河弧菌VfqI-VfqR密度感应系统对VflT6SS2附属基因簇中vgrG操纵子的调控研究

Regulation of VfqI-VfqR Quorum sensing circuit on vgrG operon in VflT6SS orphan cluster of Vibrio fluvialis

目的探究河弧菌VfqI-VfqR密度感应系统对VflT6SS2附属基因簇hcp-vgrG中vgrG操纵子的调控。方法使用荧光定量反转录聚合酶链式反应(qRT-PCR)检测野生株和VfqI-VfqR系统缺失株中vgrG的mRNA水平。利用启动子-lux报告质粒系统检测冷光值,确定vgrG启动子在河弧菌野生株、缺失株、回补株中的活性差异。在大肠埃希菌中,导入VfqR过表达质粒,检测vgrG启动子-lux报告质粒冷光值,确定VfqR可否激活vgrG启动子。结果△vfqI、△vfqR和△vfqI-vfqR缺失株中vgrG mRNA水平和启动子活性均显著低于野生株;在△vfqR中回补VfqR表达质粒pSRvfqR和在△vfqI中分别添加3-oxo-C_(10)-HSL、C_(10)-HSL和3-oxo-C_(12)-HSL AHLs分子均能恢复vgrG启动子活性,并且3种AHLs分子效果大体一致。在大肠埃希菌中过表达VfqR并添加上述3种外源AHLs,对vgrG启动子活性没有影响。结论VfqI-VfqR密度感应系统在启动子水平正性调控vgrG操纵子,并且该调控作用是间接的。

Objective To explore the regulation of the vgrG operon in the VflT6SS2 orphan clusters by the Quorum sensing system VfqI-VfqR of Vibrio fluvialis.Methods Quantitative reverse transcription PCR was used to detect the relative expression of vgrG mRNA level in wild V.fluvialis strain and VfqI-VfqR system deletion mutant strains.Luminescence of promoter-lux fusion was measured to determine the vgrG promoter activity in wild V.fluvialis strain,derivative mutants and corresponding complemented strains.VfqR-overexpressed plasmid was introduced into Escherichia coli,the luminescence of vgrG promoter-lux fusion was compared to determine the direct effect of VfqR on vgrG operon activation.Results The vgrG mRNA and its promoter activity significantly decreased in the VfqI-VfqR deletion mutants compared with wild V.fluvialis strain.Furthermore,the vgrG promoter activity of △vfqR was restored either with trans-complemented plasmid or supplemented with exogenous 3-oxo-C_(10)-HSL,C_(10)-HSL or 3-oxo-C_(12)-HSL AHL molecules.The effects of the three AHLs were similar.However,overexpression of VfqR combined with addition of AHLs had no effect on the promoter activity of vgrG in E.coli.Conclusion The VfqI-VfqR system is one of positive regulators of vgrG operator indirectly tuning the activation of its promoter.