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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI019793-02
Application #
3129209
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1984-01-01
Project End
1986-12-31
Budget Start
1985-01-01
Budget End
1985-12-31
Support Year
2
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Maryland Baltimore
Department
Type
Schools of Medicine
DUNS #
003255213
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Zhou, T; Rosen, B P (1997) Tryptophan fluorescence reports nucleotide-induced conformational changes in a domain of the ArsA ATPase. J Biol Chem 272:19731-7
Scott, D L; Ramanathan, S; Shi, W et al. (1997) Genetically engineered bacteria: electrochemical sensing systems for antimonite and arsenite. Anal Chem 69:16-20
Kuroda, M; Dey, S; Sanders, O I et al. (1997) Alternate energy coupling of ArsB, the membrane subunit of the Ars anion-translocating ATPase. J Biol Chem 272:326-31
Sanders, O I; Rensing, C; Kuroda, M et al. (1997) Antimonite is accumulated by the glycerol facilitator GlpF in Escherichia coli. J Bacteriol 179:3365-7
Chen, Y; Rosen, B P (1997) Metalloregulatory properties of the ArsD repressor. J Biol Chem 272:14257-62
Bruhn, D F; Li, J; Silver, S et al. (1996) The arsenical resistance operon of IncN plasmid R46. FEMS Microbiol Lett 139:149-53
Bhattacharjee, H; Rosen, B P (1996) Spatial proximity of Cys113, Cys172, and Cys422 in the metalloactivation domain of the ArsA ATPase. J Biol Chem 271:24465-70
Dey, S; Ouellette, M; Lightbody, J et al. (1996) An ATP-dependent As(III)-glutathione transport system in membrane vesicles of Leishmania tarentolae. Proc Natl Acad Sci U S A 93:2192-7
Chen, Y; Dey, S; Rosen, B P (1996) Soft metal thiol chemistry is not involved in the transport of arsenite by the Ars pump. J Bacteriol 178:911-3
Li, J; Liu, S; Rosen, B P (1996) Interaction of ATP binding sites in the ArsA ATPase, the catalytic subunit of the Ars pump. J Biol Chem 271:25247-52

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