M. tuberculosis copper-sensitive operon repressor (Mtb CsoR) is the founding member of a new metalloregulatory protein family in prokaryotes that regulates the transcription of the cso operon in response to copper toxicity. Mtb CsoR tetramer binds 1 monomer mol equiv of Cu(I) with very high affinity (log KCu=18.0) via three conserved residues, Cys36, His61' and Cys65'. Binding of Cu(I) allosterically inhibits the CsoR binding to the DNA operator (CsoO) overlapping the cso promoter (DeltaGc=+3.6 kcal/mol, pH 7.0, 25 oC). These findings are consistent with a role of CsoR as a transcriptional repressor with Cu(I) binding inducing transcriptional derepression. To explore the mechanism of this regulation, His61 was substituted with 1-methylhistidine (MeH) or Beta- (2-thiazolyl)-alanine (Thz) using a native chemical ligation strategy. The CsoO binding affinities of the resultant H61MeH and H61Thz CsoRs are both refractory to inhibition by Cu(I) binding despite the fact that each forms a high affinity 3-coordinate complex with Cu(I). This suggests that while Cu(I) is coordinated by the N?11 atom of His61, the N?22 atom plays an critical role in driving this allosteric switch. Evidence in support of a formation of a hydrogen bonding network involving the N?1 face of His61 and two conserved "second coordination shell" residues, Glu81' and Tyr35, is presented. Remarkably, this mechanism is analogous to that proposed for the Zn(II) sensor CzrA from S. aureus. To test this, we employed the same native chemical ligation approach to substitute the key Zn(II) ligand His97 with 1-methylhistidine; with the preliminary findings fully consistent with an intersubunit allosteric switch involving the N?2 face of this key His97 residue in CzrA. Two predicted homologs of Mtb CsoR were also biochemically characterized to obtain additional support for the hypothesis that CsoR is a key Cu(I) regulatory protein in many bacterial species. B. subtilis CsoR, known to regulate the transcription of the copZA operon, was found to have biochemical properties similar to those of Mtb CsoR as to Cu(I) binding, DNA binding and Cu(I)-dependent allosteric regulation. Interestingly, Bsu CsoR also binds other divalent metal ions (Zn, Ni) with high affinity but with metal coordination geometries distinct from that of Cu(I). Binding of these divalent metal ions only weakly regulates copZA operator binding in vitro, suggesting that coordination number and geometry are most closely related to the allosteric regulation. Finally, a putative CsoR from the pathogenic S. aureus Newman strain was identified and characterized, and was found to exhibit biochemical properties similar to those of Mtb and Bsu CsoRs. Parallels between Cu(I)-sensing CsoRs and functional orthologs in the CsoR/RcnR family are further discussed in the context of the mechanism and evolutionary divergence of this new family of regulatory proteins.