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Transcription Activation In Vitro by the Bradyrhizobium japonicum Regulatory Protein FixK₂

Institute of Microbiology, Eidgenössische Technische Hochschule, CH-8093 Zürich, Switzerland

Received 28 October 2004/ Accepted 4 February 2005

In Bradyrhizobium japonicum, the N₂-fixing root nodule endosymbiont of soybean, a group of genes required for microaerobic, anaerobic, or symbiotic growth is controlled by FixK₂, a key regulator that is part of the FixLJ-FixK₂ cascade. FixK₂ belongs to the family of cyclic AMP receptor protein/fumarate and nitrate reductase (CRP/FNR) transcription factors that recognize a palindromic DNA motif (CRP/FNR box) associated with the regulated promoters. Here, we report on a biochemical analysis of FixK₂ and its transcription activation activity in vitro. FixK₂ was expressed in Escherichia coli and purified as a soluble N-terminally histidine-tagged protein. Gel filtration experiments revealed that increasing the protein concentration shifts the monomer-dimer equilibrium toward the dimer. Purified FixK₂ productively interacted with the B. japonicum ⁸⁰-RNA polymerase holoenzyme, but not with E. coli ⁷⁰-RNA polymerase holoenzyme, to activate transcription from the B. japonicum fixNOQP, fixGHIS, and hemN₂ promoters in vitro. Furthermore, FixK₂ activated transcription from the E. coli FF(–41.5) model promoter, again only in concert with B. japonicum RNA polymerase. All of these promoters are so-called class II CRP/FNR-type promoters. We showed by specific mutagenesis that the FixK₂ box at nucleotide position –40.5 in the hemN₂ promoter, but not that at –78.5, is crucial for activation both in vivo and in vitro, which argues against recognition of a potential class III promoter. Given the lack of any evidence for the presence of a cofactor in purified FixK₂, we surmise that FixK₂ alone is sufficient to activate in vitro transcription to at least a basal level. This contrasts with all well-studied CRP/FNR-type proteins, which do require coregulators.

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