C. Olano et al., A 2ND ABC TRANSPORTER IS INVOLVED IN OLEANDOMYCIN RESISTANCE AND ITS SECRETION BY STREPTOMYCES-ANTIBIOTICUS, Molecular microbiology, 16(2), 1995, pp. 333-343
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.