title: Transcription activation in vitro by the Bradyrhizobium japonicum regulatory protein FixK2.
abstract: 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 sigma80-RNA polymerase holoenzyme, but not with E. coli sigma70-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.
authors.affiliation: Institute of Microbiology, Eidgenössische Technische Hochschule, CH-8093 Zürich, Switzerland. mesam@micro.biol.ethz.ch
authors.forename: Socorro
authors.initials: S
authors.lastname: Mesa
authors.forename: Zöhre
authors.initials: Z
authors.lastname: Ucurum
authors.forename: Hauke
authors.initials: H
authors.lastname: Hennecke
authors.forename: Hans-Martin
authors.initials: HM
authors.lastname: Fischer
mesh_terms: D000332:Aerobiosis; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D020369:Bradyrhizobium / Q000235:genetics* / Q000378:metabolism; D003304:Coproporphyrinogen Oxidase / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D066298:In Vitro Techniques; D009586:Nitrogen Fixation / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000502:physiology; D015533:Transcriptional Activation / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D003304:Coproporphyrinogen Oxidase; C066628:FixK protein, Bacteria; C109194:HemN protein, Bacteria