Corral Lugo Andrés, Daddaoua Abdelali, Ortega Alvaro, Morel Bertrand, Díez Peña Ana Isabel, Espinosa-Urgel Manuel, Krell Tino
Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/ Prof. Albareda, 1, 18008 Granada, Spain.
Department of Physical Chemistry, Faculty of Chemistry, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, 30071 Murcia, Spain.
Protein Expr Purif. 2017 Feb;130:107-114. doi: 10.1016/j.pep.2016.10.007. Epub 2016 Oct 15.
Quorum sensing systems are essential for bacterial communication. We report here the purification and characterization of the Pseudomonas aeruginosa LasR quorum sensing regulator purified from lysates of E. coli cultures grown in the absence of added acyl-homoserine lactones (AHL). We show by isothermal titration calorimetry that LasR recognizes different AHLs with an affinity of approximately 1 μM. The affinity of LasR for its cognate 3-Oxo-C12-AHL was similar to that of other AHLs, indicating that this regulator has not evolved to preferentially recognize its cognate AHL. The α-helical content as determined by CD spectroscopy was found to be in agreement with the corresponding value derived from the homology model. Analytical ultracentrifugation studies show that LasR is a mixture of monomers and dimers and that AHL binding does not alter its oligomeric state. Thermal unfolding studies indicate that LasR has a significant thermal stability and that AHL binding does not significantly alter the unfolding temperature. Two LasR-DNA complexes were observed in electrophoretic mobility shift assays using the hcnABC promoter that has two lux boxes. Taken together, data indicate that the presence of AHLs is not a requisite for correct LasR protein folding. The protein is able to bind AHL ligands in a reversible manner, revising initial concepts of this regulator. The availability of AHL-free protein will permit further studies to determine more precisely its mode of action.
群体感应系统对于细菌通讯至关重要。我们在此报告了从在未添加酰基高丝氨酸内酯（AHL）的情况下培养的大肠杆菌裂解物中纯化并鉴定的铜绿假单胞菌LasR群体感应调节因子。我们通过等温滴定量热法表明，LasR以约1μM的亲和力识别不同的AHL。LasR对其同源3-氧代-C12-AHL的亲和力与其他AHL相似，表明该调节因子并未进化到优先识别其同源AHL。通过圆二色光谱法测定的α-螺旋含量与同源模型得出的相应值一致。分析超速离心研究表明，LasR是单体和二聚体的混合物，并且AHL结合不会改变其寡聚状态。热变性研究表明，LasR具有显著的热稳定性，并且AHL结合不会显著改变变性温度。在使用具有两个lux框的hcnABC启动子的电泳迁移率变动分析中观察到两种LasR-DNA复合物。综上所述，数据表明AHL的存在不是LasR蛋白正确折叠的必要条件。该蛋白能够以可逆方式结合AHL配体，修正了对该调节因子的最初概念。无AHL蛋白的可得性将允许进行进一步研究以更精确地确定其作用模式。
