- 學位論文
以定點突變探討根瘤菌Azorhizobium caulinodans ORS571 ParA蛋白質在其細胞週期與莖瘤形成所扮演的角色
Site-directed mutagenesis of ParA protein in Azorhizobium caulinodans ORS571: effect on cell cycle progression and stem-nodule development
摘要
根瘤菌Azorhizobium caulinodans ORS571的染色體分配蛋白 ParA 不僅參與細菌的細胞週期，並且與莖瘤的成熟發育息息相關。ParA 蛋白質是 Walker 型的 ATP 水解酶，具有 Walker A、A’ 與 B 等保守功能區 (motif)，其分別與 ATP 結合、ATP水解活性以及鎂離子結合有關。在 ATP 存在下，ParA 蛋白能夠形成雙聚體，具有與 DNA 結合的能力。本研究針對 ParA 蛋白的 A 與 A’ motif 進行點突變，試圖影響 ParA 蛋白質 ATP 結合能力 (ParA K33A)、雙聚體形成能力 (ParA G29V)，以及 ATP 水解酶活性 (ParA D57A)。利用孔雀石綠磷酸法分析 ATP 水解酶活性，發現上述三種點突變的水解活性皆下降。藉由凝膠遷移法探討 ParA 蛋白在 ATP 存在下與 DNA 結合的能力，發現ParA K33A 與 ParA G29V 皆不能與 DNA 結合。在非共生條件下，ParA 點突變菌株其細胞型態顯著變長，並有部分菌體呈分支狀，亦有多套核樣體，表示染色體分配缺失。根據乙炔還原法所測定之非共生態固氮活性，所有點突變株固氮能力皆顯著降低。在共生條件下，點突變株所形成莖瘤較小，固氮活性也顯著較低。因此推測，ParA 蛋白質三個必要的胺基酸 (G29, K33以及D57) 對於調控根瘤菌的固氮活性及莖瘤發育過程扮演重要角色。
並列摘要
Chromosome partitioning protein, ParA, of Azorhizobium caulinodans ORS571 not only plays crucial roles in bacterial cell cycle when the microbe is under free-living state, but also negatively regulates bacteroid formation in Sesbania rostrata stem nodule. ParA has been regarded as a Walker type ATPase and contains three active sites, including Wallker A, A’ and B motifs, which participate in ATP binding, ATP hydrolysis and magnesium ion binding, respectively. In the presence of ATP, the wild-type ParA formed dimmer and showed DNA-binding activity. Three residues (G29, K33, and D57) previously shown to be functionally important in ParA proteins were altered by site-directed mutagenesis in this study. The dimerization-deficient ParA G29V, the ATP-binding-deficient ParA K33A, and the ATP hydrolysis-deficient ParA D57A all showed significantly reduced ATPase activity by using Malachite green phosphate assay. Furthermore, ParA K33A and ParA G29V failed to bind to DNA in the presence of ATP by electrophoretic mobility shift assay (EMSA), indicating that ATP-dependent dimerization is necessary for DNA binding. The phenotype of the A. caulinodans parA mutant derivatives harboring the site-specific mutations showed pleiomorphic cell shapes and polyploid, indicating defects in chromosome partitioning. Besides, markedly reduced nitrogenase activities of the mutants were determined by acetylene reduction assay, suggesting that the ParA is involved in nitrogen fixation in the free-living state. Upon inoculation of the mutants onto the stem of S. rostrata, immature nodule-like structures with impaired nitrogen-fixing activity were generated. Taken together, it suggests that the essential amino acid (G29, K33 and D57) in ParA protein play important roles in cellular development, nitrogen fixation and stem nodule development.
參考文獻
延伸閱讀
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