JOURNAL OF BACTERIOLOGY, June 1992, p. 3684-3694 Vol. 174, No. 11 0021-9193/92/113684-11$02.00/0 Copyright X 1992, American Society for Microbiology Regulation and Expression of the Arsenic Resistance Operon from Staphylococcus aureus Plasmid p1258 GUANGYONG JI* AND SIMON SILVER Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, Illinois 60680 Received 16 December 1991/Accepted 26 March 1992 The arsenic resistance operon from Staphylococcus aureus plasmid p1258 was cloned and sequenced. The DNA sequence contains three genes in the order arsR, arsB, and arsC. The predicted amino acid sequences of the gene products are homologous with those of the products of the ars operons of plasmids pSX267 from Staphylococcus xylosus and R773 from Escherichia cofi. The cloned staphylococcal ars operon confers resistances to arsenate, arsenite, and antimonite in S. aureus and BaciUus subtiis. The same operon was also expressed in E. coli and conferred resistance to arsenite but less resistance to arsenate and antimonite. Regulation of the p1258 ars operon was studied by using a translational arsB-blaZ fusion in S. aureus and a transcriptional arsB-luxAB fusion in E. coli. The ars operon was induced by arsenate [As(V)J, arsenite [As(III)J, and antimonite [Sb(III)], to which the strains were resistant, plus Bi(IHI) in S. aureus. Only arsenate and arsenite induced the operon in E. coli. Northern (RNA) blot DNA-RNA hybridization analysis showed inducible synthesis of a fuill-length ars mRNA, about 2.1 kb in size, both in S. aureus and in E. coli. S. aureus ars proteins were expressed in E. coli from the T7 phage promoter under the control of the T7 RNA polymerase. Primer extension (reverse transcriptase) analysis showed that the ars mRNA started at the same position (nucleotides 17 and 18 upstream from the arsR ATG) both in S. aureus and in E. coli. An internal deletion mutation in arsB resulted in decreased resistance to arsenate and total loss of arsenite and antimonite resistances. Partial deletion of 56 bp from the 3' end of the arsC gene resulted in loss of resistance to arsenate; the determinant retained arsenite and antimonite resistances. Plasmid-determined bacterial resistances to arsenic and antimony have been known since the work of Novick and Roth (20) and Hedges and Baumberg (7). Silver et al. (32) showed that the resistances for plasmids from both Esche- richia coli and Staphylococcus aureus resulted from reduced cellular accumulation. Resistances to arsenate [As(V)], ar- senite [As(III)], and antimonite [Sb(III)] have been found, and the resistance systems of both species are inducible by these three oxyanions and also by Bi(III). Subsequently, Silver and Keach (33) and Mobley and Rosen (18) found that the reduced uptake resulted from accelerated energy-depen- dent arsenate efflux, which in the case of the E. coli system was shown to be ATP dependent. The arsenic-antimony resistance determinant of E. coli plasmid R773 was cloned and sequenced (1, 30). The ars operon of plasmid R773 consists of at least four genes (Fig. 1). After the operator-promoter region, the first gene tran- scribed is arsR, the determinant of the repressor protein (30, 43) of the ars system. There then follows 430 bp before the three structural genes arsA (the determinant of the catalytic subunit, membrane-associated polypeptide, whose ATPase activity is stimulated by arsenite and antimonite [8, 25]); arsB (the integral membrane protein with which ArsA asso- ciates [39], forming the oxyanion-translocating ATPase [35, 36, 40]); and arsC (which encodes a small soluble protein that was thought to expand the substrate range of the arsenic ATPase [12, 35] but has recently been found to confer enzymatic activity converting arsenate to arsenite, which is subsequently effluxed [9, 10]. This report presents the cloning and sequencing of the ars determinant from the most thoroughly studied staphylococ- * Corresponding author. cal multiple-resistance plasmid, pI258 (19, 34). The DNA sequences are identical in gene order and 90% identical in base positions with those of S. xylosus plasmid pSX267 (26). The staphylococcal ars operons (see Results) start with operator-promoter regions, followed by arsR (the gene for the repressor protein) (Fig. 1). A gene homologous with arsA of the E. coli plasmid R773 is missing from the staphylococ- cal systems, and arsR and arsB abut directly (Fig. 1). Both the R773 and the staphylococcal ars operons end with an arsC gene. The resistance patterns, gene regulation, and transcript start point and length have been determined. MATERIALS AND METHODS Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1. Cells were grown in LB broth (29) or 2XNY broth (17). When required, ampicillin (50 ,ug/ml), kanamycin (40 ,ug/ml), chlorampheni- col (5 ,g/ml for S. aureus and 40 ,ug/ml for E. coli), or erythromycin (10 pg/ml) was added to the medium as indi- cated. The P-lactamase gene translational fusion vector pWN1818 (41) was a gift from R. P. Novick; plasmids pSK265 and pSK270 (11, 13) were from S. A. Khan; and the lux4B transcriptional fusion vector pQF70 (4) was from Mark A. Farinha and Andrew M. Kropinski. Materials. Sodium arsenate, sodium arsenite, and anti- mony potassium tartrate (Sigma Chemical Co., St. Louis, Mo.) were used as oxyanions. Bismuth sodium tartrate was obtained from RSA Corp. (Ardsley, N.Y.). Other salts and chemicals were either from Sigma Chemical Co. or other commercial sources. Nitrocefin was from Becton Dickinson (Hunt Valley, Md.) and was initially obtained from J. P. Quinn (Michael Reese Hospital, Chicago, Ill.). [a-32P]dATP or [a-3 P]dCTP for labeling DNA probes and [35S]a-thio- 3684