Fsihi H, Kottwitz B, Bremer E
Department of Biology, University of Konstanz, Germany.
J Biol Chem. 1993 Aug 15;268(23):17495-503.
The Tsx protein from the Escherichia coli outer membrane is a channel-forming protein containing a nucleoside-specific binding site. The antibiotic albicidin enters the cell via this substrate-specific channel. Because albicidin is toxic for E. coli at a very low external substrate concentration, the Tsx channel is likely to contain a binding site for this antibiotic. To identify residues involved in the Tsx substrate-specific channel activity, we devised a selection scheme to isolate albicidin-resistant tsx mutants synthesizing Tsx proteins with defects in their nucleoside uptake function. We recovered seven distinct albicidin-resistant tsx alleles, six point mutations and a 39-base pair duplication. The mutants with a duplication of residues 21-33 of Tsx or with single amino acid substitutions of residue Gly28 (to Arg) and Ser217 (to Arg) are completely deficient in nucleoside uptake at a low substrate concentration. Substitutions of Phe27 to Leu, Gly28 to Glu, Gly239 to Asp, and Gly240 to Asp result in a Tsx protein partially defective in nucleoside transport. These mutant proteins still permit nonspecific diffusion of serine indicating that the mutations do not result in a block of the Tsx channel. Our results are discussed in terms of a model for the topological organization of the Tsx protein within the outer membrane of E. coli.
来自大肠杆菌外膜的Tsx蛋白是一种形成通道的蛋白，含有一个核苷特异性结合位点。抗生素杀稻瘟菌素通过这个底物特异性通道进入细胞。由于杀稻瘟菌素在极低的外部底物浓度下对大肠杆菌有毒，Tsx通道可能含有这种抗生素的结合位点。为了鉴定参与Tsx底物特异性通道活性的残基，我们设计了一种筛选方案，以分离出对杀稻瘟菌素具有抗性的tsx突变体，这些突变体合成的Tsx蛋白在核苷摄取功能上存在缺陷。我们获得了7个不同的对杀稻瘟菌素具有抗性的tsx等位基因，6个点突变和一个39个碱基对的重复。Tsx的21-33位残基重复或Gly28（突变为Arg）和Ser217（突变为Arg）的单个氨基酸取代的突变体在低底物浓度下的核苷摄取完全缺陷。Phe27突变为Leu、Gly28突变为Glu、Gly239突变为Asp和Gly240突变为Asp导致Tsx蛋白在核苷转运方面部分存在缺陷。这些突变蛋白仍然允许丝氨酸的非特异性扩散，这表明这些突变不会导致Tsx通道的阻断。我们根据大肠杆菌外膜中Tsx蛋白的拓扑组织模型对结果进行了讨论。
