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探討十字花科黑腐病菌中 flhA 與 flhB 基因之功能 Functionl study of flhA and flhB genes in Xanthomonas campestris pv.campestris
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| 作者 |
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劉又維 |
| 學校系所 |
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亞洲大學生物科技學系碩士班 |
| 地點 |
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全臺 全部
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| 研究內容 |
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[ 摘要 ]
Xanthomonas campestris pv. campestris ( Xcc ) 為革蘭氏陰性菌，具單極性單鞭毛，且能感染十字花科植物而導致黑腐病。根據文獻所載鞭毛細菌與第三類型分泌系統（T3SS）關係密切，T3SS 對許多病源體的致病毒素相當重要，包括 Xcc。在相似序列中，我們在 Xcc 查尋超過 40 幾個基因為鞭毛的合成相關。rpoN、fleQ、fliA、flgM、fleN、flhF 等6個基因，在我們實驗室已證明為調控許多鞭毛基因的表現。本研究中，我們報告FlhA與FlhB的功能於相似性的T3SS 蛋白InvA與SpsS 於過去報導Salmonlla中為鞭毛構造的輸出。 Xcc flhA 與 flhB 突變株經崁入突變後分析其表現型。 flhA 或 flhB 突變株具致病性，但沒有運動的能力。兩個突變株在FleQ 與 RpoN 蛋白的表現量為正常。flhA突變株在 FliA 蛋白的表現量稍有減少，但 flhB 突變株沒有。兩個突變株分別不能合成主要的鞭毛蛋白 FliC。 fliC 基因的啟動子是包含一個供選擇的 sigma 因子RNA 聚合酶轉錄與fliA基因編碼。FlgM 蛋白是一個多重 sigma 因子抑制 FliA 。成熟鞭毛的 T3SS 從細胞質釋放 FlgM 可恢復 FliA 的功能。因為 FlhA 與 FlhB 在鞭毛 T3SS 為不可缺的成分， flhA 與 flhB 突變株在 FliC 蛋白的表現量可能是由細胞質的 FlgM 蛋白堆積所抑制。
第二部分的研究使用酵母菌雙雜交系統所測試 RpoN、 FleQ、 FleN、 FlhF、 FlgM、 FliA 和 NtrC 蛋白之間的交互作用，結果推測 RpoN2 和 FlhF; RpoN2和 FleQ; RpoN2 和 NtrC 之間有交互作用。
[ 英文摘要 ]
Xanthomonas campestris pv. campestris ( Xcc ) is a Gram negative, rod-shaped bacterium, and is a causative agent of black rot disease in cruciferous plants. According to previously reports, bacterial flagella and the type III secretion systems (TTSS) are highly related. The TTSS is essential for the virulence in many pathogens, including Xcc. Based on sequence homology, our search in Xcc revealed that more than 40 genes might be involved in the flagellar biogenesis. Six genes, rpoN, fleQ, fliA, flgM, fleN and flhF, have been demonstrated in our laboratory to be involved in the regulation of expression of many flagellar genes. In this work, we report the function of FlhA and FlhB which are homologs of TTSS proteins InvA and SpaS reported to be involved in the exportation of flagellar structure in Salmonlla. Xcc flhA and flhB mutants were obtained by insertional mutagenesis and were used for phenotypical assays. The flhA and flhB mutants are virulent, but immotile. Both mutants expressed a normal quantity of FleQ and RpoN protein. The expression of FliA protein was slightly decreased in an flhA mutant but not in an flhB mutant. Both mutants cannot synthesis the major flagellin protein FliC. Induction of plasmid-born wild-type flhA or flhB genes can successfully complement the motility defect, and restore the biosynthesis of FliC protein in flhA and flhB mutants, respectively. The promoter of fliC gene was transcribed by RNA polymerases containing an alternative sigma factor encoded by fliA gene. The FlgM protein is an anti-sigma factor acts against FliA. A mature flagellar TTSS expels FlgM from the cytoplasm and recover the function of FliA. As FlhA and FlhB are essential components of flagellar TTSS, the expression of fliC in flhA and flhB mutants might be blocked by an accumulation of cytoplasmic FlgM protein.
The second part of this work is to use a yeast two-hybrid system to detect the protein-protein interaction between RpoN, FleQ, FleN, FlhF, FlgM, FliA and NtrC. The results suggested that RpoN2 and FlhF; RpoN2 and FleQ; RpoN2 and NtrC can
interact with each other. |
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