UNUSUAL REGULATORY MECHANISM FOR A STREPTOMYCES MULTIDRUG-RESISTANCE GENE, PTR, INVOLVING 3 HOMOLOGOUS PROTEIN-BINDING SITES OVERLAPPING THE PROMOTER REGION

A promoter controlling expression of the pristinamycin multidrug resis tance gene (ptr), originally isolated from Streptomyces pristinaespira lis, is inducible by many toxic compounds in various Streptomyces spec ies. Studies of pfr promoter control were carried out in the heterolog ous host. Streptomyces lividans. In S. lividans, a regulatory protein or a protein complex (Pip), identified by its ability to bind to the p tr promoter in gel-retardation experiments, was induced by pristinamyc in I (PI). In situ copper-phenanthroline footprinting analysis identif ied three (A, B, and C) similar Pip-binding sites having the sequence GTACA(C/G)CGTA(C/T). These sites overlapped with functionally importan t regions of the promoter: the 'A' site overlapped with the -35 hexame r, 'B' overlapped with the -10 hexamer and 'C' was located between the transcription start site and the Shine-Dalgarno sequence, A GT-AG din ucleotide mutation was introduced at positions 8-9 of the consensus se quence to generate seven variant promoters: three mutated in one of th e three sites, three mutated in two sites, and one mutated in all thre e sites, Whereas these promoters had reduced antibiotic (PI)-induced a ctivity, their levels of expression in the absence of PI was higher. T his suggested an unusual regulatory mechanism in which Pip could act e ither as an activator or repressor, Gel shift experiments revealed Pip or its homologues in many other Streptomyces species, suggesting that it is widely employed in the regulation of antibiotic resistance gene s and perhaps secondary metabolism.