Metalloregulatory properties of the ArsD repressor

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 Kd values for Sb(III) and As(III) of 2 and 60 microM, respectively, were obtained. These results demonstrate that the arsR-arsD pair provide a sensitive mechanism for sensing a wide range of environmental heavy metals.