Regulation of high affinity nickel uptake in bacteria - Ni2+-dependent interaction of NikR with wild-type and mutant operator sites

Escherichia coli actively imports nickel via the ATP-dependent NikABCDE per mease. NikR, a protein of the ribbon-helix-helix family of transcription fa ctors, represses expression of the nikABCDE operon in the presence of exces sive concentrations of intracellular nickel. Here, the NikR operator site i s identified within the nikABCDE promoter by footprinting and mutational an alyses. The operator consists of two dyad-symmetric 5'-GTATGA-3' recognitio n sequences separated by 16 base pairs. Mutations in the GTATGA sequences r educe NikR binding affinity in vitro and reduce repression of a P-nik-lacZ fusion in vivo. Moreover, NikR is shown to be a direct sensor of nickel ion s. Strong operator binding requires the continual presence of 20-50 mu M ni ckel, indicating the presence of a low affinity nickel-binding site, and Ni kR dimers also contain two high affinity nickel-binding sites. In addition to both GTATGA sites and nickel, high affinity operator binding also requir es the C-terminal domain of NikR.