title: Mutational analysis of GlnB residues critical for NifA activation in Azospirillum brasilense.
abstract: PII proteins are signal transduction that sense cellular nitrogen status and relay this signals to other targets. Azospirillum brasilense is a nitrogen fixing bacterium, which associates with grasses and cereals promoting beneficial effects on plant growth and crop yields. A. brasilense contains two PII encoding genes, named glnB and glnZ. In this paper, glnB was mutagenised in order to identify amino acid residues involved in GlnB signaling. Two variants were obtained by random mutagenesis, GlnBL13P and GlnBV100A and a site directed mutant, GlnBY51F, was obtained. Their ability to complement nitrogenase activity of glnB mutant strains of A. brasilense were determined. The variant proteins were also overexpressed in Escherichia coli, purified and characterized biochemically. None of the GlnB variant forms was able to restore nitrogenase activity in glnB mutant strains of A. brasilense LFH3 and 7628. The purified GlnBY51F and GlnBL13P proteins could not be uridylylated by GlnD, whereas GlnBV100A was uridylylated but at only 20% of the rate for wild type GlnB. Biochemical and computational analyses suggest that residue Leu13, located in the α helix 1 of GlnB, is important to maintain GlnB trimeric structure and function. The substitution V100A led to a lower affinity for ATP binding. Together the results suggest that NifA activation requires uridylylated GlnB bound to ATP.
authors: Department of Chemistry, Universidade Estadual de Ponta Grossa, Av. Gal. Carlos Cavalcanti, 4748, CEP 84030-900 Ponta Grossa, PR, Brazil. Electronic address: jinaba@uepg.br.
authors: Juliana
authors: J
authors: Inaba
authors: Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom. Electronic address: eremy.thornton@jic.ac.uk.
authors: Jeremy
authors: J
authors: Thornton
authors: Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, CP 19046, CEP 81531-990 Curitiba, PR, Brazil. Electronic address: huergo@ufpr.br.
authors: Luciano Fernandes
authors: LF
authors: Huergo
authors: Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, CP 19046, CEP 81531-990 Curitiba, PR, Brazil. Electronic address: rose.adele@terra.com.br.
authors: Rose Adele
authors: RA
authors: Monteiro
authors: Department of Basic Pathology, Universidade Federal do Paraná, CP 19046, CEP 81531-990 Curitiba, PR, Brazil. Electronic address: giseli@ufpr.br.
authors: Giseli
authors: G
authors: Klassen
authors: Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, CP 19046, CEP 81531-990 Curitiba, PR, Brazil. Electronic address: fpedrosa@ufpr.br.
authors: Fábio de Oliveira
authors: Fde O
authors: Pedrosa
authors: Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom. Electronic address: mike.merrick@jic.ac.uk.
authors: Mike
authors: M
authors: Merrick
authors: Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, CP 19046, CEP 81531-990 Curitiba, PR, Brazil. Electronic address: souzaem@ufpr.br.
authors: Emanuel Maltempi
authors: EM
authors: de Souza
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D015806:Azospirillum brasilense / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004252:DNA Mutational Analysis; D015870:Gene Expression; D009154:Mutation; D009591:Nitrogenase / Q000235:genetics; D051922:PII Nitrogen Regulatory Proteins / Q000737:chemistry / Q000235:genetics*; D011485:Protein Binding; D011487:Protein Conformation; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C061338:NifA protein, Bacteria; D051922:PII Nitrogen Regulatory Proteins; D014157:Transcription Factors; C404143:PIID regulatory protein, Bacteria; D000255:Adenosine Triphosphate; D009591:Nitrogenase
keywords: GlnB activity; Mutagenesis; NifA activation