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標題: 十字花科黑腐病菌的 Clp 參與轉錄調控
Transcriptional Regulation by Clp in Xanthomonas campestris pv. campestris
作者: 陳智華
Chen, Chih-Hua
關鍵字: Xanthomonas campestris
transcriptional regulation
出版社: 分子生物學研究所
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摘要: 革蘭氏陰性的植物致病菌 Xanthomonas campestris pv. campestris(Xcc)是十字花科植物黑腐病的病原菌,此菌會產生的大量胞外多醣,稱為 xanthan gum,也會分泌多種胞外酵素,如蛋白酶、果膠酶以及纖維素酶等,胞外多醣與胞外酵素長期以來被視為重要的致病因子,這些重要的致病因子的生產會受到多元轉錄活化因子 Clp(cyclic AMP receptor protein-like protein)的調控,此蛋白為 Escherichia coli CRP 的同源蛋白。Clp 會與 engA、 pehA、 pelA1 及 xpsE 等基因的啟動子區域結合,對其轉錄活化進行正向調控;Clp 也會對 fliC、groESL、prt1 及 manA 等基因的轉錄進行活化,但不會與這些基因上游區域結合。 本研究的目標在於闡明 Clp 對於 Xcc 基因體的轉錄調控情形,為了達成這個目標,使用 PromScan 程式( 於 Xcc strain ATCC 33913 基因體(GenBank accession no. AE008922)內搜尋可能的 Clp-binding sites(CBSs),程式所須的 frequency matrices 則分別使用彙整的典型與非典型 CBSs 製成。 首先,對 clp、 gumB 及 xpsE 等基因的上游區域進行探討。利用 electrophoretic mobility shift assay (EMSA) 和 transcriptional fusion assay,證明 Xcc clp 的轉錄受到 Clp 正向調控,Clp 會與位於其轉錄起始點(transcription initiation site)上游的典型 CBS 進行結合作用。根據 reporter assay 和 EMSA的結果,得知 Clp 會與 gum operon 上游區域中的 2 個非典型 CBSs 結合後,對 gum operon 的轉錄進行正向調控,這 2 個 CBSs 的右臂都具有較高的序列保守性,於結合作用發生時,可以彌補缺乏序列保守性的左臂所喪失的結合能力;另外,中間區域的 6 個鹼基對為高 GC 含量是有助於結合作用的,而且是迴文結構的 GC 鹼基對能增強結合能力。transcriptional fusion assays 的結果顯示:Clp 會活化 xpsEF operon 的轉錄,但對 xpsG 沒有直接調控的情形,雖然在 xpsE 上游區域中有 2 個 CBSs,不過只有鄰近 xpsE 啟動子的CBS 才是轉錄活化所必要的。 將 gumB CBS I、CBS II 與 xpsF CBS 三個 atypical CBSs 的序列比例轉換成 frequency matrix,利用 PromScan 程式找出 Xcc 基因體所有基因啟動子區域中與 atypical CBS 相似的序列,這些序列與 microarray 試驗的結果重複的基因有 21 個,但彼此推測的 CBS 並不相同,從其中挑選 5 個進行驗證,經 competitive EMSA 證實本研究的推測無誤。將此 8 個 atypical CBSs 的鹼基組合加以統計,歸納出 consensus atypical CBS,序列為 5’-AnAGGCGA-ACGCnG-TCACAnAA-3’。同時把 8 個已知的 typical CBSs 的鹼基組合加以統計,歸納出 consensus typical CBS,序列為 5’-TnGTGTGn-nnnAnn-TCnCATCG-3’,也把這 8 個 Xcc typical CBSs 序列組合比例轉換成另一個 frequency matrix,執行 PromScan 程式,也找到 30 個與 microarray 試驗的結果重複的基因,這些 CBSs 有待將來的研究加以證實。 由本研究的結果可知有 typical 與 atypical CBSs 這兩類的 CBSs 存在於 Xcc 基因上游區域中,而且 Clp 保有與這兩類 CBSs 進行結合作用的能力。本研究另有 2 個重要的發現:(一) xpsE 的轉錄受到 RpfF 的正調控;(二)透過 EMSA 證明 Clp 與 Xcc engA 上游區域片段於 in vitro 情況下的結合作用會受到 cyclic di-GMP 的抑制。
The Gram-negative plant pathogenic Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers. It is capable of producing large amounts of an exopolysaccharide, xanthan gum, and secreting an array of extracellular enzymes, including proteases, pectinases, and endoglucanases, which have long been considered important virulence determinants. Production of these extracellular products is regulated by the global transcription factor Clp (cyclic AMP receptor protein-like protein, a homolog of the Escherichia coli CRP) and DSF whose biosynthesis involves RpfF protein. It is also known that Xcc encodes no adenylate cyclase required for the production of cyclic AMP, an effector that is involved in activation of CRP for binding to CRP-regulated promoters. Furthermore, it has previously been demonstrated in our laboratory that Clp 1) in vitro can bind to the regulated promoters in the absence of cyclic AMP, and 2) exerts transcriptional activation by binding to the promoter of some regulated genes (i.e., engA, pehA, and pelA1), but without direct binding to others (i.e., fliC, groESL, prt1, and