THE ATP-COUPLED ARSENICAL PUMP OF ESCHERICHIA COLI

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The overall goals of this research are elucidation of the molecular mechanisms of an ion pump whose genes are natural components of a bacterial resistance plasmid, and second, the role of this transport system in bacterial resistance to antibiotics and toxic compounds. The clinical resistance plasmid R773 carries the arsenical resistance (ars)operon, which produces resistance to arsenate, arsenite, and antimonite. The operon encodes an oxyanion-translocating ATPase which functions as an ATP-coupled extrusion pump for the toxic oxyanions. The catalytic subunit of the pump is the ArsA protein, an arsenite-stimulated ATPase. The specific aims of the project involve a structure-function analysis of the ArsA protein. In particular a) the role of residues within the two nucleotide binding sites will be examined by molecular genetic and biochemical approaches; b) the four cysteine residues will be mutagenized to examine their role in catalysis; c) the domains involved in dimerization and interaction with the other components of the pump will be determined. While plasmid-mediated antibiotic and heavy metal resistances which are due to energy-dependent efflux systems may be wide spread in nature, anion pumps appear to be rather rare. The arsenical efflux system provide a good model system for the study of transmissible bacterial antibiotic resistances. The plasmid encoded ars system also provides a bacterial model for the study of multidrug resistance in mammalian cells. The Ars ATPase exhibits structural and functional similarity to the P-glycoprotein, the protein which produces multiple drug resistance in tumor cells.