Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.
Braz J Med Biol Res. 2012 Feb;45(2):113-7. doi: 10.1590/s0100-879x2012007500006. Epub 2012 Jan 26.
Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ(54) co-factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH(4)Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.
巴西固氮螺菌是一种与水稻、小麦和玉米等重要农作物相关的固氮细菌。该细菌中负责固氮的基因（nif 基因）的表达依赖于转录激活因子 NifA。该蛋白包含三个结构域：N 端结构域负责固定氮的负调控；中央结构域与 RNA 聚合酶 σ(54)辅助因子相互作用，C 端结构域参与 DNA 结合。中央和 C 端结构域由结构域间接头（IDL）连接。中央结构域末端和 IDL 中包含的保守四半胱氨酸基序可能参与 NifA 的氧敏感性。在本研究中，我们表达、纯化并表征了巴西固氮螺菌 NifA 的 N 端截断形式。在大肠杆菌中进行蛋白表达，使用亲和金属螯合树脂进行色谱纯化，然后使用肝素结合树脂进行纯化。通过密度计分析确定蛋白质的均一性。N 端截断蛋白在体内激活 nifH::lacZ 转录，而与固定氮浓度（存在或不存在 20mM NH(4)Cl）无关，但仅在低氧水平下。另一方面，通过电泳迁移率变动分析证明，需氧纯化的 N 端截断 NifA 蛋白与 nifB 启动子结合，这意味着 DNA 结合活性不受氧水平的严格控制。我们的数据表明，虽然 N 端截断的 NifA 在有氧条件下体内无活性，但它仍然保留 DNA 结合活性，这表明与 DNA 结合的氧化形式的 NifA 不能激活转录。
