title: Effect of ATP and 2-oxoglutarate on the in vitro interaction between the NifA GAF domain and the GlnB protein of Azospirillum brasilense.
abstract: Azospirillum brasilense is a diazotroph that associates with important agricultural crops and thus has potential to be a nitrogen biofertilizer. The A. brasilense transcription regulator NifA, which seems to be constitutively expressed, activates the transcription of nitrogen fixation genes. It has been suggested that the nitrogen status-signaling protein GlnB regulates NifA activity by direct interaction with the NifA N-terminal GAF domain, preventing the inhibitory effect of this domain under conditions of nitrogen fixation. In the present study, we show that an N-terminal truncated form of NifA no longer required GlnB for activity and lost regulation by ammonium. On the other hand, in trans co-expression of the N-terminal GAF domain inhibited the N-truncated protein in response to fixed nitrogen levels. We also used pull-down assays to show in vitro interaction between the purified N-terminal GAF domain of NifA and the GlnB protein. The results showed that A. brasilense GlnB interacts directly with the NifA N-terminal domain and this interaction is dependent on the presence of ATP and 2-oxoglutarate.
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.
authors.forename: P
authors.initials: P
authors.lastname: Sotomaior
authors.forename: L M
authors.initials: LM
authors.lastname: Araújo
authors.forename: C Y
authors.initials: CY
authors.lastname: Nishikawa
authors.forename: L F
authors.initials: LF
authors.lastname: Huergo
authors.forename: R A
authors.initials: RA
authors.lastname: Monteiro
authors.forename: F O
authors.initials: FO
authors.lastname: Pedrosa
authors.forename: L S
authors.initials: LS
authors.lastname: Chubatsu
authors.forename: E M
authors.initials: EM
authors.lastname: Souza
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D015806:Azospirillum brasilense / Q000201:enzymology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D005822:Genetic Vectors; D007656:Ketoglutaric Acids / Q000378:metabolism*; D010957:Plasmids; D014157:Transcription Factors / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D007656:Ketoglutaric Acids; C061338:NifA protein, Bacteria; D014157:Transcription Factors; D000255:Adenosine Triphosphate; D001616:beta-Galactosidase