西瓜食酸菌抗铜基因cueR的生物信息学分析及功能验证

Bioinformatics analysis and functional verification of copper resistance gene cueR in Acidovorax citrulli

摘要:

【背景】CueR被证实在模式细菌大肠杆菌的Cue抗铜系统中参与转录调控，西瓜食酸菌(Acidovorax citrulli)中是否有类似的机制尚不清楚。【目的】鉴定西瓜食酸菌中的cueR基因、分析其编码蛋白的特点与功能，可以为进一步探究类Cue系统在西瓜食酸菌铜稳态中的作用机制奠定基础。【方法】以大肠杆菌等4个模式细菌中已经鉴定的CueR为参照，运用生物信息学手段对西瓜食酸菌的CueR (AcCueR)与大肠杆菌的EcCueR、铜绿假单胞菌的PaCueR、沙门氏菌的SeCueR、霍乱弧菌的VcCueR蛋白进行结构、性质、亚细胞定位、互作因子等特征分析；利用同源重组插入突变技术构建西瓜食酸菌FC440菌株cueR基因的突变体，并制备突变体基因功能互补菌株，比较分析各菌株抗铜性表型。【结果】西瓜食酸菌和铜绿假单胞菌的CueR序列相似性最高；5个细菌的CueR蛋白均属于HTH-MerR-SF超家族，三级结构主要由α-螺旋和无规则卷曲构成；5种蛋白结构相似；AcCueR可以与西瓜食酸菌中P型ATP酶(即CopA)、多铜氧化酶CueO产生互作，且copA启动子中存在一个与CueR结合的回文结构。在含Cu2+培养基上，突变菌株FC440(?cueR)生长能力明显减弱，基因功能互补菌株FC440(?cueR-cueR)的生长能力则完全恢复。【结论】西瓜食酸菌中的cueR基因与菌的抗铜性相关，其AcCueR蛋白与大肠杆菌等菌中的CueR具有相似的结构与功能，在西瓜食酸菌中可能存在类似于大肠杆菌的Cue抗铜系统。

Abstract:

[Background] CueR is involved in transcription al regulation of Cue copper-resistant system in model bacteria Escherichia coli. However, if a similar system is present in Acidovorax citrulli, a bacterial plant pathogen, remains unclear. [Objective] Analyzing the cueR gene and its protein in Acidovorax citrulli can facilitate the mechanism study of copper resistance for this destructive phytopathogen. [Methods] AcCueR of Acidovorax citrulli was identified and compared with CueRs from four representative bacteria, i.e. EcCueR of E. coli, PaCueR of Pseudomonas aeruginosa, SeCueR of Salmonella and VcCueR of Vibrio cholerae. The characteristics of the structure, physicochemical properties, subcellular localization, and interaction proteins of these CueRs were analyzed by bioinformatics methods. The cueR gene mutant was prepared through homologous recombination with Acidovorax citrulli strain FC440. The phenotypes of copper resistance of wild type strain, cueR gene mutant, and gene functional complementary strain were also assayed. [Results] Comparative sequence analysis of CueR proteins revealed that AcCueR and PaCueR have the highest sequence similarity. All these five CueR proteins belong to HTH-MerR-SF super family. The tertiary structure is mainly composed of alpha-helix and coiled-coil. The CueR proteins of different bacteria are similar. AcCueR can interact with P-type ATPase (CopA) and multi-copper-oxidase (CueO) in Acidovorax citrulli. In the promoter of copA, there is a typical palindrome motif that binds to CueR. When challenged with Cu2+, the cueR mutated strain FC440(?cueR) exhibited a significantly reduced in growth. Consistently, the growth capacity of the complementary strains completely recovered. [Conclusion] The cueR gene contributes to its copper resistance in Acidovorax citrulli. The AcCueR protein has a similar structure and function compared with CueR in E. coli and other bacteria. These results indicate that the Cue copper-resistance system works in Acidovorax citrulli.