- 學位論文
Xanthomonas campestris pv. campestris 抗銅基因的表現與調控
Expression and regulation of copper resistance genes in Xanthomonas campestris pv. campestris
摘要
Xanthomonas campestris pv. campestris (Xcc) 為革蘭氏陰性植物病原菌，可感染十字花科植物造成黑腐病 (black rot)，引起農業上的重大損失。含銅的殺菌劑被使用在減少此疾病的散佈已經有好幾十年。在 Xcc 基因體中有一 copLAB 基因串位於染色體上，其序列與 X. axonopodis pv. vesicatoria 和 X. campestris pv. juglandis 的抗銅基因具有同源性。本研究探討 Xcc strain Xc17 的 copLAB 基因串的特性。 首先，以 insertional inactivation 的方式構築 Xc17 的 copLAB突變株，並進行金屬離子感受性分析。結果顯示在含 1.0 mM CuSO4 或 CuCl2 的 LB 培養基中，這些突變株對銅的抗性比 Xc17 低，因此推測，CopLAB 與 Xc17 的銅抗性有關。另一方面，以不同的金屬離子進行感受性試驗，如 FeSO4、FeCl3 和 ZnCl2，由於不同菌株間不具明顯差異，表示 copLAB 為專一性抗銅基因。其次，將 copL 和 copA 上游區域進行啟動子活性分析。由 reporter assays 的結果得知，copL 為持續表現的基因；copA 則為銅誘導表現之基因。此外，copL 轉譯起始點上游 -151 至 -187 的位置有一疑似 repressor 的結合區域，而 -151 至 -126 的位置則為 copL 與 copA 表現所必須；至於 copA 轉譯起始點上游 -28 至 +147 的位置以及完整的 copL 基因片段，則為 copA 受銅誘導表現所必須。 關鍵詞：黑腐病菌，抗銅性，銅誘導
並列摘要
The Gram-negative plant pathogenic Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers, a disease causing tremendous loss in agriculture. Copper-containing bactericides have been used for several decades to reduce the severity of this disease. In the fully sequenced Xcc genome, there is a chromosomal locus, copLAB, which shares nucleotide sequence homology with copper resistance genes from X. axonopodis pv. vesicatoria and X. campestris pv. juglandis. In this study, the copLAB gene cluster in Xcc strain Xc17 was characterized. First, the copLAB mutants were isolated from Xcc wild type strain Xc17 by insertional inactivation and metal ion susceptibility was evaluated. The resultant mutant strains exhibited reduced copper resistance on LB medium containing CuSO4 or CuCl2 at 1 mM using Xc17 for comparison. It was suggested that CopLAB are responsible for copper resistance in this bacterium in our assay condition. Furthermore, there were no differences when other ions were used such as FeSO4, FeCl3, or ZnCl2. It was indicated that the resistance medicated via copLAB is copper specific. Second, nested fragments of the copL and copA upstream region were made to map the location of the promoter elements. Reporter assays indicated that copL is constitutive while copA is copper-inducible. In addition, there is a putative repressor binding site located at -151 to -187 bp relative to the translation start codon of copL and the fragment containing -151 to -126 is essential for their expression. It was also indicated that the fragment containing -28 to +147 relative to the copA translation start site and the presence of full length copL are necessary for copper induction. Keyword: Xanthomonas campestris pv. campestris, copper resistance, copper induction.