicted product of the chl gene is a polypeptide of 452 a mino acids, and that the chl gene was preceded by two open reading fra mes, which could endode polypeptides of 50 kDa and 32 kDa, respectivel y. A search for sequences homologous to these ORFs found that the form er product strongly resembles that of the 6-hydroxylation enzyme for o xytetracycline biosynthesis, and that the latter product has a weak bu t significant similarity to the hydroxyindole O-methyltransferase of b ovine pineal gland. By Northern blot analysis, these three genes were suggested to be polycistronically transcribed.