分離與分析Ralstonia pickettii可降解訊號分子3-hydroxypalmitic acid methyl ester之酵素

Abstract

青枯病 (bacterial wilt disease) 是由 Ralstonia solanacearum 引起之細菌性病害，對於全球農業經濟影響甚鉅。R. solanacearum 主要毒力因子為胞外多醣體 (exopolysaccharides) 及細胞壁分解酵素 (cell wall–degrading enzymes)，藉此造成植物維管束堵塞以及細胞壁瓦解等現象，兩毒力因子皆由化學訊號分子酯化合物 3-hydroxypalmitic acid methyl ester (3-OH PAME) 以集約反應系統 (quorum sensing system) 進行基因調控，3-OH PAME 之濃度會進一步正調控集約反應下游與毒力相關之 LysR 家族轉錄調節蛋白 PhcA。許多以集約反應調控細菌致病基因的相關研究結果顯示，藉由降解集約反應訊號分子或阻斷其受器等方式 (quorum quenching)，可降低細菌致病力，為細菌性病害管理提供了新的策略。在本研究中，以具有 3-OH PAME 降解活性菌株 Ralstonia pickettii RP4510 為實驗目標，為了篩選出 RP4510 中目標酵素基因與探討其生化特性，以轉座子 (transposon) 隨機突變為策略篩選影響 3-OH PAME 降解活性相關之基因，結果顯示，細菌的第二型蛋白質分泌系統及調節因子、與數種營養代謝能力均可影響 RP4510 3-OH PAME 降解活性，並由此闡明目標酵素之分泌路徑。後續以蛋白質純化技術分離目標酵素，以陰、陽離子交換層析法、FPLC 蛋白質層析儀分離純化 RP4510 之外泌蛋白質，將純化後之蛋白質以液相層析串聯式質譜儀 (LC/MS/MS) 分析，並與資料庫進行胺基酸序列比對；比對結果顯示，候選蛋白 RPD0809、RPD1992 具有 alpha/ beta hydrolase 特性，可能具有 3-OH PAME 降解酵素之活性，因此將 rpd0809 與 rpd1992 基因分別進行突變，結果顯示 rpd0809 突變株3-OH PAME 降解活性顯著減少，而 rpd1992 突變株則與野生型菌株無明顯差異。以 PCR 增幅rpd0809 基因並以蛋白質表現系統於大腸桿菌 BL21(DE3) 表現候選蛋白質，結果顯示，過量表現蛋白質 RPD0809 形成不溶體 (inclusion body)，因此不利於後續酵素純化與特性分析。分析 RP4510 野生型、rpd0809 突變株、互補菌株與 rpd0809 過表現菌株分解 3-OH PAME 之活性，結果顯示互補菌株可恢復 rpd0809 基因缺失株之活性，且 rpd0809 過表現菌株分解 3-OH PAME 之活性顯著增加。本研究闡明了具有 lipase 特性的蛋白 RPD0809 為 R. pickettii RP4510 中主要降解 3-OH PAME 之酵素，此酵素未來可應用於開發青枯病菌複合式防治策略。

Bacterial wilt disease, caused by Ralstonia solanacearum, is the most destructive bacterial disease on plants. The main virulence factors of R. solanacearum are exopolysaccharides (EPS) and cell wall–degrading enzymes (CWDE), which are positively regulated by a LysR family transcriptional regulator PhcA, and phcA is positively regulated by the 3-hydroxypalmitic acid methyl ester (3-OH PAME)-inducing quorum sensing (QS) system. Interfering the QS signaling pathway by the prevention of signal recognition and the degradation of quorum sensing signaling molecules (quormones), i.e. quorum quenching (QQ), may be a potential approach to control bacterial wilt disease. In this study, R. pickettii RP4510, isolated from suppressive soils was screened for their capability of degrading 3-OH PAME. The genome of RP4510 was mutagenized by a mini-transposon to genetically characterize the proteins associated with 3-OH PAME-degradion. The transposon-inserted mutations that exhibited reduced 3-OH PAME degrading activity were found to reside in the genes involved in general secretion pathway, global regulation, and substrate hydrolysis for catabolizing 3-OH PAME. To further characterize the biochemical properties of the proteins, they were purified by ion exchange chromatography and FPLC, and identified by LC/MS/MS. The results showed that two proteins, RPD0809 and RPD1992, exhibited alpha/beta hydrolase activity and contained the functional motifs in the deduced amino acid sequences. Genetic analyses revealed the rpd0809 knockout mutant was defective in 3-OH PAME degrading activity, whereas the rpd1992 mutant retained the wild-type activity. Overexpression of RPD0809 in E. coli led to the production of non-functional inclusion bodies. Therefore, the wild type, rpd0809 mutant, a complement strain, and an rpd0809-overexpressing strain were compared for their enzymatic activities in degrading 3-OH PAME and lipids. The results showed that the 3-OH PAME degrading activity of rpd0809 mutant was restored by complementation, and it was significantly increased in the rpd0809-overexpressing strain. The data indicated that RPD0809 was predominantly involved in the degradation of 3-OH PAME in R. pickettii RP4510, which can be applied in formulating a combinatorial strategy for managing bacterial wilt disease.