Sotomaior P, Araújo L M, Nishikawa C Y, Huergo L F, Monteiro R A, Pedrosa F O, Chubatsu L S, Souza E M
Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.
Braz J Med Biol Res. 2012 Dec;45(12):1135-40. doi: 10.1590/s0100-879x2012007500146. Epub 2012 Sep 18.
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.
巴西固氮螺菌是一种与重要农作物共生的固氮菌，因此有潜力成为一种氮生物肥料。巴西固氮螺菌转录调节因子NifA似乎是组成型表达的，它激活固氮基因的转录。有人提出，氮状态信号蛋白GlnB通过与NifA的N端GAF结构域直接相互作用来调节NifA的活性，从而在固氮条件下防止该结构域的抑制作用。在本研究中，我们发现NifA的N端截短形式不再需要GlnB来发挥活性，并且失去了铵的调节作用。另一方面，N端GAF结构域的反式共表达会响应固定氮水平抑制N端截短的蛋白。我们还使用下拉实验来展示纯化的NifA N端GAF结构域与GlnB蛋白之间的体外相互作用。结果表明，巴西固氮螺菌GlnB与NifA的N端结构域直接相互作用，并且这种相互作用依赖于ATP和2-氧代戊二酸的存在。
