甲基化受体蛋白MCP2923在睾丸酮丛毛单胞菌CNB-1趋化过程中的作用

Chemotactic responses towards various organic compounds mediated by MCP2923 in Comamonas testosteroni CNB-1

【目的】MCP2923是睾丸酮丛毛单胞菌(Comamonas testosteroni)CNB-1的一种甲基化趋化受体蛋白,本研究旨在阐明其在CNB-1菌株趋化过程中的作用。【方法】利用游动平板法(swimming plate)检测了CNB-1及其MCP突变菌株CNB-1?20、CNB-1?MCP2923、CNB-1?20/p DSK519-MCP2923、CNB-1?20/p BBR1MCS-2-MCP2923和CNB-1?20/p BBR1MCS-2-MCP2923?LBD对35种芳香族化合物以及9种小分子有机酸的趋化性;进一步利用Agarose-in-plug法表征了MCP2923介导的对芳香族化合物的趋化表型;结合生物信息学分析,对MCP2923配体结合结构域(MCP2923LBD)进行了克隆、表达和纯化,利用等温滴定量热法(ITC)检测了MCP2923LBD与原儿茶酸等11种化合物的相互作用。【结果】CNB-1对原儿茶酸、4-羟基苯甲酸等12种芳香族化合物以及顺乌头酸等9种TCA循环中间代谢产物具有强、中强和弱3个层次的趋化表型;敲除MCP2923基因减弱了菌株对上述趋化诱导物的表型;将MCP2923基因回补到CNB-1?20菌株中可回补菌株对15种趋化效应物的表型,敲除MCP2923基因的配体结合区丧失了对15种趋化物的表型回补能力。虽然Agarose-in-plug法检测到了菌株对原儿茶酸和4-羟基苯甲酸的趋化表型,但ITC未能检测到原儿茶酸和4-羟基苯甲酸等11种化合物与MCP2923LBD直接的相互作用。【结论】MCP2923可引发CNB-1菌株对多种芳香族化合物以及小分子有机酸的趋化表型,且MCP2923LBD在这个过程中起关键作用;由于ITC的结果不能证明MCP2923LBD与芳香化合物和小分子有机酸等效应物的直接结合,推测MCP2923引发CNB-1趋化作用的机制不同于已报道的MCP2201和MCP2901的作用方式,其确切机制还有待于进一步深入研究。

[Objective] MCP2923 is a putative of Comamonas testosteroni CNB-1. The objective of this study was to experimentally characterize MCP2923 for chemotactic response. [Methods] Using swimming plate assay, we determined chemotaxis towards 35 aromatic compounds and 9 Tricarboxylic Acid Cycle intermediates. Agrose-in-plug was used to screen aromatic chemoattractants that might bind to MCP2923 directly. To study the ligand of MCP2923, Isothermal Titration Calorimetry experiment was done to 11 chemoattractants. [Results] Swimming plate assay showed that CNB-1 responded to 12 aromatic compounds and 9 Tricarboxylic Acid Cycle intermediates that were defined as strong, medium and weak chemoattractants. Knockout MCP2923 gene reduced chemotactic responses to these chemoattractants. Complemented with MCP2923 gene to CNB-1？20, chemotaxis toward 15 chemoattractants was restored. Deletion of the ligand binding domain of MCP2923, chemotaxis failed to complement. Isothermal Titration Calorimetry experiment showed no response to the tested 11 compounds, including protocatechuic acid and 4-hydroxybenzoic acid. [Conclusion] MCP2923 mediates chemotaxis towards both aromatic compounds and Tricarboxylic Acid Cycle cycle intermediates by CNB-1. The ligand binding domain of MCP2923 is necessary for triggering chemotaxis toward these chemoattractants.