State Key Laboratory of Agrobiotechnology and College of Biological Sciences, China Agricultural University, Beijing 100193, PR China.
Microbiol Res. 2011 Sep 20;166(6):458-67. doi: 10.1016/j.micres.2010.08.006. Epub 2010 Sep 23.
We here report the sequence and functional analysis of org35 of Azospirillum brasilense Sp7, which was originally identified to be able to interact with NifA in yeast-two-hybrid system. The org35 encodes a hybrid two-component system protein, including N-terminal PAS domains, a histidine kinase (HPK) domain and a response regulator (RR) domain in C-terminal. To determine the function of the Org35, a deletion-insertion mutant in PAS domain [named Sp7353] and a complemental strain Sp7353C were constructed. The mutant had reduced chemotaxis ability compared to that of wild-type, and the complemental strain was similar to the wild-type strain. These data suggested that the A. brasilense org35 played a key role in chemotaxis. Variants containing different domains of the org35 were expressed, and the functions of these domains were studied in vitro. Phosphorylation assays in vitro demonstrated that the HPK domain of Org35 possessed the autokinase activity and that the phosphorylated HPK was able to transfer phosphate groups to the RR domain. The result indicated Org35 was a phosphorylation-communicating protein.
我们在此报告巴西固氮螺菌 Sp7 中 org35 的序列和功能分析，该基因最初被鉴定为能够在酵母双杂交系统中与 NifA 相互作用。org35 编码一种混合双组分系统蛋白，包括 N 端 PAS 结构域、组氨酸激酶 (HPK) 结构域和 C 端响应调节子 (RR) 结构域。为了确定 Org35 的功能，构建了一个 PAS 结构域缺失插入突变体 [命名为 Sp7353] 和一个互补菌株 Sp7353C。与野生型相比，突变体的趋化能力降低，而互补菌株与野生型菌株相似。这些数据表明，巴西固氮螺菌 org35 在趋化性中起着关键作用。表达了含有不同 org35 结构域的变体，并在体外研究了这些结构域的功能。体外磷酸化实验表明，Org35 的 HPK 结构域具有自激酶活性，磷酸化的 HPK 能够将磷酸基团转移到 RR 结构域。结果表明 Org35 是一种磷酸化通讯蛋白。
