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

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。

Abstract:

[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 and ΔmcpU 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.