精氨酸代谢调控蛋白ArgR调控嗜热链球菌胞外多糖合成

目的] 研究精氨酸代谢调控蛋白ArgR对嗜热链球菌胞外多糖（EPS）合成的调控作用。方法] 利用大肠杆菌异源表达嗜热链球菌ArgR蛋白，通过尿素变性-复性和Ni²⁺亲和层析纯化。采用凝胶电泳迁移（EMSA）和生物膜层干涉（BLI）分析ArgR和eps基因簇中P_epsA启动子的相互作用和动力学信息。构建过表达和弱化argR基因菌株，利用苯酚-硫酸法测定其合成EPS差异。结果] 大肠杆菌异源表达的ArgR为包涵体，使用尿素变性-复性纯化可获得2.95 mg/mL可溶性蛋白；EMSA和BLI结果显示ArgR和启动子P_epsA有特异性结合，且结合因解离水平低而稳定；过表达argR基因可显著降低嗜热链球菌EPS合成，而弱化argR基因则提高EPS合成。结论] 本研究表明ArgR能特异性结合嗜热链球菌eps基因簇启动子，并负调控EPS生物合成。

Arginine regulator ArgR regulates exopolysaccharides biosynthesis of Streptococcus thermophilus

Objective] The regulatory effect of arginine regulator ArgR on the biosynthesis of exopolysaccharides (EPS) was studied in Streptococcus thermophilus. Methods] ArgR from S. thermophilus was heterologously expressed by Escherichia coli, and purified by urea denaturation refolding and Ni²⁺ affinity chromatography. The interaction and kinetic information between ArgR and eps promoter P_epsA were detected by electrophoretic mobility shift assays (EMSA) and biolayer interferometry (BLI). The yield alteration of EPS was determined by phenol-sulfuric acid assay when the gene argR overexpressed or repressed. Results] Heterologous expression of ArgR was formed inclusion body, and 2.95 mg/mL of soluble protein was achieved by urea denaturation refolding. EMSA and BLI analysis showed that ArgR can specifically bind with the promoter P_epsA and their affinity was high because of the low dissociation. Increased expression of argR gene reduced EPS synthesis and the suppression raised. Conclusion] It is the first time to report that ArgR can specifically bind the promoter of eps gene cluster and negatively regulate EPS synthesis in S. thermophilus.