The overall goals of this research are first, 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. A bacterial anion-translocating TPase has been identified as the product of the arsenical resistance (ars) operon of resistance plasmid R773. When expressed in Escherichia coli the ATP-driven pump catalyzes extrusion of the oxyanions arsenite, antimonite, and arsenate, thus providing resistance to the toxic compounds. This anion-translocating ATPase serves as an excellent model system for ion-translocating ATPases. The ars operon has been cloned and sequenced, and most of the protein components have been identified-and purified. Two of the structural genes, the arsA and arsB genes, encode the two subunits of the pump. This two-component inner membrane complex binds and transports arsenite and antimonite, oxyanions with the +III oxidation state of arsenic or antimony.
Specific aims of the project include determination of the structural elements of the ArsA and ArsB proteins which contribute toward their function as ATPase and ion transport system. Domains of interest in the ArsA protein are a) nucleotide binding sites, b) anion binding sites, and c) sites of self interaction during dimerization. The ArsB protein will be characterized by a) purification and b) development of an in vitro assay for 73AsO2- transport. The ArsA and ArsB proteins form a complex. The methodology for reconstitution of a functional pump from isolated ArsA and ArsB proteins will be optimized. The stoichiometry of the subunits in the pump will be determined, and the contact regions of the two proteins will be identified. The operon is metalloregulated by a diverse set of inducers, including various salts of arsenic, antimony and bismuth. Characterization of the transcriptional regulators, the ArsR and ArsD proteins, will be carried out with the following aims: a) purification of the two proteins, b) determination of their role in regulation, including binding sites on the proteins for DNA and ionic substrates, and c) determination of the repressor binding sites on the DNA.
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