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標題: Xanthomonas campestris β-lactamase的訊號序列與運送系統之探討
Investigation of the signal peptide and translocation system of Xanthomonas campestris β-lactamase
作者: 李振維
Lee, Chen-Wei
關鍵字: Xanthomonas campestris;十字花科蔬菜黑腐病菌;beta-lactamase;Tat;beta-lactamase;Tat
出版社: 分子生物學研究所
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Xanthomonas campestris pv. campestris (Xcc) 為十字花科蔬菜黑腐病菌,其分離株多可產生 β-lactamase (Bla),以分解含 β-lactam ring 之抗生素。野生株 Xc11、Xc17 與 Xc85 的 bla 基因序列均已定序完成。依 signal peptide 序列推測 Xcc 可能利用 twin-arginine translocation (Tat) pathway 進行 Bla 之運送。本論文中首先構築了 Xc11 與 Xc17 的 tatA 與 tatC 基因突變株供探討。經以 Western blotting 測試,發現只有在 Xcc 野生株的胞質週緣區才能偵測到 Bla 的成熟蛋白,在tatA 與 tatC 突變株則否。 Xcc Tat 系統一旦被破壞,抗 β-lactam 抗生素的能力明顯減弱,MIC 值降低,顯示 Xcc Bla 的確藉由 Tat 系統運送。此外,經序列比對分析,發現 Bla 的第七個胺基酸 (Phe-7) 之變異會影響酵素產量。 Phe-7 為 signal peptide 的 Tat motif 中保守序列 Arg-Arg 下游的第二個胺基酸,在 Bacillus subtilis 該位置胺基酸具疏水性傾向。本論文中,將 Xcc Bla 蛋白的 Phe-7 突變為 Pro 、 Leu 、Arg 與 Tyr 後,其活性分別為原來的 5.3、24.5、47.5 及 107.1 %。在 Western blotting 結果中,所有突變株的 Bla 由細胞質運送至胞質週緣區的量皆減少。此外,突變為 Arg 後大量訊號胜肽未被切除的 pre-Bla 蛋白被發現累積在細胞質中。將 Phe-7 突變為親水性的Tyr-7 後,蛋白運送及訊號胜肽被辨識切除的效率稍微降低;而該位置突變為同屬疏水性胺基酸的 Leu 時,卻嚴重影響蛋白的運送與修飾。突變為 Pro-7,則可能因為訊號序列構形產生劇烈轉折,導致蛋白不穩定而容易被降解,是所有突變株中 Bla 表現最低者。上述研究結果顯示在不同物種中,Tat 系統辨識受質訊號序列之機制不盡相同,Xcc Bla 蛋白的 Phe-7 對於蛋白的運送及訊號胜肽的切除相當重要。此外,Xcc 野生株與 tatA、tatC 突變株在生長曲線、胞外多醣 (EPS) 的合成、運動能力以及 endoglucanase、protease、phospholipase C 等胞外酵素之表現上均無明顯差異,顯示影響這些性狀的蛋白主要並非經由 Tat系統進行分泌。然而,在 tatA 與 tatC 突變株的致病性測試中發現, tatA 突變株已無致病力,tatC 突變株則仍具致病性。由 RT-PCR的結果推測,tatA、tatB 與 tatC 屬同一個 operon。tatA 突變株之 tatA 無法被表現,tatB、tatC 表現量降低。 Tat 系統影響致病性的真正機制尚不清楚,有待繼續探討。
其他識別: U0005-1808201014522900
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