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铜绿假单胞菌二鸟苷酸环化酶SiaD突变体的功能研究
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国家自然科学基金(32170178,31900121)


Functions of mutants of diguanylate cyclase SiaD from Pseudomonas aeruginosa
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    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SiaDR119M蛋白表达水平;利用GST-pull down实验检测SiaC蛋白与SiaDR119M蛋白在体外的结合能力;针对siaDR119M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SiaDR119M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,siaDR119M的回补菌株生物被膜产量增加,siaDR119A的回补菌株生物被膜产量低于siaDR119M的回补菌株,siaDR201A回补菌株生物被膜含量显著增加,siaDR119M R201A双突变回补菌株生物被膜的含量低于siaDR201A回补菌株。Western blotting和GST-pull down实验证明,与野生型SiaD蛋白相比,SiaDR119M蛋白表达水平无差别,SiaC和SiaDR119M蛋白之间存在特异的相互作用。高效液相色谱结果显示SiaDR119M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,siaDR119M回补菌株运动能力减弱,siaDR201AsiaDR119M R201A回补菌株运动能力无差别。【结论】siaDR119M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 SiaDR119M,and GST-pull down assay to measure the binding ability of SiaC to SiaDR119M in vitro.The fusion expression vector was constructed for the point mutation gene siaDR119M,and the protein was expressed and purified.The enzyme activity of SiaDR119M 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 siaDR119A increased,while the biofilm yield of siaDR119A was lower than that of siaDR119M;the biofilm yield of siaDR201A significantly increased and was higher than that of siaDR119M R201A.Western blotting showed no difference in the expression level between SiaDR119M and wild-type SiaD,and the GST-pull down assay indicated there was a specific interaction between SiaC and SiaDR119M.The HPLC results showed that the activity of SiaDR119M decreased.Compared with wild-type siaD complementary strain,siaDR119M showed weakened motility,and siaDR201A and siaDR119M 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.

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霍卫萍,刘智猛,陈韦,贾佳,王媛媛,张亚妮,陈谷奎. 铜绿假单胞菌二鸟苷酸环化酶SiaD突变体的功能研究. 微生物学报, 2022, 62(10): 3997-4007

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  • 收稿日期:2022-02-24
  • 最后修改日期:2022-04-29
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  • 在线发布日期: 2022-09-24
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