Biosensor-based assays for PQS, HHQ and related 2-alkyl-4-quinolone quorum sensing signal molecules
Biosensor-based assays for PQS, HHQ and related 2-alkyl-4-quinolone quorum sensing signal molecules
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DOI：
10.1038/nprot.2007.158
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Williams, Paul
中科院分区：
文献类型：
--
作者：
Fletcher, Matthew P.;Diggle, Stephen P.;Williams, Paul
2-烷基-4-喹诺酮（AHQ）如2-庚基-3-羟基-4-喹诺酮（PQS）和2-庚基-4-喹诺酮（HHQ）是群体感应信号分子。在这里，我们描述了AHQ检测，初步鉴定和定量的方法，其中采用了基于LUX的铜绿假单胞菌AHQ生物传感器菌株。该方案描述了薄层色谱法（TLC）和微量滴定板测定法，其使用生物发光或绿脓菌素的绿色颜色作为检测终点。将细菌细胞或无细胞培养物上清液的有机溶剂提取物在TLC板上进行色谱分析，将其干燥并用AHQ生物传感器覆盖。AHQ显示为发光和绿色斑点。对于微量滴定测定，将用过的细菌培养物上清液或提取物添加到含有AHQ生物传感器的生长培养基中。光输出与样品的AHQ含量成比例。所描述的测定需要大约2天才能完成，操作简单，不需要复杂的仪器，并且非常适合筛选大量的细菌样品。然而，除了铜绿假单胞菌中的PQS和HHQ外，AHQ的最终鉴定还需要额外的MS和NMR分析。
2-Alkyl-4-quinolones ( AHQs) such as 2-heptyl-3-hydroxy-4-quinolone ( PQS) and 2-heptyl-4-quinolone ( HHQ) are quorum sensing signal molecules. Here, we describe methods for AHQ detection, tentative identification and quantification, which employ a lux-based Pseudomonas aeruginosa AHQ biosensor strain. The protocol describes both thin-layer chromatography ( TLC) and microtiter plate assays, which use bioluminescence or the green color of pyocyanin as detection end points. Organic solvent extracts of bacterial cells or cell-free culture supernatants are chromatographed on TLC plates, which are dried and overlaid with the AHQ biosensor. AHQs appear as both luminescent and green spots. For the microtiter assay, either spent bacterial culture supernatants or extracts are added to a growth medium containing the AHQ biosensor. Light output is proportional to the AHQ content of the sample. The assays described take approximately 2 days to complete, are simple to perform, do not require sophisticated instrumentation and are highly amenable to screening large numbers of bacterial samples. However, apart from PQS and HHQ in P. aeruginosa, definitive AHQ identification will require additional MS and NMR analyses.