Functional characterization of a csoR-cueA divergon in Bradyrhizobium liaoningense CCNWSX0360, involved in copper, zinc and cadmium cotolerance

Random mutagenesis in a symbiotic nitrogen-fixing Bradyrhizobium liaoningense CCNWSX0360 (Bln0360) using Tn 5 identified five copper (Cu) resistance-related genes. They were functionally sorted into three groups: transmembrane transport ( cueA and tolC ); oxidation ( copA ); and protection of the membrane barrier ( lptE and ctpA ). The gene cueA , together with the upstream csoR (Cu-sensitive operon repressor), constituted a csoR-cueA divergon which plays a crucial role in Cu homeostasis. Deletion of cueA decreased the Cu tolerance of cells, and complementation of this mutant restored comparable Cu resistance to that of the wild-type. Transcriptional and fusion expression analysis demonstrated that csoR-cueA divergon was up-regulated by both the monovalent Cu + and divalent Zn 2+ /Cd 2+ , and negatively regulated by transcriptional repressor CsoR, via a bidirectional promoter. Deletion of csoR renders the cell hyper-resistant to Cu, Zn and Cd. Although predicted to encode a Cu transporting P-type ATPase (CueA), cueA also conferred resistance to zinc and cadmium; two putative N-MBDs (N-terminal metal binding domains) of CueA were required for the Cu/Zn/Cd tolerance. Moreover, cueA is needed for nodulation competitiveness of B. liaoningense in Cu rich conditions. Together, the results demonstrated a crucial role for the csoR-cueA divergon as a component of the multiple-metal resistance machinery in B. liaoningense .