Biochimica et Biophysica Acta, 1018 (1990) 203-205 203 Elsevier BBAEBC 00017 A plasmid-encoded anion-translocating ATPase Barry P. Rosen, Ching-Mei Hsu, Cyrus E. Karkaria, Parjit Kaur, Joshua B. Owolabi and Louis S. Tisa Department of Biochemistry, Wayne State Unioersity, School of Medicine, Detroit, M1 (U.S.A.) (Received 1 May 1990) Key words: ATPase; Ion transport; Anion pump; Arsenic; Antimony; Plasmid resistance An anion-translocating ATPase has been identified as the product of the arsenical resistance operon of resistance plasmid R773. When expressed in Escherichia coil this ATP-driven oxyanion pump catalyzes extrusion of the oxyanions arsenite, antimonite and arsenate. Maintenance of a low intracellular concentration of oxyanion produces resistance to the toxic agents. The pump is composed of two polypeptides, the products of the arsA and arsB genes. This two-subunit enzyme produces resistance to arsenite and antimonite. A third gene, arsC, expands the substrate specificity to allow for arsenate pumping and resistance. Two superfamilies of ion-translocating ATPases have been identified [1]. The F0F1 enzymes of bacterial, mito- chondrial and chloroplast membranes are proton pumps [1]. A group of related proton pumps are found in the plasma membrane of archeabacteria [2] and in plant [3] and fungal [4] tonoplasts. The EzE 2 cation-translocating ATPases form the second family of ion pumps [1]. We have recently identified a unique anion pump, the first recognized member of a family of anion-trans- locating ATPases [5,6]. All other ion-translocating ATPases transport only cations. The genes for this pump are carried on a naturally occurring resistance plasmid. It is clear from analyses of the sequences of the genes for this pump that they evolved separately from the genes for cation-translocating ATPases. The anion pump provides bacterial resistance to toxic oxyanions, including arsenite, antimonite and arsenate. The mecha- nism of resistance is simply to lower the intracellular concentration of the anions by pumping them out of the ceils [7-9]. Although there are a number of means by which plasmid-encoded genes produce resistance to toxic agents such as antibiotics and heavy metals, the most frequently utilized stratagem appears to be simply via extrusion systems (for a review see Ref. 10).The ars operon which encodes the pump genes has been cloned and sequenced [5]. Fig. 1 summarizes information in- ferred from the nucleotide sequence. The operon has four genes: arsR is a regulatory gene [6]. The arsA gene Correspondence: B.P. Rosen, Department of Biochemistry, Wayne State University, School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201, U.S.A. encodes the catalytic subunit of the pump [11-13]. The arsB gene product is a hydrophobic protein localized in the inner membrane of E. coli [14]. These two p01y- peptides are both necessary and sufficient for transport of arsenite and antimonite, which contain the (+ III) oxidation state of the metals [15,16]. The two form a membrane-bound complex with oxyanion-stimulated ATPase activity [17]. The operon also encodes resis- tance to arsenate, which has the (+ V) oxidation state of arsenic. This requires the ArsA and ArsB proteins, but in addition, the arsC gene product is needed. The mechanism by which the 16 kDa ArsC protein alters the substrate specificity of the oxyanion pump is as yet unknown, although it is intriguing to speculate that it ar~ an~l ar~ arsC I 2 3 4 Bl~ I I l I II I I I II II E H P P B B K KH n Rr~ AreR Ar~q Rr~ I--I I IJ ,"--1 117 583 4L~ 141 ota IL~I3 83180 45577 15811 13o Fig. 1. Physical map of the ars operon. In the top line the four genes of the operon are shown with the direction of transcription indicated by the arrow. The length of the DNA is indicated in kilobase pairs (kb). Restriction endonuelease sites are B, BamHI; E, EcoRI; H, HindIII; K, Kpnl; P, PstI. In the bottom portion of the figure, the four gene products are listed with the number of amino acid residues and molecularmasses in daltons (Da). 0005-2728/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)