- 學位論文
Xanthomonas campestris pv. campestris dsbA 與 dsbC 特性之研究
Charaterization of dsbA and dsbC of Xanthomonas campestris pv. campestris
摘要
Xanthomonas campestris pv. campestris (簡稱Xcc) 會造成十字花科植物黑腐病，屬於革蘭氏陰性植物病原菌，Xcc 會產生胞外黏多醣(exopolysaccharide，簡稱EPS)，以及分泌多種胞外酵素，如 a-澱粉? ( -amylase)、果膠? (pectate lyase)、纖維素? (endoglucanase) 及蛋白? (protease)。胞外酵素及胞外黏多醣是造成植物致病性所必需。在 Xcc 基因體上有一群能調控致病因子的的基因稱rpf(regulation of pathogenicity factors)。目前已知 rpf 基因串與多種致病因子 (virulence factor) 合成有關。其中，RpfF 經比對與其他生物的enoyl-CoA hydratase 有相當的相似度，RpfF 會直接影響訊號小分子DSF (small diffusible regulatory molecule) 的合成。已知 DSF 所引發的訊號傳導 (signal transduction) 會影響致病因子合成。先前，本實驗室得到rpfF 突變株 P20H(rpfF::Gm)，利用 2D-PAGE 比較野生株與 rpfF 突變株的胞內及細胞膜蛋白，初步發現12 個蛋白點有明顯的 差異。其中第五號的蛋白點，經 LC-MS-MS 分析與比對之結果是Xcc 染色體上 (菌株 ATCC33913) 的 XCC3400 (DsbA)，DsbA 經比對類似 periplasmic thiol：disulfide oxidoreductase。在 XCC3400 的上游基因為 XCC3399 與 XCC3400 同屬於 DsbA family，為了方便區分，我們將 XCC3400 命名為 dsbA1，XCC3399 命名為 dsbA2。為3 了解在 Xcc 中，RpfF 如何去調控 dsbA1 與 dsbA2 基因表現，以及DsbA1 與 DsbA2 是否藉由影響各種致病因子的蛋白質摺疊，進而影響Xcc 的致病性。於是進行一系列實驗，得到以下結果：(1) dsbA1 基因與 dsbC 基因發生缺失時，都會造成致病力的喪失，但 dsbA2 基因缺失卻不影響致病力，(2) 在蛋白? (protease) 分泌測試中，dsbA1與 dsbC 發生缺失時都沒有十分明顯的下降，但dsbA1 與 dsbC 同時發生缺失時，protease 分泌明顯降低，(3) dsbA1 與 dsbC 基因缺失，造成線狀噬菌體 fLf 感染受影響，dsbA2 則不明顯，(4) dsbA1與 dsbC 發生缺失都會降低鞭毛的 swimming motility 型式之運動，(5) 在transcription 表現的分析中，初步推測 dsbA1 的轉錄作用可能受 RpfF 正向調控；DsbA1 與 DsbA2 對 fliC 轉錄作用都具正向調控作用，推測可能是間接的調控作用；pilA1 的轉錄與轉譯作用都不受 DsbA1 與 DsbA2 之調控，推測 PilA1 在蛋白質折疊過程受影響所致。
並列摘要
Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in cruciferous plants. Xcc produces exopolysaccharide (EPS) and various extracellular enzymes, such as a-amylase, pectate lyase, endoglucanase and proteases, which are collectively essential for pathogenesis. Pathogenicity of Xcc is regulated by rpf gene cluster (for regulation of pathogenicity factors). Previous studies have shown that production of these virulence factors is positive regulated by rpf gene cluster. The amino acid sequence of RpfF has highly identity to enoyl-CoA hydratase and is demonstrated directly leading DSF synthesis. DSF regulate various virulence factors synthesis by signal transduction. Twelve protein spots difference was found by 2D-PAGE which was compared between wild type and P20H(rpfF::Gm), rpfF mutant obtained in previous study. The number 5 protein spot was identified as XCC3400 (DsbA) (ATCC33913 strain) which is periplasmic thiol: disulfide oxidoreducatase by LC/MS/MS. XCC3399, upstream gene of XCC3400, also belong to DsbA family. We named those two genes as dsbA1 and dsbA2 to make XCC3400 and XCC3399 could be distinguishable. Serial experiments were carried out for understanding how RpfF regulate dsbA1 and dsbA2, and whether DsbA1 and DsbA2 influence the Xcc virulence by catalyses folding of various factors. The results of above-mentioned study: (1) Mutation in dsbA1 or dsbC abolish ability of virulence, but mutation in dsbA2 shows no difference with wild type. (2) Secretion of protease slightly decreases while mutate in dsbA1 or dsbC, but decreases while mutate of dsbA1 and dsbC. (3) The deletion of dsbA1 and dsbC brings filamentous phage fLf resistibility to host, while mutation in dsbA2 is no effect. (4) Swimming motility abolish when mutation in 5 dsbA1 and dsbC. (5)We suppose possible inferences about the assay of transcription expression: the transcription of dsbA1 is upregulated by RpfF and fliC is indirectly upregulated by DsbA1 and DsbA2; transcription of pilA1 is not regulated by DsbA1 and DsbA2, these two genes probably effect on protein folding.