Purification and characterization of CopR, a transcriptional activator protein that binds to a conserved domain (cop box) in copper-inducible promoters of Pseudomonas syringae.

The copper resistance (cop) operon promoter (Pcop) of Pseudomonas syringae is copper-inducible, and requires the regulatory genes copRS. Sequence analysis revealed that CopR has significant homology with other known activator proteins from bacterial two-component regulatory systems. In the present study we characterized Pcop and its interaction with CopR. We found that crude protein extracts from copper-resistant and -sensitive strains of P. syringae contain a Pcop-specific DNA-binding protein. We hypothesized that this DNA-binding protein was the product of copR. A 27-kDa protein, which corresponded to the predicted copR product, was expressed from this gene in Escherichia coli. CopR was purified, and the first eight amino acids were sequenced to confirm its relationship to copR. Specific binding of purified CopR to the plasmid-borne Pcop and the chromosomally encoded cop homolog promoter (PcopH), identified in this report, was demonstrated using specific and non-specific promoter competitors in DNA mobility shift assays. DNAse I footprinting identified a conserved CopR binding region (cop box) on Pcop and PcopH. The cop box contains an inverted repeat within a stretch of 16 bp, which shares approximately 75% identity with the PhoB binding region from several phosphate regulon gene promoters in E. coli. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of copA, and the transcriptional initiation site of PcopH 88 bp 5' to the translational start site of the chromosomal homolog of copA. The cop box was localized to between positions -54 and -35 relative to the transcriptional initiation site of Pcop and PcopH. Deletion analysis of Pcop delimited copper-inducible activity to a 104-bp region. Pcop and PcopH do not share a sequence consensus with other characterized promoters from P. syrinagae or E. coli. The results presented delineate important regions on two copper-inducible promoters form P. syringae.