The long-term goal of the project is to develop a bacterial system for the study of the biochemical mechanism of an ion transport ATPase. The plasmid resistance factor R773 confers inducible resistance to high concentrations of arsenate, arsenite, and antimony upon its bacterial host, Escherichia coli. Resistance results from extrusion of arsenate. We have demonstrated that the electrochemical proton gradient (proton motive force) is not involved in extrusion, but, rather, protection against arsenate is due to and ATP-coupled efflux pump. Arsenate which gets into the cell is rapidly expelled by an ATP-utilizing transport process which we have postulated to be an anion-tranlocating ATPase.
The specific aims of this project are to 1) characterize further the energy coupling mechanism of this anion extrusion system by studying arsenate transport and arsenate-stimulataed ATPase activity in everted (inside-out orientation) membrane vesicles; 2) identify the protein(s) components of the anion pump; and 3) isolate the plasmid encoded gene product(s) necessary for bacterial arsenated resistance. The latter two aims will involve a) cloning of the arsenate resistance operon from resistance factor R773 and insertion of the genes into a well-characterized plasmid vector, and b) detection of plasmid-coded, [35S]-methionine-labeled proteins in E. coli minicells induced with arsenate. The project is significant for several reasors. First, this may represent one of the few bacterial ion transport ATPase and probably the best from the point of view of availability of the DNA and gene products. Second, it is one of the very few anion pumps thus far identified. Third, the mechanism of arsenate resistance appears to very similar to that several antibiotics such as tetracycline, making the arsenate efflux system a good model for the study of transmissible bacterial resistances.
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