The long-term objectives of this research is determination of the molecular mechanisms of plasmid-encoded ATP-driven anion pumps and the role of these transport systems in bacterial resistance to antibiotics and toxic compounds. The clinical resistance plasmid R773 carries the arsenical resistance (ars) operon, which codes for an ATP-linked arsenical extrusion pump for arsenate (AsO43-) and arsenite (AsO2-). Analysis will be extended to determine the biochemical properties of the systems, with the following specific aims. 1. GENETICS AND MOLECULAR BIOLOGY: As a preliminary to biochemical studies of the transport systems, the physical and genetic properties of the plasmid DNA will be characterized. 2. IDENTIFICATION AND PURIFICATION OF GENE PRODUCTS OF THE ARS OPERON: The ArsA and C have been purified. The ArsB protein, predicted from the nucleic acid sequence to be a membrane protein, will be identified by expression in the appropriate vectors, isolated, and purified. 3. STRUCTURE-FUNCTION RELATIONSHIPS: The position of amino acyl residues of potential functional importance in the Ars proteins will be predicted. Indentifiable residues will be changed by site-directed mutagenesis in order to map functional domains within the subunits. 4. DETERMINATION OF IN VITRO ACTIVITY: The catalytic activities of the arsenical pumps will be examined in vitro. ATP binding studies will be performed with the ArsA protein, which, from its nucleotide sequence, is predicted to have two nucleotide binding sites.
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