Operator Binding of the CbbR Protein, Which Activates the Duplicate Cbb CO2 Assimilation Operons of Alcaligenes Eutrophus

The regulatory protein CbbR, which activates the transcription of the duplicate, chromosomally and megaplasmid pHG1-borne cbb CO2 assimilation operons of Alcaligenes eutrophus H16, was purified to homogeneity from Escherichia coli after heterologous expression of the cloned cbbR gene. The pure protein occurred as either a 63-kDa dimer at room temperature or a 125-kDa tetramer at 4 degrees C. CbbR bound to the 167-bp cbb control region separating the divergently oriented cbbR gene (defective copy on pHG1) from the cbb operon. DNase I footprinting revealed binding of the protein between position -29 and -74 relative to the transcriptional start point of the cbb operon, with a hypersensitive site at positions -47 and -48, suggesting potential DNA bending. Hydroxyl radical footprinting disclosed the same central binding region. The region was found to consist of two subsites to which the activator apparently bound in a cooperative manner. At higher CbbR concentrations, the binding region extended to position +13. The overlapping arrangement of the operon promoter and CbbR-binding region (operator) suggests an interaction between CbbR and RNA polymerase to cause transcription activation. Transcriptional fusions with fragments carrying 1- or 2-bp insertions within the central region showed no operon promoter activity, although CbbR binding was not prevented by these mutations. Dissection of the central region enabled the differentiation of two apparently independent binding subsites. Strongly increased cbbR promoter activity originating from a fragment that contained only a part of the central region indicated negative autoregulation of cbbR transcription.