N-acyl homoserine lactone binding to the CarR receptor determines quorum-sensing specificity in Erwinia
N-acyl homoserine lactone binding to the CarR receptor determines quorum-sensing specificity in Erwinia
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DOI：
10.1093/emboj/19.4.631
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发表时间：
2000-02-15
期刊：
影响因子：
11.4
通讯作者：
Salmond, GPC
中科院分区：
文献类型：
--
作者：
Welch, M;Todd, DE;Salmond, GPC
通过N-酰基高丝氨酸内酯（HSL）信息素的群体感应控制胡萝卜软腐欧文氏菌中碳青霉烯类抗生素的生物合成。碳青霉烯生物合成基因的转录依赖于LuxR型激活蛋白卡尔R。使用荧光猝灭、DNA带移分析、有限蛋白水解和报告基因测定，检测了一系列HSL分子的平衡结合，这些HSL分子被认为激活卡尔R与其DNA靶序列结合，卡尔R结合最生理相关的配体，N-（3-氧代己酰基）-L-高丝氨酸内酯，具有两个配体分子/蛋白质二聚体的化学计量和1.8 μ M的解离常数，与体内激活碳青霉烯产生所需的HSL浓度良好一致。在HSL存在下，卡尔R与其靶DNA形成非常高分子量的复合物，表明配体引起蛋白质多聚化。化学交联分析支持这一解释。我们的数据表明，给定的HSL促进卡尔R与其靶DNA序列结合的能力与HSL对蛋白质的亲和力成正比。
Quorum sensing via an N-acyl homoserine lactone (HSL) pheromone controls the biosynthesis of a carbapenem antibiotic in Erwinia carotovora. Transcription of the carbapenem biosynthetic genes is dependent on the LuxR-type activator protein, CarR, Equilibrium binding of a range of HSL molecules, which are thought to activate CarR to bind to its DNA target sequence, was examined using fluorescence quenching, DNA bandshift analysis, limited proteolysis and reporter gene assays, CarR bound the most physiologically relevant ligand, N-(3-oxohexanoyl)-L-homoserine lactone, with a stoichiometry of two molecules of ligand per dimer of protein and a dissociation constant of 1.8 mu M, in good agreement with the concentration of HSL required to activate carbapenem production in vivo. In the presence of HSL, CarR formed a very high molecular weight complex with its target DNA, indicating that the ligand causes the protein to multimerize. Chemical cross-linking analysis supported this interpretation. Our data show that the ability of a given HSL to facilitate CarR binding to its target DNA sequence is directly proportional to the affinity of the HSL for the protein.