A second ABC transporter is involved in oleandomycin resistance and its secretion by Streptomyces antibioticus.
A 3.2 kb Sstl-Sphl DNA fragment of Streptomyces antibioticus, an oleandomycin producer, conferring resistance to oleandomycin was sequenced and found to contain an open reading frame of 1710 bp (oleB). Its deduced gene product (OleB) showed a high degree of similarity with other proteins belonging to the ABC-transporter superfamily including the gene product of another oleandomycin-resistance gene (OleC). The OleB protein contains two ATP-binding domains, each of approximately 200 amino acids in length, and no hydrophobic transmembrane regions. Functional analysis of the oleB gene was carried out by deleting specific regions of the gene and assaying for oleandomycin resistance. These experiments showed that either the first or the second half of the gene containing 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 amylose resin and polyclonal antibodies were raised against the fusion protein. These were used to monitor the biosynthesis and physical location of OleB during growth. By Western analysis, the OleB protein was detected both in the soluble and in the membrane fraction and its synthesis paralleled oleandomycin biosynthesis. It was also shown that a Streptomyces albus strain, containing both a glycosyltransferase (OleD) able to inactivate oleandomycin and the OleB protein, was capable of glycosylating oleandomycin and secreting the inactive glycosylated molecule. It is proposed that OleB constitutes the secretion system by which oleandomycin or its inactive glycosylated form could be secreted by S. antibioticus.
Olano
C
C
Departamento de Biología Funcional e Instituto Universitario de Biotecnologia (I.U.B.A.), Universidad de Oviedo, Spain.
Rodríguez
A M
AM
Méndez
C
C
Salas
J A
JA
D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D006019:Glycolysis; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009827:Oleandomycin / Q000378:metabolism* / Q000494:pharmacology; D016366:Open Reading Frames; D010957:Plasmids / Q000032:analysis; D011817:Rabbits; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013303:Streptomyces antibioticus / Q000235:genetics* / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; C095776:OleB protein, Streptomyces antibioticus; C461599:maltose transport system, E coli; D008320:Maltose; D009827:Oleandomycin