AI-2通过甲基化趋化受体McpU调控恶臭假单胞菌KT2440的趋化运动及生物膜形成  

Autoinducer-2 regulates the chemotaxis and biofilm formation of Pseudomonas putida KT2440 by the methyl-accepting chemotaxis protein McpU

作　　者：谢来工 赵文瑾 张磊[1] XIE Laigong;ZHAO Wenjin;ZHANG Lei(College of Life Sciences,Northwest A&F University,Yangling 712100,Shaanxi,China)

机构地区：[1]西北农林科技大学生命科学学院,陕西省杨凌示范区,陕西杨凌712100

出　　处：《微生物学报》2022年第2期628-639,共12页Acta Microbiologica Sinica

基　　金：国家重点研发计划(2018YFA0901200)。

摘　　要：【背景】广泛存在于革兰氏阴性菌和革兰氏阳性菌中的自诱导物autoinducer-2(AI-2)能够介导细菌种内和种间通讯,并调节细菌的多种生理过程。然而恶臭假单胞菌KT2440能否感知AI-2信号还未见报道。【目的】挖掘介导恶臭假单胞菌KT2440对AI-2趋化反应的趋化受体,检测AI-2信号通过趋化受体对恶臭假单胞菌KT2440生物膜形成的调控作用。【方法】本研究首先检测恶臭假单胞菌KT2440对AI-2信号的趋化反应,随后表达纯化了与铜绿假单胞菌AI-2受体高度同源的甲基化趋化受体McpU的配体结合结构域(ligand-binding domain,LBD),利用哈维氏弧菌的生物发光实验和等温滴定量热法(ITC)分析McpU-LBD与AI-2的相互作用;软琼脂平板法和毛细管定量分析法分析KT2440及mcpU敲除菌株(ΔmcpU)对AI-2的趋化反应;结晶紫染色法检测AI-2对KT2440及ΔmcpU生物膜形成能力的影响。【结果】软琼脂平板法和毛细管定量分析发现KT2440对AI-2信号表现出明显的正趋向性。哈维氏弧菌的生物发光实验和ITC分析发现AI-2与McpU-LBD具有高亲和力相互作用。进一步研究发现KT2440对AI-2的趋化反应是通过McpU介导的。生物膜测定结果显示,AI-2能通过其受体McpU显著增强KT2440的生物膜形成能力(P<0.05)。【结论】以上结果表明,甲基化趋化受体McpU介导了恶臭假单胞菌KT2440对AI-2的趋化作用,并且AI-2通过作用于该受体显著提高恶臭假单胞菌KT2440的生物膜形成能力。[Background]Autoinducer-2(AI-2),ubiquitous in Gram-negative bacteria and Gram-positive bacteria,can mediate intraspecific and interspecific communication and regulate a variety of physiological processes.However,whether Pseudomonas putida KT2440 can sense AI-2 signal has not been reported yet.[Objective]To explore the chemotactic receptor mediating the chemotaxis of KT2440 to AI-2,and to detect the regulatory role of AI-2 in the biofilm formation of KT2440.[Methods]Firstly,soft agar plate assay and quantitative capillary assay were employed to detect the chemotactic response of KT2440 to AI-2.Then,the ligand binding domain(LBD)of the methyl-accepting chemotaxis protein McpU which had a high homology with that of the known AI-2 receptor TlpQ in Pseudomonas aeruginosa was expressed and purified.Vibrio harveyi MM32 bioluminescence and isothermal titration calorimetry(ITC)were adopted to detect the interaction between McpU-LBD and AI-2.Soft agar assay and quantitative capillary assay were carried out to evaluate the chemotaxis of the mcpU knockout strain(ΔmcpU)to AI-2.The effect of AI-2 on the biofilm formation of KT2440 andΔmcpU was detected by crystal violet staining.[Results]KT2440 exhibited chemotaxis to AI-2.The bioluminescence and ITC assays showed that AI-2 bound to McpU-LBD with high affinity.The chemotaxis of KT2440 to AI-2 was mediated by McpU,and AI-2 significantly enhanced the biofilm formation in KT2440 via its receptor McpU(P<0.05).[Conclusion]McpU mediates the chemotaxis of P.putida KT2440 to AI-2,and AI-2 significantly enhances the biofilm formation of P.putida KT2440 by engaging McpU.

关 键 词：恶臭假单胞菌 甲基化趋化受体蛋白 AI-2 趋化作用 生物膜 

分 类 号：Q935[生物学—微生物学]