细菌菌群传感效应信号分子高丝氨酸内酯平板分析法的优化

目的利用指示菌紫色杆菌CV026菌株,建立和优化稳定的细菌菌群传感效应信号分子高丝氨酸内酯平板分析方法。方法优化受测铜绿假单胞菌高丝氨酸内酯的提取方法,分析铜绿假单胞菌培养时间对信号分子检测的影响。另一方面,优化指示菌株CV026的培养基成分,并优化紫色菌素的提取方法,以完善信号分子的检测反应。结果恒温水浴挥发法可有效提取高丝氨酸内酯,并且发现当铜绿假单胞菌的培养时间为3d时,高丝氨酸内酯最易被检出。进一步,试验结果显示添加L-Try可提高指示菌株紫色菌素的产量,并利于高丝氨酸内酯的检测。此外,比较发现采用乙醇溶解紫色菌素的方法可更高效的测定紫色菌素的量。结论本课题建立的检测方法将更有效的检测细菌菌群传感系统信号分子高丝氨酸内酯,将有利于细菌菌群传感机制研究和抑制细菌菌群传感药物的开发工作。

Optimization of plate assay for signal molecules, N-acyl homoserine lactones, of quorum sensing system of bacteria

Abstract: Objective To build and optimize the plate assay for the signal molecules, N-acyl homoserine lactones (AHLs), of quorum sensing system of bacteria using Chromobacterium violaceum strain CV026. Methods First, we optimized the extraction method of AHLs of Pseudomonas aeruginosa, and analyzed the effect of cultivation time of P. aeruginosa on the detection of signal molecules. Second, we optimized the constituents of the media for strain CV026 and the extraction method of violacein to improve the detection reaction of AHLs. Results The method of Volatilization in thermostatic waterbath extracted AHLs effectively, and AHL was detected the most easily when the cultivation time of P. aeruginosa was three days. Furthermore, we found that adding L-Try in the media for CV026 cultivation increased the production of violacein by CV026, which facilitated the detection of AHLs more effectively. In addition, dissolving violacein with alcohol enabled more efficient quantification of violacein. Conclusion Our optimized method can detect the AHLs of quorum sensing more efficiently, and would benefit the research of bacterial quorum sensing mechanism and development of discovery of quorum quenching drugs.