High Specificity in CheR Methyltransferase Function CheR2 OF PSEUDOMONAS PUTIDA IS ESSENTIAL FOR CHEMOTAXIS, WHEREAS CheR1 IS INVOLVED IN BIOFILM FORMATION
High Specificity in CheR Methyltransferase Function CheR2 OF PSEUDOMONAS PUTIDA IS ESSENTIAL FOR CHEMOTAXIS, WHEREAS CheR1 IS INVOLVED IN BIOFILM FORMATION
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DOI：
10.1074/jbc.m113.472605
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发表时间：
2013-06-28
影响因子：
4.8
通讯作者：
Krell, Tino
中科院分区：
文献类型：
--
作者：
Garcia-Fontana, Cristina;Antonio Reyes-Darias, Jose;Krell, Tino
化学感受途径是细菌中的主要信号转导机制。CheR甲基转移酶催化化学感受器的胞质信号结构域的甲基化，并且是化学感受级联的核心蛋白质之一。这些酶主要在大肠杆菌和鼠伤寒沙门氏菌中研究，它们具有参与趋化性的单一CheR。许多其他细菌拥有多个cheR基因。由于化学感受器信号传导结构域的序列是高度保守的，因此仍有待确定CheR旁系同源物发挥其活性的特异性程度。我们在这里报告的三个CheR旁系同源的恶臭假单胞菌的比较分析。等温滴定量热法研究表明，这些旁系同源物与甲基化反应产物S-腺苷高半胱氨酸的结合亲和力（K-D为0.14-2.2 μ M）远高于与底物S-腺苷甲硫氨酸的结合亲和力（K-D为22-43 μ M），这表明产物反馈抑制。对于CheR 2，产物结合特别紧密。分析超离心实验表明，在S-腺苷甲硫氨酸或S-腺苷高半胱氨酸的存在和不存在下，CheR 2是单体的。甲基化分析表明，CheR 2，而不是其他旁系同源物，甲基化的McpS和McpT趋化性受体。CheR 2的突变体在趋化性上是缺陷的，而CheR 1和CheR 3的突变对趋化性没有或几乎没有影响。相比之下，生物膜形成的CheR 1突变体在很大程度上受损，但在其他突变体中不受影响。我们的结论是，CheR 2形成的趋化性途径的一部分，和CheR 1形成的一部分，控制生物膜形成的化学感受途径。数据表明，CheR甲基转移酶作用于其同源化学受体具有高特异性。
Chemosensory pathways are a major signal transduction mechanism in bacteria. CheR methyltransferases catalyze the methylation of the cytosolic signaling domain of chemoreceptors and are among the core proteins of chemosensory cascades. These enzymes have primarily been studied Escherichia coli and Salmonella typhimurium, which possess a single CheR involved in chemotaxis. Many other bacteria possess multiple cheR genes. Because the sequences of chemoreceptor signaling domains are highly conserved, it remains to be established with what degree of specificity CheR paralogues exert their activity. We report here a comparative analysis of the three CheR paralogues of Pseudomonas putida. Isothermal titration calorimetry studies show that these paralogues bind the product of the methylation reaction, S-adenosylhomocysteine, with much higher affinity (K-D of 0.14-2.2 mu M) than the substrate S-adenosylmethionine (K-D of 22-43 mu M), which indicates product feedback inhibition. Product binding was particularly tight for CheR2. Analytical ultracentrifugation experiments demonstrate that CheR2 is monomeric in the absence and presence of S-adenosylmethionine or S-adenosylhomocysteine. Methylation assays show that CheR2, but not the other paralogues, methylates the McpS and McpT chemotaxis receptors. The mutant in CheR2 was deficient in chemotaxis, whereas mutation of CheR1 and CheR3 had either no or little effect on chemotaxis. In contrast, biofilm formation of the CheR1 mutant was largely impaired but not affected in the other mutants. We conclude that CheR2 forms part of a chemotaxis pathway, and CheR1 forms part of a chemosensory route that controls biofilm formation. Data suggest that CheR methyltransferases act with high specificity on their cognate chemoreceptors.