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Biochimie. 2012 Apr;94(4):1041-7. doi: 10.1016/j.biochi.2012.01.007. Epub 2012 Jan 15.

Interaction of GlnK with the GAF domain of Herbaspirillum seropedicae NifA mediates NH₄⁺-regulation.

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Department of Biochemistry and Molecular Biology, Universidade Federal do Parana, PO Box 19046, Curitiba PR 81531-990, Brazil.


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.

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