Effect of ATP and 2-oxoglutarate on the in vitro interaction between the NifA GAF domain and the GlnB protein of Azospirillum brasilense

Abstract

Azospirillum brasilense is a diazotroph that associates with important agricultural crops and thus has potential to be a nitrogen biofertilizer. The A. brasilense transcription regulator NifA, which seems to be constitutively expressed, activates the transcription of nitrogen fixation genes. It has been suggested that the nitrogen status-signaling protein GlnB regulates NifA activity by direct interaction with the NifA N-terminal GAF domain, preventing the inhibitory effect of this domain under conditions of nitrogen fixation. In the present study, we show that an N-terminal truncated form of NifA no longer required GlnB for activity and lost regulation by ammonium. On the other hand, in trans co-expression of the N-terminal GAF domain inhibited the N-truncated protein in response to fixed nitrogen levels. We also used pull-down assays to show in vitro interaction between the purified N-terminal GAF domain of NifA and the GlnB protein. The results showed that A. brasilense GlnB interacts directly with the NifA N-terminal domain and this interaction is dependent on the presence of ATP and 2-oxoglutarate.

Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense

“…The reduction in the intracellular glutamine levels would affect the nitrogen sensory cascade in such way that GlnD would maintain the uridylylation of GlnB despite the presence of ammonium in the medium. Uridylylated GlnB activates the NifA protein (Sotomaior et al, 2012) thereby allowing the transcription of the genes for nitrogen fixation (nif) (Pedrosa and Yates, 1984). Under nitrogen-fixing conditions, the low GS activity of HM053 reduces the ability to assimilate the NH 4 + produced by nitrogenase thereby facilitating ammonia release by diffusion to the cell membrane to the culture medium (Santos et al, 2017).…”

“…The GlnB paralogue, named GlnK in E. coli and GlnZ in A. brasilense, undergoes a similar cycle of modification by the UTase, but has distinct cell targets. In A. brasilense, GlnB in addition of controlling the ATase activity also controls the NifA transcriptional activator (Sotomaior et al, 2012) and the inactivation of nitrogenase by ADP-ribosylation (Moure et al, 2014) whereas GlnZ controls the reactivation of nitrogenase by the removal of the ADP-ribosyl moiety (Moure et al, 2014).…”

Molecular Mechanism and Agricultural Application of the NifA–NifL System for Nitrogen Fixation

“…GlnB can interact with the GAF domain to activate NifA in nitrogen–limiting conditions. GlnZ may play a critical role in prohibiting the expression of the nitrogenase gene [ 69 ]. In contrast, NifA can be inhibited via interacting with the PII protein to adapt to the nitrogen–excess condition in Rhodobacter capsulatus [ 70 ].…”

Redox-Based Transcriptional Regulation in Prokaryotes: Revisiting Model Mechanisms

“…The activity of the NifA protein is highly regulated in response to redox and fixed nitrogen through interaction with the antiactivator protein NifL. Binding of NifL to NifA inhibits the ATPase activity of NifA, and this interaction is controlled by the amino-terminal GAF domain of NifA that binds 2-oxoglutarate (348). as carbon and nitrogen fixation, electron transport chain configuration, photosynthesis, and aerotaxis (87,320,358,397).…”