title: Functional analysis of the GAF domain of NifA in Azospirillum brasilense: effects of Tyr-->Phe mutations on NifA and its interaction with GlnB.
abstract: Regulation of NifA activity in Azospirillum brasilense depends on GlnB (a PII protein), and it was previously reported that the target of GlnB activity is the N-terminal domain of NifA. Furthermore, mutation of the Tyr residue at position 18 in the N-terminal domain resulted in a NifA protein that did not require GlnB for activity under nitrogen fixation conditions. We report here that a NifA double mutant in which the Tyr residues at positions 18 and 53 of NifA N-were simultaneously replaced by Phe (NifA-Y1853F) displays high nitrogenase activity, which is still regulatable by ammonia, but not by GlnB. The yeast two-hybrid technique was used to investigate whether GlnB can physically interact with wild-type and mutant NifA proteins. GlnB was found to interact directly with the N-terminal GAF domain of wild-type NifA, but not with its central or C-terminal domain. GlnB could still bind to the single NifA mutants Y18F and Y53F. In contrast, no interaction was detected between GlnB and the double mutant NifA-Y18/53F or between GlnB and NifA-Y43.
authors.affiliation: Department of Microbiology and National Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing, 100094, China.
authors.forename: Sanfeng
authors.initials: S
authors.lastname: Chen
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Zhou
authors.forename: Claudine
authors.initials: C
authors.lastname: Elmerich
authors.forename: Ji-Lun
authors.initials: JL
authors.lastname: Li
mesh_terms: D016949:Azospirillum / Q000378:metabolism; D015806:Azospirillum brasilense / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009591:Nitrogenase / Q000378:metabolism; D051922:PII Nitrogen Regulatory Proteins; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013696:Temperature; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000737:chemistry; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C061338:NifA protein, Bacteria; D051922:PII Nitrogen Regulatory Proteins; D014157:Transcription Factors; D014443:Tyrosine; C404143:PIID regulatory protein, Bacteria; D009591:Nitrogenase; D001616:beta-Galactosidase