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.
