Department of Microbiology, University of Washington School of Medicine, Seattle, WA 98195-7242, USA.
J Bacteriol. 2011 Jan;193(2):421-8. doi: 10.1128/JB.01041-10. Epub 2010 Nov 19.
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.
铜绿假单胞菌转录因子 QscR 对各种脂肪酸酰基高丝氨酸内酯 (HSL) 有反应，包括 N-3-氧代十二酰基高丝氨酸内酯 (3OC12-HSL)，这是由铜绿假单胞菌群体感应回路 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，为了进行纯化，需要在合适的酰基 HSL 存在下培养重组细菌，才能产生足够数量的可溶性 QscR。与 LasR 相比，QscR 被认为与 3OC12-HSL 的结合较弱，与 LasR 不同的是，纯化的 QscR 与靶 DNA 的结合强烈依赖于外源添加的 3OC12-HSL。我们表明，在足够高的浓度下，纯化的 QscR 是二聚体，而在较低的浓度下则是单体。此外，QscR 与 3OC12-HSL 的结合比以前认为的更紧密。纯化的 QscR 保留了 3OC12-HSL，并且在足够高的浓度下，它在没有添加 3OC12-HSL 的情况下结合靶 DNA。我们还从在 N-3-氧代己酰基高丝氨酸内酯 (3OC6-HSL) 存在下生长的重组大肠杆菌中获得了可溶性 QscR，而不是 3OC12-HSL，由于 3OC6-HSL 与 QscR 的结合比我们测试的其他酰基 HSL 松散得多，我们能够在体外交换 3OC6-HSL 与其他酰基 HSL，并估计 QscR 对不同酰基 HSL 和靶 DNA 的结合亲和力。我们的数据支持这样一种模型，即 QscR 多肽在没有酰基 HSL 的情况下正确折叠，但可溶性、无酰基 HSL 的 QscR 不会积累，因为它容易快速聚集或蛋白水解。
