| 摘要: |
| 【目的】筛选泰国伯克霍尔德氏菌环二鸟苷酸（c-di-GMP）代谢关键酶，并对其活性以及突变体的运动性、生物膜形成能力和毒性等进行分析，为相关致病性伯克霍尔德氏菌的防治提供科学依据。【方法】以泰国伯克霍尔德氏菌E264菌株为研究对象，通过氨基酸多序列比对筛选泰国伯克霍尔德氏菌中的c-di-GMP代谢酶的关键活性位点。采用同源重组方法构建单基因缺失突变体，利用泳动试验和生物膜形成试验筛选c-di-GMP代谢关键酶，并通过体外酶促反应和高效液相色谱分析其酶活性。通过大蜡螟幼虫感染试验检测c-di-GMP代谢关键酶基因缺失对E264菌株毒性的影响。【结果】共筛选到14个c-di-GMP代谢酶，其中BTH_Ⅰ0751和BTH_Ⅰ1393的EAL结构域以及BTH_Ⅰ2871的GGDEF结构域活性位点发生了突变；成功构建了14个c-di-GMP代谢酶基因缺失突变体，并筛选出PdcA（BTH_Ⅱ2363）为c-di-GMP代谢关键酶。结构域分析结果显示，PdcA含有Per-ARNT-Sim（PAS）传感结构域（N-端）和GGDEF结构域（C-端）；酶活性分析结果显示，PdcA具有c-di-GMP合成酶活性，能够调节胞内c-di-GMP水平，而且其PAS结构域对酶活性的发挥至关重要。大蜡螟幼虫感染试验发现，pdcA的缺失增强了泰国伯克霍尔德氏菌E264的毒力。【结论】PdcA是泰国伯克霍尔德氏菌E264 c-di-GMP代谢的关键酶，能够调节其运动性、生物膜形成能力以及毒力。PdcA的c-di-GMP合成活性依赖于自身的PAS结构域。 |
| 关键词: 泰国伯克霍尔德氏菌 环二鸟苷酸 PdcA 运动性 生物膜形成能力 |
| DOI：10.13207/j.cnki.jnwafu.2025.10.016 |
| 分类号: |
| 基金项目:国家自然科学基金项目（32330004，32300111）;运城市基础研究计划项目（YCKJ-2023030） |
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| Screening and enzymatic activity analysis of a key c-di-GMP metabolizing enzyme PdcA in Burkholderia thailandensis |
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WANG Zhuo1, LI Xin1, SHEN Xihui2
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1.Shanxi Key Laboratory of Yuncheng Salt Lake Ecological Protection and Resource Utilization,Yuncheng University,Yuncheng,Shanxi 044000,China;2.College of Life Sciences,Northwest A&F University,Yangling,Shaanxi 712100,China
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| Abstract: |
| 【Objective】This research aimed to screen key c-di-GMP metabolizing enzyme in Burkholderia thailandensis and investigate its enzymatic activity.In addition,the motility,biofilm formation ability,and virulence of this c-di-GMP metabolizing enzyme gene-deleted mutant were analyzed to provide a new idea for treating related pathogenic Burkholderia.【Method】With B.thailandensis E264 strain as material,the key active sites of c-di-GMP metabolizing enzymes were identified by amino acids multiple sequence alignment.Single-gene deletion mutants of c-di-GMP metabolism enzymes were constructed by homologous recombination and the key c-di-GMP metabolizing enzyme was screened via motility assay and biofilm formation assay.Its enzyme activity was analyzed by in vitro enzyme-catalyzed reactions and high performance liquid chromatography.The effect of this key c-di-GMP metabolizing enzyme gene deletion on the virulence of E264 strain was analyzed by Galleria mellonella larvae infection assay.【Result】14 c-di-GMP metabolizing enzymes were identified,in which mutation was observed at the active sites in the EAL domain of BTH_Ⅰ0751 and BTH_Ⅰ1393 and GGDEF domain of BTH_Ⅰ2871.A total of 14 single-gene deletion mutants of c-di-GMP metabolizing enzymes were constructed,and PdcA (BTH_Ⅱ2363) was identified as the key c-di-GMP metabolism enzyme in B.thailandensis.Domain analysis showed that PdcA contains the Per-ARNT-Sim (PAS) sensory domain (N-terminus) and the GGDEF domain (C-terminus).Enzymatic activity analysis revealed that PdcA has c-di-GMP synthesis enzyme activity,and the PAS domain plays a crucial role in enzyme activity.Furthermore,the deletion of pdcA enhanced the virulence of B.thailandensis E264.【Conclusion】PdcA is a key c-di-GMP metabolism enzyme in B.thailandensis E264,regulating its motility,biofilm formation ability,and virulence.Additionally,the enzymatic activity of PdcA depends on its PAS domain. |
| Key words: Burkholderia thailandensis c-di-GMP PdcA motility biofilm formation ability |
