Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20711
標題: Xanthomonas campestris pv. campestris β-lactamase的運送與表現
Translocation and expression of β-lactamase in Xanthomonas campestris pv. campestris
作者: 鄧福勝
Deng, Fu-Sheng
關鍵字: 十字花科黑腐菌;Xanthomonas campestris pv. campestris
出版社: 分子生物學研究所
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摘要: 
Xanthomonas campestris pv. campestris (Xcc)為感染十字花科植物病原細菌,造成十字花科植物的黑腐病。大部分Xcc菌株具有抵抗β-lactam類抗生素能力。為了瞭解其抗菌機制,本研究針對β-lactamase運送路徑與調控機制進行探討。本研究證明Xc17之β-lactamae運送是透過Tat pathway。將Tat pathway組成蛋白tatA、tatB、tatC以及tatABC各別破壞後,Tat 突變株會失去對ampicillin抗性。根據Westen blotting結果顯示,tat突變株仍可正常表現Bla,但會以具酵素活性的前驅物形式滯留於胞內。在Xc17生理上,破壞Tat pathway除了造成菌體對於鹽類離子的耐受度改變外,在生長曲線、移動力、胞外酵素分泌、生物膜形成及病原性等性狀並無明顯的差異。實驗結果顯示,Xc17之β-lactamae運送為依賴Tat pathway系統,當Tat pathway組任一基因被破壞後,β-lactamase以前驅物型式滯留於胞內,無法分泌至胞質週緣區,導致菌體失去抗藥性。
Xc17之β-lactamase表現,主要是受到其上游ampR調控。本實驗中發現將ampR突變後,突變株仍可分泌少量之β-lactamase至胞質週緣區,推測缺失AmpR時,Xc17仍有基底量 (basal level)之Bla表現;而提升AmpR表現則會促進Bla表現。在啟動子活性測試上,Xc17的bla啟動子活性會受到ampicillin誘導而上升,而將bla上游啟動子區域一段預測為LysR-motif區域剔除,則會大幅降低bla啟動子活性,推測此為活化態AmpR結合區,供正向調控β-lactamase表現。此外,ampR突變株中,ampR啟動子活性受ampR基因缺失下降,認為AmpR具自我調控ampR基因功能。在clp (CRP-like protein gene)突變株TC817,推測anhMurNAc-tripeptide及UDP-MurNAc-pentapeptide含量極低,AmpR無法調控bla與ampR表現,因此,TC817的Bla表現轉變為誘導型。
Xc17染色體中,包含五個預測為β-lactamase之基因,除了受AmpR所調控之Xcc3039外,其它功能則尚未被證實。本實驗中構築了Xc17Aps突變株,利用互補質體在Xc17Aps菌株中表現Xcc2636、Xcc2873、Xcc3039、Xcc3197以及Xcc3922。經MIC以及酵素活性測試結果顯示,Xcc3039為Xc17主要抗藥性基因,可明顯回復Xc17Aps的抗藥性。其它預測為bla基因,除了Xcc3197具極微弱活性外,Xcc2636、Xcc2873以及Xcc3922並不具有β-lactamase活性。結果說明,除Xcc3039外,其他預測的bla基因在Xc17之抗藥性上不具有重要功能。
URI: http://hdl.handle.net/11455/20711
其他識別: U0005-2008201215350100
Appears in Collections:分子生物學研究所

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