The Atp-Coupled Arsenical Pump of Escherichia coli

Rosen, Barry P.

Abstract

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.

Funding Agency

• Agency: National Institute of Health (NIH)
• Institute: National Institute of Allergy and Infectious Diseases (NIAID)
• Type: Research Project (R01)
• Project #: 5R01AI019793-07
• Application #: 3129213
• Study Section: Physical Biochemistry Study Section (PB)
• Project Start: 1987-08-01
• Project End: 1992-07-31
• Budget Start: 1990-08-01
• Budget End: 1991-07-31
• Support Year: 7
• Fiscal Year: 1990

Institution

• Name: Wayne State University
• Type: Schools of Medicine
• City: Detroit
• State: MI
• Country: United States
• Zip Code: 48202

Publications

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

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

Xu, C; Shi, W; Rosen, B P (1996) The chromosomal arsR gene of Escherichia coli encodes a trans-acting metalloregulatory protein. J Biol Chem 271:2427-32