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PMID:22269934

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Citation

Oliveira, MA, Aquino, B, Bonatto, AC, Huergo, LF, Chubatsu, LS, Pedrosa, FO, Souza, EM, Dixon, R and Monteiro, RA (2012) Interaction of GlnK with the GAF domain of Herbaspirillum seropedicae NifA mediates NH₄⁺-regulation. Biochimie 94:1041-7

Abstract

Nitrogen fixation in Herbaspirillum seropedicae is transcriptionally regulated by NifA, a σ(54) transcriptional activator with three structural domains: an N-terminal GAF domain, a catalytic AAA+ domain and a C-terminal DNA-binding domain. NifA is only active in H. seropedicae when cultures are grown in the absence of fixed nitrogen and at low oxygen tensions. There is evidence that the inactivation of NifA in response to fixed nitrogen is mediated by the regulatory GAF domain. However, the mechanism of NifA repression by the GAF domain, as well as the transduction of nitrogen status to NifA, is not understood. In order to study the regulation of NifA activity by fixed nitrogen independently of oxygen regulation, we constructed a chimeric protein containing the GAF domain of H. seropedicae NifA fused to the AAA+ and C-terminal domains of Azotobacter vinelandii NifA. This chimeric protein (NifAQ1) lacks the cysteine motif found in oxygen sensitive NifA proteins and is not oxygen responsive in vivo. Our results demonstrate that NifAQ1 responds to fixed nitrogen and requires GlnK protein for activity, a behavior similar to H. seropedicae NifA. In addition, protein footprinting analysis indicates that this response probably involves a protein-protein contact between the GAF domain and the GlnK protein.

Links

PubMed Online version:10.1016/j.biochi.2012.01.007

Keywords

Bacterial Proteins/chemistry; Bacterial Proteins/isolation & purification; Bacterial Proteins/metabolism; Gene Expression Regulation, Bacterial; Genes, Reporter; Herbaspirillum/metabolism; Kinetics; Nitrogen Fixation; Peptide Fragments/chemistry; Protein Binding; Protein Interaction Domains and Motifs; Proteolysis; Quaternary Ammonium Compounds/metabolism; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/isolation & purification; Recombinant Fusion Proteins/metabolism; Transcription Factors/chemistry; Transcription Factors/isolation & purification; Transcription Factors/metabolism; beta-Galactosidase/biosynthesis; beta-Galactosidase/genetics

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

See also

References

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