Functional and Expression Analyses of the cop Operon, Required for Copper Resistance in Agrobacterium tumefaciens

Abstract

The copper resistance determinant copARZ, which encodes a CPx-type copper ATPase efflux protein, a transcriptional regulator, and a putative intracellular copper chaperone, was functionally characterized for the phytopathogenic bacterium Agrobacterium tumefaciens. These genes are transcribed as an operon, and their expression is induced in response to increasing copper and silver ion concentrations in a copR-dependent fashion. Analysis of the copARZ promoter revealed a putative CopR binding box located within the spacer of the ؊35 and ؊10 promoter motifs. In vitro, purified CopR could specifically bind to the box. The inactivation of the copARZ operon or copZ reduces the level of resistance to copper but not to other metal ions. Also, the copARZ operon mutant shows increased sensitivity to the superoxide generators menadione and plumbagin. In addition, the loss of functional copZ does not affect the ability of copper ions to induce the copARZ promoter, indicating that CopZ is not involved in the copper-sensing mechanism of CopR. Altogether, the results demonstrate a crucial role for the copARZ operon as a component of the copper resistance machinery in A. tumefaciens.

The interplay of the metallosensor CueR with two distinct CopZ chaperones defines copper homeostasis in Pseudomonas aeruginosa

“…A slightly more pronounced phenotype was observed in the double mutant ⌬copZ1/⌬copZ2, whereas the susceptibility to Cu 2ϩ was abrogated in the ⌬copZ1::copZ1-complemented strain. This altered tolerance to Cu 2ϩ in ⌬copZ1 was similar to that observed for deletion mutants of homologous genes in Bacillus subtilis, Agrobacterium tumefaciens, and Enterococcus hirae (22,(33)(34)(35); albeit, mutation of CopZ in Listeria monocytogenes and Pseudomonas fluorescens did not lead to a diminished metal tolerance (36,37). The distinct growth phenotypes of ⌬copZ1 and ⌬copZ2 under Cu 2ϩ stress were supported by determinations of whole cell Cu levels ( Fig.…”

Copper-responsive gene regulation in bacteria

“…tumefaciens is a plant-pathogenic bacterium that induces tumour formation in dicotylic plants. A. tumefaciens contains the copARZ operon, which encodes the copper-ATPase CopA, the CueR-like activator CopR, and the putative copper chaperone CopZ (Nawapan et al, 2009). CopR induces expression of the copARZ operon in response to copper and silver by binding a palindromic promoter sequence similar to the motif recognized by E. coli CueR.…”

A comprehensive phylogenetic analysis of copper transporting P1B ATPases from bacteria of the Rhizobiales order uncovers multiplicity, diversity and novel taxonomic subtypes

“…We found CopZ in four rhizobial genomes, previously surveyed in Ridge's work (Ridge, Zhang, & Gladyshev, 2008) but undetected by them; the same study was also unable to detect the presence of the CopZ homolog present in the A. tumefaciens plant pathogen which was characterized later on (Nawapan et al, 2009), indicating that Ridge′s method, based on BLAST searches, was not sensitive enough to detect distant CopZ homologs.The absence of CopZ homologs in fourteen rhizobial genomes from different species implies that an alternative protein should carry out its function. We found eight putative S. pneumoniae CupA homologs, five of them in strains without CopZ.…”