信号分子自诱导物-2 (AI-2)通过Tar1调节伸长盐单胞菌的趋化运动和生物膜形成

The signaling molecule autoinducer-2 regulates the chemotaxis and biofilm formation of Halomonas elongata via Tar1

摘要:

自诱导物-2 (autoinducer-2, AI-2)是一种广泛存在于细菌中的种间群体感应信号分子，参与调解生物发光、趋化运动以及生物膜形成等许多重要的生理过程。然而，AI-2对伸长盐单胞菌(Halomonas elongata)的影响及其作用机制尚未见报道。目的 揭示伸长盐单胞菌中信号分子AI-2的受体蛋白，并检测AI-2通过受体蛋白对伸长盐单胞菌趋化运动和生物膜形成能力产生的影响。方法 采用毛细管定量分析法检测伸长盐单胞菌对AI-2的趋化响应；通过对甲基趋化受体蛋白进行结构域分析、序列比对和分子对接找到AI-2的潜在受体蛋白Tar1及其关键氨基酸位点；表达并纯化Tar1的配体结合结构域(ligand-binding domain, LBD)蛋白及其点突蛋白，通过哈维氏弧菌发光试验检测Tar1-LBD是否结合信号分子AI-2；运用同源重组技术构建tar1基因缺失突变体，并通过毛细管定量分析和生物膜形成试验检测AI-2对伸长盐单胞菌趋化运动和生物膜形成的影响。结果 毛细管定量分析法显示伸长盐单胞菌对信号分子AI-2表现出趋向性。在伸长盐单胞菌中共找到4个甲基受体趋化蛋白，结构域分析、序列比对、分子对接以及哈维氏弧菌发光试验表明Tar1-LBD能够结合信号分子AI-2。利用同源重组技术成功构建了伸长盐单胞菌tar1基因缺失突变体，tar1基因的缺失会抑制伸长盐单胞菌对AI-2的趋化响应，而互补菌株的趋化响应可恢复到接近野生型的水平。此外，生物膜形成试验检测显示，AI-2能够通过Tar1促进伸长盐单胞菌的生物膜形成。结论 伸长盐单胞菌对信号分子AI-2具有趋化性，AI-2通过结合甲基趋化受体蛋白Tar1的配体结合结构域来调控伸长盐单胞菌的趋化运动和生物膜形成。

Abstract:

As a widely conserved interspecies quorum sensing signaling molecule, autoinducer-2 (AI-2) is involved in regulating various crucial physiological processes such as bioluminescence, chemotaxis, and biofilm formation. However, the effects of AI-2 on Halomonas elongata and its underlying mechanisms remain unreported.Objective To reveal the receptor that regulates the chemotaxis and biofilm formation of H. elongata in response to AI-2.Methods The quantitative capillary assay was employed to examine the chemotactic response of H. elongata to AI-2. We conducted protein domain identification, sequence alignment, and molecular docking of methyl-accepting chemotaxis proteins to identify the key amino acid sites in Tar1, the potential AI-2 receptor. The ligand-binding domain (LBD) of Tar1 and single-point mutants were expressed and purified, and the binding between Tar1-LBD and AI-2 was measured by the Vibrio harveyi MM32 bioluminescence assay. tar1 was deleted by homologous recombination, and the effects of AI-2 on the chemotaxis and biofilm formation of H. elongata were evaluated by quantitative capillary and biofilm formation assays.Results The quantitative capillary assay revealed that H. elongata exhibited chemotaxis to AI-2. Four methyl-accepting chemotaxis proteins were identified in H. elongata. Protein domain identification, sequence alignment, molecular docking, and V. harveyi MM32 bioluminescence assay demonstrated that Tar1-LBD bound to AI-2. The tar1-deleted mutant of H. elongata was successfully constructed by homologous recombination. The deletion of tar1 impaired the chemotaxis of H. elongata to AI-2, whereas the complementation of this gene restored the chemotaxis to level comparable to that in the wild-type. Furthermore, biofilm formation assay revealed that AI-2 enhanced the biofilm formation in H. elongata via Tar1.Conclusion H. elongata exhibits chemotaxis to AI-2, and this signal molecule binds to the LBD of Tar1, thereby modulating chemotaxis and biofilm formation.