探討黑腐病菌具組胺酸-天門冬胺酸輸出區的反應調控者 XCC3315 的功能與表現

Function and expression of XCC3315, a novel response regulator with an histidine-aspartate output domain in Xanthomonas campestris pv. campestris

宋宛玲(Wan-Ling Song)

指導教授 ： 柯俊良

中山醫學大學/醫學院/醫學研究所/碩士(2012年)

摘要

Xanthomonas campestris pv. campestris (Xcc) 會引起十字花科植物黑腐病 (black rot)，造成農業上的重大損失。Xcc 之毒力取決於多種因子，包括胞外酵素、胞外多醣與運動能力。近來有研究指出 Xcc 有一反應調控者 (response regulator，基因編號 XCC3315) 與本菌的壓力反應有關，係由一個CheY-like 接收區 (REC domain) 與一個功能未知的組胺酸-天門冬胺酸輸出區 (histidine-aspartate-related output domain, HDOD domain) 所組成。本研究的目的在於探討 XCC3315 的功能與表現，共分三個部份進行。第一部份：進行 XCC3315 突變株與其互補株的表型分析，探討 XCC3315 的功能。發現 XCC3315 突變會降低本菌的運動能力，但對於致病性與胞外酵素的產生無顯著差異。蛋白質體學分析顯示數種蛋白的表現量因 XCC3315 的突變而改變，其中一個在 XCC3315 突變株中表現量降低的蛋白質被鑑定為由 fliC 所編碼的鞭毛蛋白 (flagellar protein)。報導基因分析也顯示 fliC 之轉錄受到 XCC3315 的正調控。進一步研究發現，XCC3315 調控鞭毛基因 flhF 與 flhB 的表現。第二部分：訂定 XCC3315 重要的高保留性胺基酸。定點突變結果顯示，REC 接收區之 R100 與 HDOD 輸出區之 H287 與 W298對於XCC3315 調控運動性上是很重要的。第三部份：探討 XCC3315 的表現。利用 cDNA 末端快速增幅 (Rapid Amplification of cDNA Ends, RACE) 技術訂定 XCC3315 之轉錄起始點，位於轉譯起始點上游第 45 個核苷酸 ‘‘A’’。啟動子活性分析與 gel retardation 的結果顯示，XCC3315 的表現受到多元調控蛋白 Clp (cAMP receptor protein-like protein) 直接正調控。報導基因分析也顯示 XCC3315 轉錄起始點上游 -156 至 +80 的片段為其表現所必需。

關鍵字

黑腐病菌 ； 反應調控者 ； 接收區 ； 組胺酸-天門冬胺酸輸出區

並列摘要

Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers, a disease causing tremendous loss in agriculture. The virulence of Xcc depends upon a number of factors, including extracellular enzymes, exopolysaccharide, and cell motility. A recent work suggests that XCC3315, a response regulator with a CheY-like receiver (REC) domain attached to an unknown-function histidine-aspartate-related output domain (HDOD domain), has a role in general stress response of Xcc. The purposes of this study were to characterize the function and expression of XCC3315. First, phenotypic evaluation of XCC3315 mutant and its complemented strain were performed. It was found that mutation of XCC3315 causes reduced cell motility but not pathogenicity and extracellular enzyme production. Proteomic analysis revealed that several protein amounts are altered after XCC3315 mutation. One of the protein spots showing reduced amount in XCC3315 mutant was identified to be flagellar protein, encoded by fliC. Reporter assay indicated that the promoter activity of fliC is positively regulated by XCC3315. Furthermore, it was found that XCC3315 regulates the expression of flagellar genes flhF and flhB. Second, the conserved amino acid residues essential for XCC3315 function were identified. Site-directed mutagenesis revealed that R100 in REC domain and H287, W298 in HDOD domain are essential for XCC3315 function in regulating cell motility. Third, the transcriptional regulation of XCC3315 was evaluated. Using 5’-RACE (Rapid Amplification of cDNA Ends) technique, nucleotide A at 45 nt upstream of XCC3315 translation start condon was mapped as the XCC3315 transcriptional initiation site. Promoter analysis and gel retardation assay revealed that the expression of XCC3315 is positively controlled by global transcriptional regulator Clp (cAMP receptor protein-like protein) directly. Reporter assays also indicated that the fragment containing -156 to +80 relative to the XCC3315 transcriptional initiation point is required for its expression.

並列關鍵字

Xanthomonas campestris pv. campestris (Xcc) ； response regulator ； receiver domain (REC) ； histidine-aspartate output domain (HDOD)

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