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

该奖项支持芝加哥伊利诺伊大学医学院的Simon D.Silver博士在两年时间里对澳大利亚墨尔本大学进行了两次为期三个月的访问，与Barry Thomas Owen Lee博士就大肠杆菌中的铜质粒进行了实验室工作。研究人员将详细描述64Cu2+在整个细胞、右侧向外和来自野生型大肠杆菌K-12的倒膜载体中的转运，以及墨尔本小组已经分离出的一系列在铜吸收、外排和动态平衡方面存在缺陷的突变菌株。这些突变既存在于正常细胞营养所需的染色体基因(CUT)中，也存在于控制对高毒性铜水平的抗性的质粒基因(PCO)中。对于显然由染色体决定的两条铜吸收途径，将确定动力学参数(替代底物的Km、Vmax、Ki‘s)。如果囊泡分析有效，那么这些将不会出现细胞壁结合的并发症，也不会有Camakaris/Lee小组假设的铜稳态蛋白在细胞内结合的并发症。类似地，摄取动力学将用由法国出版社破坏产生的由内向外的囊泡来测量，就像在其他实验室对其他阳离子所做的那样。替代的摄取底物，特别是相关的二价阳离子，将作为64Cu2+摄取的抑制剂和运输分析中的放射性底物进行测试。从这种详细的阳离子转运动力学中，研究人员希望拼凑出一幅复杂的图景，其中可能包括两条吸收途径，以及一条由染色体和质粒决定的外流途径，以及决定细胞内铜结合蛋白功能的基因，这些蛋白在储存和将新积累的Cu2+输送到细胞内位置或外流系统的过程中发挥作用。

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.