铜绿假单胞菌PAO1中c-di-GMP代谢相关基因PA0575对表型的影响

【背景】铜绿假单胞菌PAO1中存在与环鸟苷二磷酸(cyclic-di-guanosine monophosphate,c-di-GMP)代谢相关基因PA0575。【目的】探讨铜绿假单胞菌PAO1中环鸟苷二磷酸代谢相关基因PA0575对运动能力及生物膜的影响。【方法】通过PCR对菌株遗传背景进行确认;利用刚果红结合实验及电转PcdrA-gfp质粒间接测量胞内c-di-GMP水平;利用泳动性(swimming)、蜂群泳动(swarming)、蹭行运动(twiching)和生物膜定量实验对细菌进行表型分析,并在运动培养基中添加抗生素研究其对运动能力的影响;针对PA0575基因进行融合蛋白表达载体的构建,并对蛋白进行原核诱导表达。【结果】3株突变体菌株的转座子插入突变位点不一致,胞内c-di-GMP水平检测结果显示,PA0575-1菌株的c-di-GMP含量高于野生型PAO1菌株(P0.05),PA0575-2、PA0575-3菌株胞内c-di-GMP水平与野生型PAO1菌株无差异(P0.05)。运动能力检测实验中,与野生型PAO1菌株相比,PA0575-1菌株泳动性增强(P0.05);PA0575-2、PA0575-3菌株的泳动性、蜂群运动均增强(P0.05);该基因不同位点的突变均导致氯霉素对菌株的运动能力产生抑制作用。生物膜定量结果显示,与野生型PAO1菌株相比,细菌培养18 h后PA0575-1的生物膜含量降低(P0.05),PA0575-2、PA0575-3菌株的生物膜含量升高。最后成功构建了PA0575基因不同结构域的8个表达载体,并获得了异源表达蛋白。【结论】PA0575基因降低铜绿假单胞菌胞内c-di-GMP的水平,影响表型的同时也抑制了氯霉素抗性基因的表达。以上研究为PA0575基因对表型的影响奠定了基础。

Effect of c-di-GMP metabolism related gene PA0575 on phenotype in Pseudomonas aeruginosa PAO1

Background] There is a gene PA0575 related to cyclic-di-guanosine monophosphate (c-di-GMP) metabolism in Pseudomonas aeruginosa PAO1. Objective] To investigate the effect of c-di-GMP metabolism related gene PA0575 on phenotype of PAO1. Methods] Identification of genetic background of strains by PCR method. Swimming, swarming, twiching and biofilm were used to analyze the phenotype of transposon mutant strain and wild type PAO1, and antibiotics were added to motility medium to study the effect of antibiotics on motility. The fusion protein expression vector of PA0575 gene was constructed and the prokaryotic expression of the protein was induced. Results] Inconsistency of transposon insertion mutation sites among three mutant strains, and the mutation sites are inconsistent. The results of intracellular c-di-GMP level test showed that: The c-di-GMP content of PA0575-1 strain was higher than that of wild type PAO1 strain (P<0.05). In the motility test, compared with the wild type PAO1 strain, enhanced Swimming of strain PA0575-1 (P<0.05). The swimming motility and swarming motility of PA0575-2 and PA0575-3 were enhanced (P<0.05). The effect of adding four antibiotics to motility media showed that three different insertion sites led to the inhibition of chloramphenicol on exercise ability. The results of biofilm detection showed that after 18 hours of bacterial culture, the biofilm content of PA0575-1 was significantly lower than that of wild type PAO1 (P<0.05). Eight expression vectors of PA0575 gene were successfully constructed and induced. SDS-PAGE results showed that the expression vector was induced by IPTG to obtain heterologous expression protein. Conclusion] PA0575 gene decreased the level of intracellular c-di-GMP of Pseudomonas aeruginosa, affected the phenotype and inhibited the expression of chloramphenicol resistance genes. The above studies laid the foundation for the effect of PA0575 gene on phenotype.