| The first row transition metal ions---Mn, Fe, Co, Ni, Cu and Zn are vital cofactors for proteins involved in diverse processes including photosynthesis, oxidative respiration and protein translation. Despite these essential functions, excess metal ions can lead to cell death through the generation of oxidative damage or the occupation of non-native metal sites. Acquiring metal ions from the environment while limiting possible toxic effects requires the coordinated regulation of metal uptake, trafficking and efflux, which in bacteria is often carried out by metal-responsive transcription factors (metalloregulators). Nickel homeostasis in E. coli is an ideal model system for understanding the relationships between metal physiology and metalloregulator function, as cellular nickel requirements are primarily limited to Ni-Fe hydrogenases, the levels of which can be easily controlled by different electron acceptors in the medium. While the previously studied Ni-responsive metalloregulator NikR controls Ni import, this thesis describes the identification of RcnR, a novel regulator of Ni efflux. RcnR is the founding member of the RcnR/CsoR family of metalloregulators that are found throughout the eubacterial kingdom and respond to a range of different environmental stresses including metal ions, aldehydes and oxidative stress. RcnR-dependent gene repression is directly relieved by excess intracellular Ni(II) or Co(II), which bind to the protein at similar high-spin, six-coordinate, distorted octahedral binding sites. The metal site is distinct from the four coordinate high-affinity site of NikR, and these differences lead to the sequential repression of nickel import and activation of nickel efflux with respect to increasing extracellular nickel levels. Unlike other transcription factors, RcnR/CsoR proteins contain a unique DNA binding motif that recognizes DNA sequences containing G-tracts flanked by AT-rich inverted repeats. RcnR binds to a pair of sites in the rcnA-rcnR intergenic region leading to DNA wrapping, and repression of both genes. The identification of this new metalloregulator family and subsequent investigation of the functional properties of one member, RcnR, provides a solid foundation for understanding the stress responses mediated by other members of this family and the mechanisms of DNA binding by the unique, all-helical fold. |
