铜绿假单胞菌二鸟苷酸环化酶SiaD突变体的功能研究

Functions of mutants of diguanylate cyclase SiaD from Pseudomonas aeruginosa

摘要:

【目的】铜绿假单胞菌(Pseudomonas aerugionsa)二鸟苷酸环化酶SiaD调控着铜绿假单胞菌的生物被膜形成等表型。在研究过表达siaD对生物被膜的调控作用时发现，与野生型siaD基因回补菌株相比，一株回补菌株的生物被膜产量显著升高。本文的目的即是探究该菌株生物被膜产量升高的原因，并对该菌株的其他表型进行研究。【方法】通过测序确定突变位点；利用生物被膜定性和定量实验对发生点突变的菌株表型进行分析；利用Western blotting实验检测SiaD^R119M蛋白表达水平；利用GST-pull down实验检测SiaC蛋白与SiaD^R119M蛋白在体外的结合能力；针对siaD^R119M点突变基因进行融合蛋白表达载体的构建，表达并纯化该蛋白，利用高效液相色谱检测SiaD^R119M的酶活；为了进一步研究c-di-GMP与细菌运动能力的关系，对细菌的运动能力进行检测。【结果】测序比对结果显示，序列的第119个氨基酸发生了突变，由精氨酸突变成了甲硫氨酸。生物被膜定性和定量实验显示，与野生型siaD回补菌株相比，siaD^R119M的回补菌株生物被膜产量增加，siaD^R119A的回补菌株生物被膜产量低于siaD^R119M的回补菌株，siaD^R201A回补菌株生物被膜含量显著增加，siaD^{R119M R201A}双突变回补菌株生物被膜的含量低于siaD^R201A回补菌株。Western blotting和GST-pull down实验证明，与野生型SiaD蛋白相比，SiaD^R119M蛋白表达水平无差别，SiaC和SiaD^R119M蛋白之间存在特异的相互作用。高效液相色谱结果显示SiaD^R119M蛋白酶活降低。运动能力检测实验中，与siaD野生型回补菌株相比，siaD^R119M回补菌株运动能力减弱，siaD^R201A、siaD^{R119M R201A}回补菌株运动能力无差别。【结论】siaD^R119M突变导致生物被膜合成增强，酶活降低，运动能力减弱。我们推测突变后表型的变化可能是因为突变在体内会影响SiaD蛋白与下游效应蛋白的相互作用，加强了下游效应蛋白的信号传导能力，然而具体机制有待进一步探索。

Abstract:

[Objective] Diguanylate cyclase SiaD regulates the biofilm formation of Pseudomonas aeruginosa.Our previous study about the effect of siaD overexpression on biofilm has revealed that the biofilm yield of a complementary strain is significantly higher than that of the strain overexpressing the wild-type siaD gene.This study aims to explore the reasons for the increase in biofilm production and to study other phenotypes of this strain.[Methods] The mutation sites of siaD were identified by sequencing.The qualitative and quantitative biofilm experiments were carried out to analyze the phenotype of the strain with point mutation.Western blotting was employed to determine the protein level of SiaD^R119M,and GST-pull down assay to measure the binding ability of SiaC to SiaD^R119M in vitro.The fusion expression vector was constructed for the point mutation gene siaD^R119M,and the protein was expressed and purified.The enzyme activity of SiaD^R119M was detected by high performance liquid chromatography (HPLC).Further,we studied the motility of the strain to reveal the relationship between c-di-GMP and bacterial motility.[Results] The sequencing comparison showed that the 119th amino acid was mutated from arginine to methionine (R119M).Compared with that of the wild-type siaD complementary strain,the biofilm yield of siaD^R119A increased,while the biofilm yield of siaD^R119A was lower than that of siaD^R119M;the biofilm yield of siaD^R201A significantly increased and was higher than that of siaD^{R119M R201A}.Western blotting showed no difference in the expression level between SiaD^R119M and wild-type SiaD,and the GST-pull down assay indicated there was a specific interaction between SiaC and SiaD^R119M.The HPLC results showed that the activity of SiaD^R119M decreased.Compared with wild-type siaD complementary strain,siaD^R119M showed weakened motility,and siaD^R201A and siaD^{R119M R201A} had no significant difference in motility.[Conclusion] The mutation of R119M in SiaD increased the biofilm yield,decreased the enzyme activity,and reduced the bacterial motility.This mutation may affect the interaction between SiaD and downstream effector to enhance the signal transduction of downstream effector.The underlying mechanism remains to be explored.