食异源物鞘氨醇菌类UrcA家族膜蛋白的表达及功能表征

Expression and functional characterization of UrcA-like family membrane protein from Sphingobium xenophagum

摘要:

目的 解析食异源物鞘氨醇菌(Sphingobium xenophagum) C1的类UrcA家族膜蛋白Chr1_2170的表达策略、DNA结合特性，及其在重金属响应与转录调控中的功能。方法 采用密码子优化、双信号肽引导以及同源分子伴侣共表达策略，在大肠杆菌(Escherichia coli) BL21(DE3)中对Chr1_2170膜蛋白进行表达与纯化；构建蛋白结合基因组DNA片段的功能性启动子文库，筛选Chr1_2170的互作元件；利用Chr1_2170-Luc报告系统分析Chr1_2170的重金属响应特性。结果 成功实现Chr1_2170膜蛋白在E. coli BL21(DE3)中的异源表达与纯化；筛选并鉴定出6个Chr1_2170特异性结合的启动子区域，其保守基序为5′-AATXGCGXGTA-3′；结合基因功能注释发现Chr1_2170蛋白调控β-N-乙酰氨基葡萄糖苷酶、双组分系统ATP结合蛋白、DNA拓扑异构酶IV亚基B、丝氨酸水解酶等多个基因的表达。Chr1_2170对Cu²⁺ (1-80 μmol/L)、Zn²⁺ (1-80 μmol/L)、Ba²⁺ (1-150 μmol/L)表现出剂量依赖性响应。结论 Chr1_2170不仅可作为重金属感应元件，还可作为多功能转录调控因子，通过识别特定DNA序列调控相关基因的表达在细菌环境适应性与应激响应中发挥重要作用。

Abstract:

Objective To analyze the expression strategy, DNA-binding characteristics, and the role in heavy metal responses and transcriptional regulation of the UrcA-like membrane protein Chr1_2170 from Sphingobium xenophagum C1.Methods Chr1_2170 was expressed in Escherichia coli BL21(DE3) by codon optimization, dual-signal peptide guidance, and co-expression with homologous molecular chaperones. The interacting genes of Chr1_2170 were screened by constructing a functional promoter library of protein-bound genomic DNA fragments. The heavy metal response characteristics of Chr1_2170 were analyzed via the Chr1_2170-Luc reporter system.Results Chr1_2170 was successfully expressed in E. coli BL21(DE3). Six promoter regions specifically bound by Chr1_2170 were screened out and identified, with the conserved motif of 5′-AATXGCGXGTA-3′. Gene function annotation predicted that Chr1_2170 regulated multiple genes, including those encoding β-N-acetylglucosaminidase, two-component system ATP-binding protein, DNA topoisomerase IV subunit B, and serine hydrolase. Chr1_2170 showed dose-dependent responses to Cu²⁺ (1-80 μmol/L), Zn²⁺ (1-80 μmol/L), and Ba²⁺ (1-150 μmol/L).Conclusion Chr1_2170 functions not only as a heavy metal sensing element but also as a multifunctional transcriptional regulator. It regulates the expression of related genes by recognizing specific DNA sequences, playing a key role in environmental adaptation and stress responses of bacteria.