Regulation of the cnr cobalt and nickel resistance determinant of Ralstonia eutropha (Alcaligenes eutrophus) CH34

The linked resistance to nickel and cobalt of Ralstonia eutropha-like strai n CH34 (Alcaligenes eutrophus CH34) is encoded by the cnr operon, which is localized on the megaplasmid pMOL28. The regulatory genes cnrYXH have been cloned, overexpressed, and purified in Escherichia coli, CnrY fractionated as a 10.7-kDa protein in in vitro translation assays, CnrX, a periplasmic p rotein of 16.5 kDa, was overproduced and purified as a histidine-tagged fus ion protein in E. coli, His-CnrX was found to posses a secondary structure content rich in alpha-helical and beta-sheet structures. CnrH, a sigma fact or of the extracytoplasmic function family, was purified as an N-terminally histidine-tagged fusion. In gel shift mobility assays, His-CnrH, in the pr esence of E, coil core RNA polymerase enzyme, could retard at least two dif ferent promoter DNA targets, cnrYp and cnrHp, localized within the cnrYXN l ocus. These promoters and their transcription start sites were confirmed by primer extension. Purified His-CnrX did not inhibit the DNA-binding activi ty of His-CnrH and is therefore unlikely to be an anti-sigma factor, as pre viously hypothesized (EMBL M91650 description entry). To study the transcri ptional response of the regulatory locus to metals and to probe promoter re gions, transcriptional fusions were constructed between fragments of cnrYXH and the luxCDABE, luciferase reporter genes. Nickel and cobalt specificall y induced the cnrYXH-luxCDABE fusion at optimal concentrations of 0.3 mM Ni 2+ and 2.0 mM Co2+ in a noncomplexing medium for metals. The two promoter r egions P-Y (upstream cnrY) and P-H (upstream cnrH) were probed and characte rized using this vector and were found to control the nickel-inducible regu latory response of the cnr operon, The cnrHp promoter was responsible for f ull transcription of the cnrCBA structural resistance genes, while the cnrY p promoter was necessary to obtain metal-inducible transcription from the c nrHp promoter. The zinc resistance phenotype (ZinB) of a spontaneous cnr mu tant strain, AE963, was investigated and could be attributed to an insertio n of IS1087, a member of the IS2 family of insertion elements, within the c nrY gene.