铜绿假单胞菌PAO1中c-di-GMP代谢相关基因PA2072的生物学分析

Biological characterization of a c-di-GMP metabolism-related gene PA2072 mutant from Pseudomonas aeruginosa PAO1

摘要:

【背景】铜绿假单胞菌为革兰氏阴性杆菌，是医院感染的常见条件致病菌之一。广泛存在于细菌中的第二信使分子环鸟苷二磷酸(cyclic-di-guanosine monophosphate, c-di-GMP)对细菌生理生化功能具有重要的调节作用。铜绿假单胞菌PAO1中存在参与c-di-GMP代谢的基因PA2072。【目的】探讨铜绿假单胞菌PAO1中c-di-GMP代谢相关基因PA2072的生物学功能。【方法】运用PCR及分子克隆技术构建PA2072基因及各结构域的自杀载体，运用基因敲除方法获取PA2072基因的3个突变株；利用泳动性(swimming)、蜂群运动(swarming)、蹭行运动(twitching)和生物膜定量实验对细菌进行初步的表型分析，进一步通过刚果红染色法对菌株进行分析。【结果】成功构建PA2072基因敲除突变菌株及回补菌株；生物膜定量结果发现基因PA2072的敲除会影响细菌生物膜的形成，PA2072蛋白的不同结构域对生物膜的合成也起到了重要作用；细菌运动能力检测中发现PA2072相关基因的敲除对细菌运动能力也有一定影响。刚果红平板检测结果显示，与野生型PAO1菌株相比，PA2072敲除菌株菌苔呈红色，提示其胞内c-di-GMP含量升高。【结论】基因PA2072的敲除影响了铜绿假单胞菌胞的表型，可能由于铜绿假单胞菌胞内c-di-GMP水平变化引起，该结果为进一步研究基因PA2072的生物学功能奠定了基础。

关键词:铜绿假单胞菌PAO1;环鸟苷二磷酸;PA2072敲除突变菌株

Abstract:

Abstract:[Background] Pseudomonas aeruginosa, a Gram-negative bacterium, is a common opportunistic pathogen of nosocomial infection. Cyclic di-guanosine monophosphate (c-di-GMP), a secondary messenger ubiquitous in bacteria, plays a role in regulating the physiological and biochemical functions of bacteria. PA2072 is a gene involved in c-di-GMP metabolism in P. aeruginosa PAO1. [Objective] To investigate the biological functions of PA2072 in P. aeruginosa PAO1. [Methods] The suicide vectors carrying the full length and different domains of PA2072 were constructed by common molecular cloning technology. Three mutant strains of PA2072 were obtained and studied. The swimming, swarming, and twitching motility, as well as the biofilm formation, of the strains was studied. Congo Red staining was further employed to determine the c-di-GMP levels of the strains. [Results] PA2072 mutant strains and complement strains were successfully constructed. The deletion of PA2072 affected the biofilm formation, and the individual domains of PA2072 also played a role in biofilm formation. The motility tests showed that PA2072 was involved in the regulation of bacterial motility. Compared with the wild-type PAO1 strain, PA2072-delected strains displayed red lawn, which indicated an increased level of intracellular c-di-GMP. [Conclusion] The deletion of PA2072 affects the phenotypes of P. aeruginosa, which might be because of the changes in c-di-GMP level. Our work lays a foundation for further characterization of the biological functions of PA2072.

Key words:Pseudomonas aeruginosa PAO1;c-di-GMP;PA2072-deleted mutant