Ren Dacheng, Bedzyk Laura A, Ye Rick W, Thomas Stuart M, Wood Thomas K
Department of Chemical Engineering and Molecular & Cell Biology, University of Connecticut, 191 Auditorium Road, U-3222, Storrs, Connecticut 06269-3222, USA.
Biotechnol Bioeng. 2004 Dec 5;88(5):630-42. doi: 10.1002/bit.20259.
The quorum sensing disrupter (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) of the alga Delisea pulchra was previously found by us (Environ Microbiol 3:731-736, 2001) to inhibit quorum sensing in Escherichia coli via autoinducer-2 (AI-2, produced by LuxS). In this study, DNA microarrays were used to study the genetic basis of this natural furanone inhibition of AI-2 signaling (significant values with p < 0.05 are reported). Using DNA microarrays, the AI-2 mutant Escherichia coli DH5alpha was compared with the AI-2 wild-type strain, E. coli K12, to determine how AI-2 influenced gene expression. Escherichia coli K12 was also grown with 0 and 60 microg/mL furanone to study the inhibition of quorum sensing gene expression. It was found that 166 genes were differentially expressed by AI-2 (67 were induced and 99 were repressed) and 90 genes were differentially expressed by furanone (34 were induced and 56 were repressed). Importantly, 79% (44 out of 56) of the genes repressed by furanone were induced by AI-2, which indicated that furanone inhibited AI-2 signaling and influenced the same suite of genes as a regulon. Most of these genes have functions of chemotaxis, motility, and flagellar synthesis. Interestingly, the aerotaxis genes aer and tsr were discovered to be induced by AI-2 and repressed by furanone. Representative microarray results were confirmed by RNA dot blotting. Furthermore, the E. coli air-liquid interface biofilm formation was repressed by furanone, supporting the results that taxis and flagellar genes were repressed by furanone. The autoinducer bioassay indicated that 100 microg/mL furanone decreased the extracellular concentration of AI-2 2-fold, yet luxS and pfs transcription were not significantly altered. Hence, furanone appeared to alter AI-2 signaling post-transcriptionally.
我们之前发现(《环境微生物学》3:731 - 736,2001),藻类美丽德尔藻的群体感应干扰物(5Z)-4-溴-5-(溴亚甲基)-3-丁基-2(5H)-呋喃酮(呋喃酮)可通过自诱导物-2(AI-2,由LuxS产生)抑制大肠杆菌中的群体感应。在本研究中,利用DNA微阵列研究了这种天然呋喃酮对AI-2信号传导抑制作用的遗传基础(报告了p < 0.05的显著值)。使用DNA微阵列,将AI-2突变型大肠杆菌DH5α与AI-2野生型菌株大肠杆菌K12进行比较,以确定AI-2如何影响基因表达。大肠杆菌K12还分别在0和60μg/mL呋喃酮条件下培养,以研究群体感应基因表达的抑制情况。结果发现,有166个基因因AI-2而差异表达(67个被诱导,99个被抑制),90个基因因呋喃酮而差异表达(34个被诱导,56个被抑制)。重要的是,呋喃酮抑制的基因中有79%(56个中的44个)被AI-2诱导,这表明呋喃酮抑制AI-2信号传导,并影响作为一个调节子的同一组基因。这些基因大多具有趋化性、运动性和鞭毛合成的功能。有趣的是,发现趋氧性基因aer和tsr被AI-2诱导而被呋喃酮抑制。代表性的微阵列结果通过RNA斑点印迹法得到证实。此外,呋喃酮抑制了大肠杆菌气液界面生物膜的形成,支持了趋化性和鞭毛基因被呋喃酮抑制这一结果。自诱导物生物测定表明,100μg/mL呋喃酮使AI-2的细胞外浓度降低了2倍,但luxS和pfs转录没有显著改变。因此,呋喃酮似乎在转录后改变AI-2信号传导。