U.S.-Australia Cooperative Research on Copper Resistance Plasmid in Escherichia coli

该奖项支持西蒙·D博士两次为期三个月的访问。银 位于芝加哥的伊利诺伊大学医学院的 为期两年的澳大利亚墨尔本大学， 与巴里托马斯欧文李博士一起进行铜的实验室工作 大肠杆菌中的抗性质粒。 研究人员将 详细描述64 Cu 2+在整个细胞中的转运，右- 侧出，并且来自野生型E的倒置膜载体。杆菌 K-12和一系列铜缺陷突变株 摄取、外排和体内平衡， 初步以墨尔本群为特征。 这些 突变都是在染色体基因（切）所需的正常 细胞营养和质粒基因（PCO）控制 抗高毒性铜水平。 动力学参数 (Km，Vmax，Ki的替代基板）将被确定 对于明显由两个染色体决定的铜吸收， 途径。 如果囊泡分析起作用，那么这些将是免费的 细胞壁结合和细胞内结合的并发症 由Camakaris/Lee假设的铜稳态蛋白质 组 类似地，将使用内部- 法国新闻中断产生的囊泡，因为已经 在其他实验室对其他阳离子进行了测试。 替代 吸收的底物，特别是相关的二价阳离子， 作为64 Cu 2+吸收的抑制剂和放射性 转运试验中的底物。 从如此详细的阳离子 研究人员希望将一种 可能有两个摄取途径，加上一个单一的 染色体和质粒决定的外排途径， 决定细胞内铜结合蛋白的附加基因 既能储存又能传递新积累的 Cu 2+的细胞内位点或外排系统。

This award supports two three-month visits by Dr. Simon D. Silver of the University of Illinois College of Medicine at Chicago over a two-year period to the University of Melbourne, Australia, to perform laboratory work with Dr. Barry Thomas Owen Lee on copper resistance plasmid in Escherichia coli. The researchers will characterize in detail 64Cu2+ transport in whole cells, right- side out, and inverted membrane vehicles from wild type E. coli K-12 and with a series of mutant strains defective in copper uptake, efflux and homeostasis that already have been isolated and preliminary characterized by the Melbourne group. These mutations are both in chromosomal genes (cut) required for normal cellular nutrition and in plasmid genes (pco) governing resistance to high toxic copper levels. The kinetic parameters (Km, Vmax, Ki's for alternative substrates) will be determined for the apparently two chromosomally-determined copper uptake pathways. If the vesicle assays work, then these will be free of complications of cell-wall binding and also intracellular binding by copper-homeostasis proteins hypothesized by the Camakaris/Lee group. Similarly uptake kinetics will be measured with inside- out vesicles produced by French Press disruption, as has been done in other laboratories for other cations. Alternative substrates for uptake, especially related divalent cations, will be tested both as inhibitors of 64Cu2+ uptake and as radioactive substrates in transport assays. From such detailed cation transport kinetics the researchers expect to piece together a complex picture of perhaps two uptake pathways, plus a single chromosomal- and plasmid-determined efflux pathway, and in addition genes determining intracellular copper-binding proteins that function both in storage and in passage of newly accumulated Cu2+ to either its intracellular sites or to the efflux system.