Bacillus subtilis CsoR (Bsu CsoR) is a copper-sensing transcriptional repressor that regulates the expression of the copZA operon encoding a copper chaperone and a Cu efflux P-type ATPase, respectively. Bsu CsoR is a homologue of Mycobacterium tuberculosis CsoR (Mtb CsoR), representative of a large Cu(I)-sensing regulatory protein family. We show here that Bsu CsoR binds approximately 1 mol equiv of Cu(I) per monomer in vitro with an affinity >or=10(21) M(-1). X-ray absorption spectroscopy shows Cu(I) adopts a trigonal S(2)N coordination like Mtb CsoR. Both apo and Cu(I)-bound Bsu CsoR are stable tetramers in the low micromolar monomer concentration range by sedimentation velocity and equilibrium ultracentrifugation. Apo-Bsu CsoR binds to a pseudopalindromic 30 bp copZA operator-promoter DNA with a stoichiometry of two tetramers per DNA and stepwise affinities of K(1)(apo) = 3.1(+/-0.8) x 10(7) M(-1) and K(2)(apo) = 8.3 (+/-2.2) x 10(7) M(-1) (0.4 M NaCl, 25 degrees C, pH 6.5). Cu(I) Bsu CsoR binds to the same DNA with greatly reduced affinities, K(1)(Cu) = 2.9(+/-0.4) x 10(6) M(-1) and K(2)(Cu) <or= 1.0 x 10(5) M(-1) consistent with a copper-dependent derepression model. This Cu-dependent regulation is abrogated by a "second shell" Glu90-to-Ala substitution. Bsu CsoR binds Ni(II) with very high affinity but forms a non-native coordination geometry, as does Co(II) and likely Zn(II); none of these metals strongly regulates copZA operator DNA binding in vitro. The implications of these findings on the specificity of metal-sensing sites in CsoR/RcnR proteins are discussed.