The natural furanone (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone disrupts quorum sensing-regulated gene expression in Vibrio harveyi by decreasing the DNA-binding activity of the transcriptional regulator protein luxR
The natural furanone (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone disrupts quorum sensing-regulated gene expression in Vibrio harveyi by decreasing the DNA-binding activity of the transcriptional regulator protein luxR
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DOI：
10.1111/j.1462-2920.2007.01367.x
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发表时间：
2007-10-01
影响因子：
5.1
通讯作者：
Bossier, Peter
中科院分区：
文献类型：
--
作者：
Defoirdt, Tom;Miyamoto, Carol M.;Bossier, Peter
本研究旨在深入了解天然呋喃酮（5 Z）-4-溴-5-（溴亚甲基）-3-丁基-2（5 H）-呋喃酮干扰哈氏弧菌群体感应的分子机制。信号分子受体双突变体的生物发光实验表明，呋喃酮阻断了哈维氏弧菌群体感应系统的所有三个通道。在使用群体感应信号转导途径中具有突变的突变体的进一步实验中，发现该化合物通过与位于Hfq蛋白下游的组分相互作用来阻断群体感应调节的生物发光。此外，使用特异性引物的逆转录酶实时聚合酶链反应显示，呋喃酮对野生型哈维氏弧菌细胞中的luxR（Vh）mRNA水平没有影响。相比之下，迁移率变动测定显示，在呋喃酮的存在下，显著较低水平的LuxR（Vh）反应调节蛋白能够结合野生型哈氏弧菌中的其靶启动子序列。最后，用纯化的LuxR（Vh）蛋白进行的测试也显示用呋喃酮处理的LuxR（Vh）的偏移较小，而发现LuxR（Vh）浓度不被呋喃酮改变（如通过SDS-PAGE测定的）。因此，我们的数据表明，呋喃酮阻断群体感应哈维氏弧菌的群体感应，使群体感应主调节蛋白LuxR（VH）无法结合到群体感应调节基因的启动子序列。
This study aimed at getting a deeper insight in the molecular mechanism by which the natural furanone (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone disrupts quorum sensing in Vibrio harveyi. Bioluminescence experiments with signal molecule receptor double mutants revealed that the furanone blocks all three channels of the V. harveyi quorum sensing system. In further experiments using mutants with mutations in the quorum sensing signal transduction pathway, the compound was found to block quorum sensing-regulated bioluminescence by interacting with a component located downstream of the Hfq protein. Furthermore, reverse transcriptase real-time polymerase chain reaction with specific primers showed that there was no effect of the furanone on luxR(Vh) mRNA levels in wild-type V. harveyi cells. In contrast, mobility shift assays showed that in the presence of the furanone, significantly lower levels of the LuxR(Vh) response regulator protein were able to bind to its target promoter sequences in wild-type V. harveyi. Finally, tests with purified LuxR(Vh) protein also showed less shifts with furanone-treated LuxR(Vh), whereas the LuxR(Vh) concentration was found not to be altered by the furanone (as determined by SDS-PAGE). Therefore, our data indicate that the furanone blocks quorum sensing in V. harveyi by rendering the quorum sensing master regulator protein LuxR(Vh) unable to bind to the promoter sequences of quorum sensing-regulated genes.