氧化葡萄糖酸杆菌中一种潜在的双组分系统蛋白的功能研究

氧化葡萄糖酸杆菌(Gluconobacteroxydans)基因组编码的蛋白质中，有相当数量的传感器激酶和反应调控蛋白组成了细菌的多个双组分信号转导系统(two-componentsignaltransduction systems, TCSs)，这些系统能够介导细菌对外界环境变化做出反应。但目前对G. oxydans中潜在的双组分系统成员蛋白质结构和功能缺少必要的研究【。目的】研究菌株G. oxydans 621H中GOX0645基因序列所编码蛋白质的自磷酸化活性，探究其与细菌趋化性运动的关联，揭示其是否作为一种双组分系统成员蛋白在细胞内发挥作用。【方法】以菌株G. oxydans 621H基因组中一段可能编码双组分系统蛋白质的基因GOX0645为基础，通过生物信息学分析其保守结构域；采用体外化学发光实验证明其编码蛋白的自磷酸化活性；利用基因定点突变筛选出与自磷酸化活性相关的氨基酸位点；通过差速离心法寻找双组分蛋白的亚细胞定位；最后运用体内双分子荧光互补和体外生物大分子相互作用实验印证其与下游鞭毛马达蛋白之间的相互作用。【结果】生物信息学分析发现GOX0645编码蛋白同时具有组氨酸激...

Function of a potential two-component system protein in Gluconobacter oxydans

The genome of Gluconobacter oxydans encodes a large number of sensor kinases and response regulators, potential members of two-component signal transduction systems (TCSs), by which bacteria recognize and respond to a variety of environmental stimuli. However, little is known about the structures and functions of these proteins in G. oxydans. Objective] To study the autophosphorylation activity of a potential TCS protein encoded by GOX0645 in G. oxydans 621H, and to reveal the role of this protein in bacterial chemotaxis.Methods] We performed sequence analysis on GOX0645 from G. oxydans 621H that might encode a TCS member, detected the autophosphorylation activity of the encoded protein by in vitro luminescent kinase assay, and then identified the amino acid residues critical to the autophosphorylation by site-directed mutagenesis. We employed differential centrifugation to study the subcellular localization of the protein, and BiFC assay and Octet^® system to explore the interaction between the protein and a flagellar motor protein of G. oxydans. Results] The protein encoded by GOX0645 contained the conserved domains of both histidine kinases and response regulators, with a conserved Asp and three His residues associated with autophosphorylation activity. Furthermore, the protein, as a hybrid protein, showed the activity of autophosphorylation which was affected by the concentration of cAMP molecule. The protein was localized in the cytoplasm and had a moderate affinity with the flagellar motor protein, which implied its regulatory role in bacterial chemotaxis. Conclusion] This study analyzed a potential TCS that might be involved in the regulation of chemotaxis in G. oxydans 621H. This finding contributes to gaining insight into the molecular mechanism of two-component systems in G.oxydans.