臺灣博碩士論文加值系統

所有生物體都會對外在環境的變化產生反應，細菌為了要適應不同的生活環境變化，發展出多種不同的因應方法。 利用不同的 sigma factors 調節不同的基因即為方法之一。 而 54 是 rpoN 基因的產物，其調節的基因包含碳源及氮源的利用、鞭毛及纖毛的形成、致病性、RNA 的修飾、以及趨化性等等。 在已完成定序的Xanthomonas campestris pv. campestris (簡稱XCC) 之基因體中，發現含有兩套 rpoN 基因，它們分別調控不同的基因。 受 RpoN 調控的基因進行轉錄時，需 NtrC-like activator (也稱為 enhancer-binding protein, EBP) 一起參與，才會使轉錄開始進行。 藉由類似真核生物之磷酸化與去磷酸化的機制，而使 EBP 的活化。
藉由與 sigma 54作用之區域序列 (Pfam PF00158) 比對，發現在XCC33913 與 XC17 中，分別有八個及九個 EBPs。 FleQ 為其中之一，其位於 rpoN2 之下游，而 rpoN2 為鞭毛合成所必需的。 在 Pseudomonas aeruginosa 中， FleQ 會與 RpoN 一起調節鞭毛基因的表現。 為瞭解 FleQ 在 XCC 中所扮演的角色，則利用 insertional mutation方法，取得 XC17 的 FleQ 突變株。 此突變株不具有鞭毛與運動性，但並不影響感染力與在營養或基本培養液的生長狀況。 利用 promoter-probing 方式來說明 fleQ 會調節受 RpoN 調控的基因。 由此結果發現，FleQ 正調控鞭毛基因 (fliE, fliL, fliQ, flgB, flgG and flhF) 的表現，而這些鞭毛基因也受 rpoN2 所調控。 而破壞 fleQ 的功能後，並沒有影響受 rpoN1 調控之基因的表現，如 pilF (產物為纖毛合成蛋白), pilA1 (產物為纖毛合成蛋白), nasA (產物為運送硝酸鹽), prpB (產物為丙酮烯醇磷酸轉位酶) 及 glnA (產物為麩醯胺酸合成酶)。
利用生物資訊方法預測 FleQ 可能的結合位置。 發現每一個被分析的 rpoN 啟動子上游都擁有一特殊的序列，包括一個富含 GC 的區域，上下游分別為 oligo A 與 oligo T。 將位於 fliE 啟動子的這個區域破壞後，會使此啟動子失去活性。 這些結果顯示，這些區域可能參與 FleQ 的活化作用。

To adapt to the diverse environmental changes, bacteria develop various methods. One of the methods is to use the alternative sigma factors to regulate gene expression. The 54 factor, encoded by rpoN, regulates genes involved in utilization of nitrogen and carbon sources, synthesis of flagellum and pilus, pathogenicity, RNA modification, and chemotaxis. Two functionally independent rpoN genes have been identified in Xanthomonas campestris pv. campestris (XCC). An interaction with an NtrC-like activator (also called enhancer-binding protein, EBP) is essential for activation of the transcription initiation by a 54-containing RNA polymerase. Activation of EBP is mediated by a series of eukaryotic-like phosphorylation-dephosphorylation pathways.
Based on sequence homology to the 54-activating domains present in other bacteria (Pfam PF00158), eight and nine EBPs were identified in the genome of XCC strain 33913 and XC17, respectively, including among others the fleQ gene downstream of rpoN2 essential for flagellum biogenesis. In Pseudomonas aeruginosa, FleQ works in concert with RpoN in activating flagellar genes. To investigate the role fleQ plays in XCC, a fleQ mutant from XC17 was constructed by insertional mutation. This mutant was found to be immotile and does not possess flagellum. However, the mutation does not interfere with the pathogenicity and growth of the cell. Promoter-probing assays were carried out to elucidate the role fleQ plays in regulation of expression of the 54-dependent promoters. The results showed that FleQ is required for the expression of flagellar genes (fliE, fliL, fliQ, flgB, flgG and flhF) which are RpoN2-dependent. Nevertheless, mutation in fleQ does not interfere with the expression of RpoN1-dependent genes, such as pilF (encoding fimbrial biogenesis protein), pilA1 (encoding pilin), nasA (encoding nitrate transporter), prpB (encoding carboxyphosphonoenolpyruvate phosphonomutase) and glnA (encoding glutamine synthetase).
Putative FleQ binding sites were predicted by bioinformatics tools. A consensus sequence containing a GC-rich region flanked by an upstream oligo-A and a downstream oligo-T regions was identified immediately upstream of the RpoN-binding site in each of the analyzed flagellar promoters. Destruction of this motif in fliE gene was shown to eliminate the normal promoter function. This data suggests that this region may play an essential role in the interaction with FleQ.

縮寫字對照表 1
Abstract 2
中文摘要 4
壹、前言 5
貳、材料與方法 9
A. 材料 9
一、Xanthomonas genus 基因體序列 9
二、菌種、噬菌體、及質體 9
三、一般藥品、酵素、及放射性同位素 9
四、培養基 9
五、試劑與緩衝溶液 10
B. 方法 13
一、細菌之培養與保存 13
二、點測試法 (Spot test) 13
三、DNA 之製備 13
四、聚合酶鏈鎖反應 14
五、洋菜膠體電泳分析 15
六、質體之選殖 (cloning) 15
七、細菌之轉形作用 16
八、生長曲線 17
九、-galactosidase 活性試驗 17
十、南方轉漬法 18
參、結果與討論 20
第一部份：分析在 Xanthomonas genus 中所有與輔助 rpoN 調節基因相關的 activators 20
第二部份：FleQ 調控鞭毛基因或非鞭毛基因之研究 23
第三部份：尋找 FleQ 結合的enhancer-like element 32
第四部份：利用生物實驗證明 FleQ 可能結合的 enhancer-like element 34
肆、表與圖 37
伍、參考文獻 72

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