Functional analysis of the GAF domain of NifA in Azospirillum brasilense: effects of Tyr-->Phe mutations on NifA and its interaction with GlnB.
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.
Chen
Sanfeng
S
Department of Microbiology and National Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing, 100094, China.
Liu
Li
L
Zhou
Xiaoyu
X
Elmerich
Claudine
C
Li
Ji-Lun
JL
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
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