柑桔潰瘍病原菌第四型線毛構造基因：pilA輿pilA'之定性

Abstract

在革蘭氏陰性菌中， type IV PIlus 是一個位於菌體表面上，具有多種功能的線狀附屬物，參與的功能如菌體的運動方式，胞外蛋白質的分泌，噬菌體的威染以及調節菌體與寄主細胞間的連結等。先前實驗室利用轉位子突變法，得到無法被線狀噬菌體 cf 威染的突變株 XT501 ，轉位子 Tnstad 破壞的基因是 type IV PIlus 主要的構造基因 pilA 。同時利用 piIA 基因為探針，從柑桔潰瘍病原菌基因庫中選殖出一個 7 . 6kb 的 DNA 片段。本實驗將 7 . 6 kb 的 DNA 片段定序並分析後，從中比對出八個開放閱讀框架，其中第六個開放閱讀框架與 Pi - A 具有相當高的相同度（ 67 % ) , 因此命名為 pjlA ’基因，利用 marke 「 exchange 的方法破壞該基因，得到突變株 XcKA 一 1 ，分別利用北方墨點法及 RT 一 PCR 偵測 pilA ’基因是否有轉錄產物，結果顯示 pilA ，基因在野生型 XW47 中有微弱的表現，並且觀察該突變株被噬菌體感染情形、致病性、分泌胞外多醣體及蛋白質能力，結果與野生型 XW47 無顯著差異，最後利用電子顯微鏡觀察野生型 XW47 , 突變株 XT501 及 XcK 八一 1 皆可觀察到疑似 Pili 的構造。

Type IV pilus is a multi-functional filamentous appendage expressed on the surface of gram-negative bacteria. The functions of type IV pili include bacterial motility, secretion of extracellular proteins, phage infection and mediating bacterial adhesion to host cells. Mutant XT501 resistant to the infection of filamentous phage cf, was generated by transposon mutagenesis in the previous study. The gene inserted by transposon Tn5tac1 is pilA, the structural gene of type IV pilus in Xanthomonas campestris pv. citri. A 7.6kb DNA fragment was isolated from the genomic library of X. campestris pv. citri by using pilA DNA as probe. Eight open reading frames were identified in the 7.6 kb DNA fragment after the sequence analysis. ORF6 was named PilA’, due to its predicted amino acid sequence has 67% identity to PilA. A pilA’ mutant (XcKA-1) was generated by marker exchange. Northern hybridization and RT-PCR analysis were used to investigate the expression of pilA’. The result showed that pilA’ is only expressed at a very low level comparing to pilA. Furthermore, the mutation of pilA’ did not affect phage infection, pathogenicity, nor the secretions of extracellular proteins and polysaccharides. Pili-like structure was observed in XW47, XT501 and XcKA’-l by eletron microscopy.