Abstract: PⅡ protein is a general regulator of nitrogen metabolism in bacteria, which can sense the changes of carbon and nitrogen signals in cells and fine-tune the interaction between itself and the target proteins, so as to achieve the purpose of cascade regulation of nitrogen fixation. α-ketoglutarate is an important signal molecule of intracellular carbon status. Previous studies have found that PⅡ protein can bind α-ketoglutarate to sense intracellular carbon status, but there are differences in different bacteria. Pseudomonas stutzeri A1501 contains only one PⅡ protein GlnK, which plays an important role in the regulation of nitrogen fixation in A1501, but the specific carbon signal perception and transduction mechanisms needs to be further explored. Based on this, GlnK protein was expressed in Escherichia coli exogenously, and the in vitro interaction between GlnK protein and carbon signal molecule α-ketoglutarate was studied by microscale thermophoresis (MST) method. It was found that GlnK could interact with the signal molecule α-ketoglutarate in vitro, and it was further confirmed that the 89th glycine (G89, Gly89) of GlnK protein might be related to the interaction. The results laid a foundation for further analysis of α-ketoglutarate signal transduction in A1501, and also provided theoretical support for the in-depth analysis of the coupling mechanism of carbon and nitrogen metabolism of nitrogen-fixing bacteria.

Key words: Pseudomonas stutzeri A1501, GlnK protein, α-ketoglutarate, interaction

摘要： 细菌中PⅡ蛋白是一类氮代谢调控因子，可通过感知胞内碳氮信号变化调整自身与靶蛋白的相互作用，从而实现对下游基因的级联调控。α-酮戊二酸是胞内碳状态的重要信号分子，前期研究发现PⅡ蛋白可结合α-酮戊二酸感应细胞内的碳状态，但不同菌中二者的结合存在差异。施氏假单胞菌A1501（Pseudomononas stutzeri A1501）只含有1种PⅡ蛋白GlnK，其在A1501固氮调控中发挥重要作用，但具体碳信号感知与转导机制有待进一步探索。基于此，通过大肠杆菌外源表达GlnK蛋白，并通过微量热泳动（microscale thermophoresis，MST）方法研究GlnK蛋白与碳信号分子α-酮戊二酸的体外互作，发现二者可以体外结合，且进一步证实GlnK蛋白的第89位甘氨酸（G89，Gly89）可能与互作相关。研究结果为进一步解析α-酮戊二酸在A1501中的信号转导奠定了基础，也为深入解析固氮菌的碳氮代谢偶联机制提供了理论支持。

关键词: 施氏假单胞菌A1501, GlnK蛋白, &alpha, -酮戊二酸, 分子互作