manA). Despite these findings, prediction of the sequences for Clp binding (Clp-binding site, CBS) was merely based on similarity to the consensus E. coli CRP-binding site, which is 22-bp long (5'-AAATGTGATCTAGATCACATTT-3') exhibiting perfect twofold sequence symmetry, because Clp has been shown to have the same DNA binding specificity as CRP at positions 5, 6, and 7 (GTG motif) of the DNA half site. However, close examination of the above CBSs that have been characterized revealed that some of them possess sequences of arms and a central region deviated from the CRP consensus sequence. Therefore, this study was aimed to deduce consensus CBS sequence(s) on the basis of a larger number of characterized Xcc CBSs than what was already available. To achieve this end, PromScan program was used to search in Xcc genome. Several putative CBSs were identified, among which promoters of clp, gumB, and xpsE were then confirmed to be up-regulated by Clp, via deletion mapping in conjunction with electrophoretic mobility shift assay (EMSA) and transcriptional fusion assay. The results suggested that 1) clp is auto-regulated positively by Clp via binding to a typical CBS, 2) gum promoter possesses two atypical CBSs, in which a more conserved right arm compensates for the lack of conservation in the left arm, a high GC content in the central region (6 bp) is important for binding, and binding is enhanced by the palindromic GC-rich central region, and 3) two typical CBSs are present upstream of xpsE and both could bind to Clp in EMSA; however, only the one in proximity to the xpsE transcription initiation site is required for transcription activation. The three atypical CBSs (gumB CBS I, gumB CBS II, and xpsF CBS) were combined to create a frequency matrix which was then used in PromScan to perform genome-wide search. Twenty-one possible atypical CBSs were identified and five of them were confirmed by competitive EMSA. These CBS-containing genes have previously been shown to be up-regulated by Clp in microarray assay; however, most of the CBSs confirmed here are situated at different positions from those predicted by the microarray assay. Based on these eight atypical CBSs, a consensus atypical CBS, 5'-AnAGGCGAACGCnGTCACAnAA-3', was compiled. In the meantime, by aligning 8 known typical CBSs, a more informative consensus typical CBS (5'-TnGTGTGnnnnAnnTCnCATCG-3') was deduced. Using a frequency matrix based on this consensus sequence for search with PromScan, 30 Xcc genes, which have been previously identified by microarray assay to be regulated by Clp, were identified to possess typical CBS. Further EMSA is needed for confirmation of these typical CBSs. In conclusion, these results suggest that both typical and atypical CBSs exist in Xcc genome and that Clp has evolved to gain the capability to bind both types of CBSs. In addition to the above results, two important findings were also made here: 1) xpsE promoter is also up-regulated by RpfF, and 2) binding of Clp in vitro is inhibited by cyclic di-GMP, as demonstrated by using the Xcc engA promoter region as the probe in EMSA.
其他識別: U0005-1108201022000700
Appears in Collections:分子生物學研究所



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