The plasmid-encoded arsenical resistance (ars) operon of plasmid R773 produces resistance to trivalent and pentavalent salts of the metalloids arsenic and antimony in cells of Escherichia coli. The first two genes in the operon, arsR and arsD, were previously shown to encode trans-acting repressor proteins. ArsR controls the basal level of expression of the operon, while ArsD controls maximal expression. Thus, action of the two repressors form a homeostatic regulatory circuit that maintains the level of ars expression within a narrow range. In this study, we demonstrate that ArsD binds to the same site on the ars promoter element as ArsR but with 2 orders of magnitude lower affinity. The results of gel shift assays demonstrate that ArsD is released from the ars DNA promoter by phenylarsine oxide, sodium arsenite, and potassium antimonyl tartrate (in order of effectiveness), the same inducers to which ArsR responds. Using the quenching of intrinsic tryptophan fluorescence to measure the affinity of the repressor for inducers, apparent K d values for Sb(III) and As(III) of 2 and 60 M, respectively, were obtained. These results demonstrate that the arsR-arsD pair provide a sensitive mechanism for sensing a wide range of environmental heavy metals.The arsenical resistance (ars) operon of resistance plasmids R773 and R46 encodes an oxyanion extrusion pump that produces resistance to arsenite and antimonite (1). The operon has five genes, ArsR, -D, -A, -B, and -C (2-5). ArsR and ArsD are both trans-acting repressor proteins that homeostatically regulate the levels of ars transcript (4, 6). Although both are 13-kDa homodimers, they share no sequence similarity. ArsR is an As(III)/Sb(III)-responsive repressor with high affinity for its operator site that controls the basal level of expression of the operon (7). Binding of arsenite or antimonite produces dissociation of ArsR from the operator site, permitting transcription. As the levels of transcript rise, synthesis of the integral membrane ArsB protein becomes toxic, limiting growth. ArsD is a second regulator that controls the upper level of expression of the operon, preventing overexpression of ArsB (4). Together, ArsR and ArsD form a regulatory circuit that controls the basal and maximal levels of expression of the ars operon. Dual regulatory proteins in a single operon are rare; in metalloregulatory systems only the MerR/MerD pair has been reported (8).Previously, it was not possible to measure ArsD binding to ars promoter DNA (4). It was not clear whether ArsD interacted with DNA or mRNA. In addition, initial observations suggested that ArsD did not respond to arsenite or antimonite.In this report, we demonstrate that ArsD binds to DNA just 5Ј to ars promoter, the same site occupied by ArsR before induction. Moreover, interaction of ArsD with arsenite, antimonite, or the organic arsenical phenylarsine oxide (PAO) 1 was demonstrated by both in vivo and in vitro assays. In gel shift assays, PAO was the most effective inducer. The intrinsic tryptophan fluorescence of...