铜绿假单胞菌cntRLMN操纵子介导锌离子摄取的功能鉴定

Characterization of zinc ion uptake mediated by cntRLMN operon in Pseudomonas aeruginosa

摘要:

[目的] 本研究以铜绿假单胞菌PAO1（Pseudomonas aeruginosa PAO1，菌种编号ATCC15692）为对象，研究cntRLMN在锌离子摄取中的功能。[方法] 在ΔznuBC的基础上，以同源重组的方法构建了cntRLMN的各种突变菌株，通过质粒接合转移的方法构建其互补菌株及lacZ转录融合报告菌株，运用β-半乳糖苷酶酶活检测研究了Zur蛋白对cntRLMN的转录调控，凝胶阻滞实验（EMSA）检验Zur蛋白与cnt启动子及cnt启动子的突变片段的体外结合，并进一步通过生长曲线分析对cntRLMN中cntR、cntL、cntN等基因的锌离子摄取功能进行了分析和鉴定。最终，通过构建大蜡螟幼虫的侵染模型来研究cntRLMN对铜绿假单胞菌毒力发挥的影响。[结果] lacZ转录融合的酶活分析显示cntRLMN受Zur蛋白的负调控，其表达以Zur蛋白依赖的方式受锌离子饥饿的诱导；EMSA实验的结果显示cntRLMN的启动子可以与His-Zur结合形成DNA-蛋白质复合体，结合位点为GCGTTATAGTATATCAT；生长曲线和大蜡螟幼虫侵染实验的分析结果显示ZnuBC和CntRLMN的功能存在互补性，仅znuBC和cntRLMN双缺失突变时菌株在限锌培养条件下的生长和对大蜡螟幼虫的毒性才受到显著抑制，说明CntRLMN代表另一种独立的锌离子摄取系统。[结论] cntRLMN是受Zur直接负调控的另一种独立的铜绿假单胞菌锌离子摄取系统，对铜绿假单胞菌毒力的发挥起重要作用。

Abstract:

[Objective] We characterized the function of cntRLMN in zinc ion uptake of Pseudomonas aeruginosa PAO1 (ATCC15692). [Methods] On the basis of the mutant strain ΔznuBC, we built ΔcntRLMN using homologous recombination. We also constructed its complementary strain and lacZ fusion report strain through plasmid conjugation transfer. Transcriptional regulation of Zur protein on cntRLMN was studied by beta-galactoside enzyme activity assay. In vitro binding of Zur protein to cnt promoter and mutated fragments of cnt promoter was tested using EMSA. The zinc ion uptake function of cntR, cntL, cntN and other genes in the cntRLMN operon was examined through growth curve analysis. Finally, the influence of cntRLMN mutation on the virulence of P. aeruginosa was studied using the infection model of the wax moth larva. [Results] The enzyme activity analysis of lacZ transcription and fusion showed that cntRLMN was negatively regulated by Zur protein, and its expression was induced by zinc ion starvation in a Zur-dependent manner. The EMSA results showed that the promoter of cntRLMN could bind His-Zur to form a DNA-protein complex, where the binding site was GCGTTATAGTATATCAT. The growth curve and the infection experiment of the wax moth larva showed that ZnuBC and CntRLMN were functionally complementary; when grown in Zn-restricted culture, the toxicity of P. aeruginosa to the wax moth larve was significantly inhibited only in the znuBC and cntRLMN double-deleted strain. [Conclusion] The cntRLMN, which plays an important role in toxicity of P. aeruginosa, is likely an alternative zinc ion uptake system directly and negatively regulated by the Zur protein.