MOLECULAR CHARACTERIZATION AND TRANSCRIPTIONAL ANALYSIS OF A MULTIDRUG-RESISTANCE GENE CLONED FROM THE PRISTINAMYCIN-PRODUCING ORGANISM, STREPTOMYCES-PRISTINAESPIRALIS

A multidrug resistance gene (mdr) has been cloned from Streptomyces pr istinaespiralis, a producer of two antibiotics having synergistic acti vities together known as pristinamycin. This gene, ptr, provides resis tance not only to two structurally dissimilar compounds (pristinamycin I, PI; pristinamycin II, PII) and the natural pristinamycin mixture b ut also to rifampicin. Mutagenesis and subcloning of ptr localized it to a 2 kb region which was sequenced and analysed. It contained an ope n reading frame of 1506 bp which encoded a putative membrane protein w ith 14 hydrophobic domains, and showed sequence similarity to a superf amily of bacterial proteins that employ transmembrane electrochemical gradients to catalyse active efflux of various antibiotics and toxic c ompounds. Ptr was most similar to a subfamily which included other mdr genes and antibiotic transport genes associated with antibiotic biosy nthetic gene clusters in actinomycetes. In vitro coupled transcription -translation experiments were used to identify the ptr gene product. A nalysis of the upstream region did not reveal a divergently transcribe d repressor gene, as is the case for several related resistance determ inants involved in antibiotic transport, suggesting that ptr is regula ted by a different mechanism. Transcriptional analyses of this gene, c arried out in both S. pristinaespiralis and Streptomyces lividans, ind icated the same transcriptional start point and predicted -10 and - 35 hexamers which were somewhat similar to Streptomyces vegetative-type promoters.