Acyl-Homoserine Lactone Binding to and Stability of the Orphan Pseudomonas aeruginosa Quorum-Sensing Signal Receptor QscR
Acyl-Homoserine Lactone Binding to and Stability of the Orphan Pseudomonas aeruginosa Quorum-Sensing Signal Receptor QscR
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DOI：
10.1128/jb.01041-10
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发表时间：
2011-01-01
影响因子：
3.2
通讯作者：
Greenberg, E. Peter
中科院分区：
文献类型：
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作者：
Oinuma, Ken-Ichi;Greenberg, E. Peter
铜绿假单胞菌转录因子 QscR 对多种脂肪酰基高丝氨酸内酯 (HSL) 作出反应，包括 N-3-氧代十二烷酰基-HSL (3OC12-HSL)，它是由铜绿假单胞菌群体感应电路 LasI 和 LasR 产生和检测的。与 LasR 和许多其他酰基-HSL 依赖性转录因子一样，生产足够量的可溶性 QscR 用于纯化需要重组细菌在适当的酰基-HSL 存在下生长。与LasR相比，QscR被认为与3OC12-HSL的结合相对较弱，并且与LasR不同的是，纯化的QscR与靶DNA的结合被证明强烈依赖于外源添加的3OC12-HSL。我们证明纯化的 QscR 在足够高的浓度下是二聚体，在较低浓度下是单体。此外，QscR 与 3OC12-HSL 的结合比之前认为的更紧密。纯化的 QscR 保留了 3OC12-HSL，并且在足够高的浓度下，它在不添加 3OC12-HSL 的情况下结合靶 DNA。我们还从在 N-3-氧代己酰基-HSL (3OC6-HSL) 而不是 3OC12-HSL 存在的情况下生长的重组大肠杆菌获得了可溶性 QscR，并且由于 3OC6-HSL 与 QscR 的结合比其他测试的酰基-HSL 松散得多，因此我们能够在体外将 3OC6-HSL 与其他酰基-HSL 交换，然后估计结合 QscR 对不同酰基-HSL 和靶 DNA 的亲和力。我们的数据支持这样一个模型，即 QscR 多肽在不存在酰基-HSL 的情况下正确折叠，但可溶性、无酰基-HSL 的 QscR 不会积累，因为它会快速聚集或蛋白水解。
The Pseudomonas aeruginosa transcription factor QscR responds to a variety of fatty acyl-homoserine lactones (HSLs), including N-3-oxododecanoyl-HSL (3OC12-HSL), which is produced and detected by the P. aeruginosa quorum-sensing circuit LasI and LasR. As is true for LasR and many other acyl-HSL-dependent transcription factors, production of soluble QscR in sufficient amounts for purification requires growth of recombinant bacteria in the presence of an appropriate acyl-HSL. QscR is thought to bind 3OC12-HSL relatively weakly compared to LasR, and unlike LasR, binding of purified QscR to target DNA was shown to strongly depend on exogenously added 3OC12-HSL. We show that purified QscR is dimeric at sufficiently high concentrations and monomeric at lower concentrations. Furthermore, QscR bound 3OC12-HSL more tightly than previously believed. Purified QscR retained 3OC12-HSL, and at sufficiently high concentrations, it bound target DNA in the absence of added 3OC12-HSL. We also obtained soluble QscR from recombinant Escherichia coli grown in the presence of N-3-oxohexanoyl-HSL (3OC6-HSL) instead of 3OC12-HSL, and because 3OC6-HSL bound much more loosely to QscR than other acyl-HSLs tested, we were able to exchange 3OC6-HSL with other acyl-HSLs in vitro and then estimate binding affinities of QscR for different acyl-HSLs and for target DNA. Our data support a model whereby QscR polypeptides fold properly in the absence of an acyl-HSL, but soluble, acyl-HSL-free QscR does not accumulate because it is subject to rapid aggregation or proteolysis.