Olano C, Rodríguez A M, Méndez C, Salas J A
Departamento de Biología Funcional e Instituto Universitario de Biotecnologia (I.U.B.A.), Universidad de Oviedo, Spain.
Mol Microbiol. 1995 Apr;16(2):333-43. doi: 10.1111/j.1365-2958.1995.tb02305.x.
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.
产竹桃霉素的抗生链霉菌（Streptomyces antibioticus）的一段3.2 kb Sstl - Sphl DNA片段，该片段赋予对竹桃霉素的抗性，对其进行了测序，发现含有一个1710 bp的开放阅读框（oleB）。其推导的基因产物（OleB）与属于ABC转运蛋白超家族的其他蛋白质具有高度相似性，包括另一个竹桃霉素抗性基因（OleC）的基因产物。OleB蛋白包含两个ATP结合结构域，每个结构域长度约为200个氨基酸，且没有疏水跨膜区域。通过缺失该基因的特定区域并检测对竹桃霉素的抗性，对oleB基因进行了功能分析。这些实验表明，仅包含一个ATP结合结构域的基因的前半部分或后半部分足以赋予对竹桃霉素的抗性。使用pMal - c2载体，oleB基因在与麦芽糖结合蛋白（MBP）融合的大肠杆菌中表达。MBP - OleB融合蛋白通过在直链淀粉树脂上的亲和层析进行纯化，并制备了针对该融合蛋白的多克隆抗体。这些抗体用于监测生长过程中OleB的生物合成和物理位置。通过蛋白质免疫印迹分析，在可溶性部分和膜部分均检测到了OleB蛋白，其合成与竹桃霉素的生物合成平行。还表明，一种含有能够使竹桃霉素失活的糖基转移酶（OleD）和OleB蛋白的白色链霉菌（Streptomyces albus）菌株，能够对竹桃霉素进行糖基化并分泌无活性的糖基化分子。有人提出，OleB构成了抗生链霉菌分泌竹桃霉素或其无活性糖基化形式的分泌系统。
