A 2ND ABC TRANSPORTER IS INVOLVED IN OLEANDOMYCIN RESISTANCE AND ITS SECRETION BY STREPTOMYCES-ANTIBIOTICUS

A 3.2kb Sstl-Sphl DNA fragment of Streptomyces. antibioticus, an olean domycin producer, conferring resistance to oleandomycin was sequenced and found to contain an open reading frame of 1710 bp (oleB). Its dedu ced gene product (OleB) showed a high degree of similarity with other proteins belonging to the ABC-transporter superfamily including the ge ne product of another oleandomycin-resistance gene (OleC). The OleB pr otein contains two ATP-binding domains, each of approximately 200 amin o acids in length, and no hydrophobic transmembrane regions. Functiona l analysis of the oleB gene was carried out by deleting specific regio ns of the gene and assaying for oleandomycin resistance. These experim ents showed that either the first or the second half of the gene conta ining only one ATP-binding domain was sufficient to confer resistance to oleandomycin. The gene oleB was expressed in Escherichia coli fused to a maltose-binding protein (MBP) using the pMal-c2 vector. The MBP- OleB hybrid protein was purified by affinity chromatography on an amyl ose resin and polyclonal antibodies were raised against the fusion pro tein. These were used to monitor the biosynthesis and physical locatio n of OleB during growth. By Western analysis, the OleB protein was det ected both in the soluble and in the membrane fraction and its synthes is paralleled oleandomycin biosynthesis. It was also shown that a Stre ptomyces albus strain, containing both a glycosyltransferase (OleD) ab le to inactivate oleandomycin and the OleB protein, was capable of gly cosylating oleandomycin and secreting the inactive glycosylated molecu le. It is proposed that OleB constitutes the secretion system by which oleandomycin or its inactive glycosylated form could be secreted by S . antibioticus.