Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20903
標題: Cloning and Analysis of pil gene in Xanthomonas campestris pv. vesicatoria 36
Xanthomonas campestris pv. vesicatoria 36 pil 基因選殖與分析
作者: 柯雅雯
Ko, Ya-Wen
關鍵字: Xanthomonas campestris pv. vesicatoria 36;黃原菌;spot disease;type IV pili;pilA;Pathogenicity tests;phi Xv;pilS-pilR-pilB-pilA-pilC-pilD-orfX;primary receptor;甜椒斑點病;第四型纖毛;互補試驗;病原性測試;線狀噬菌體;電孔法;初級接受器
出版社: 分子生物學研究所
摘要: 
Xanthomonas campestris is a species of gram-negative, plant pathogenic bacterium possessing single polar flagellum. This species is divided into more than 123 pathovars on the basis of the plant hosts they infect. X. campestris pv. vesicatoria is the pathogen causing spot disease in peppers and tomatoes. Type IV pili have been found in X. campestris pv. hyacinthi and X. campestris pv. vesicatoria 3240. Recently, in our laboratory, type IV fimbrial biogenesis genes of X. campestris pv. campestris 17 (Xc17), thought to serve as the primary receptor for filamentous phage infection, have also been identified. These genes are organized into a cluster, pilS-pilR-pilB-pilA1-pilA2-pilC-pilD. To understand the role X. campestris pv. vesicatoria pili might play in filamentous phage Xv infection, the pilA gene cluster was isolated from a local strain Xv36 by chromosome walking via a procedure including i) cloning of a pilD gene segment from Xc17 into the E. coli plasmid pOK12 that cannot be maintained in Xanthomonas, ii) electroporation of the resultant plasmid into Xv36, allowing for single crossover to integrate the whole plasmid through the homologous pilD segment, and iii) cutting the chromosome of the transformant with a restriction endonuclease that does not cut the plasmid, followed by self-ligation of the fragment by treatment with T4 ligase. Using this strategy, a 13-kb BamHI fragment containing the genes adjacent to pilD was cloned. Sequence analysis revealed one incomplete and nine complete open reading frames. These genes can be divided into three groups. The first group includes the incomplete and two complete orfs whose deduced amino acid sequence shows similarity to that of NAD+ synthetase (bp 1-711), sucC (bp 2,407-1,530), and sucD (bp 3,598-2,432), that are associated with energy metabolism. The second group contains genes homologous to pil genes: pilS (bp 3,763-5,443), pilR (bp 5,769-7,163), pilB (bp 9,111-7,381), pilA (bp 9,673-9,248), pilC (bp 10,015-11,274) and pilD (bp 11,384-12,138). The third is an unknown open reading frame, orfX (bp 12,152-12,754). The deduced PilA exhibits several structural features typical of type IV fimbrillins, including the presence of the basic leader sequence MKKQNG followed by a sequence of GFTLIE recognized by prepilin leader peptidase. Since the genome organization of the Xv36 pil cluster is different from that of Xc17, it was interesting to dissect whether the other vesicatoria strains have the same pil gene organization. For this purpose, a pair of primers were synthesized according to the pilA upstream and downstream sequences and used to PCR-amplify the corresponding region in 56 other X. campestris pv. vesicatoria strains. The results showed that a PCR product of 1.3 kb, with the same size as that in the Xv36 chromosomal region, was amplified on all the templates. Mutation in pilA and pilB, generated by insertional mutation, results in a phage-resistant phenotype, and phage-sensitivity is restored by cloned wild-type gene in complementation tests. While Xc17 and Xv3240 are both resistant to infection of Xv, Xc17 and Xv3240 carrying cloned Xv36 pilA gene are sensitive to the Xv36 pilus-specific phage Xv. These data suggest PilA to be the determinant for infection of the Xanthomonas filamentous phages. Pathogenicity tests showed that mutation in pilA or pilB has no apparent effect on the ability of Xv36 to cause spot disease in peppers.

Xanthomonas campestris 為一革蘭氏陰性植物致病菌,具有單極單鞭毛。 根據不同宿主分成超過 123 小種。 X. campestris pv. vesicatoria 36 為 X. campestris 之一病原小種,會造成甜椒或蕃茄斑點病。 在 X. campestris pv. hyacinthi 及 X. campestris pv. vesicatoria 3240 已找到 type IV pili,本實驗室近來也找到 X. campestris pv. campestris 17 (Xc17) type IV pili 及有關 pili 生合成基因。 此型 pili 被認為作為線狀噬菌體感染時初級接受器,此點已被證實。 這些 pil 生合成基因組成一 cluster,其排列組合為 pilS-pilR-pilB-pilA2-pilA1-pilC-pilD。 為了了解 X. campestris pv. vesicatoria 的 pili 在線狀噬菌體 Xv 感染時所扮演的角色,利用 chromosome walking 的方式,選殖出本土 Xv36 的 pilA 鄰近基因,實驗過程包括,一、選殖一含 Xc17 pilD 基因之片段於可在 Escherichia coli 內進行複製,但無法在 Xanthomonas 中複製之質體 pOK12。 二、將此質體以電孔法送入 Xv36,利用 pilD 同質性進行 single crossover。 三、利用限制酵素將pOK12 鄰近片段作切割,並使片段進行自我粘接,得到一質體。 內含一13-kb BamHI 片段,包括 pilD 及其鄰近的基因。 經定序分析,找到一個不完整的 ORF 及九個 ORF,這些基因可分成三群,一、與能量有關之基因;NAD+ synthetase (bp 1-711) 與 sucC (bp 2,407-1,530)、sucD (bp 3,598-2,432)、第二群為與 pil 基因串相似之基因;包含 pilS (bp 3,763-5,443)、pilR (bp 5,769-7,163)、pilB (bp 9,111-7,381)、pilA (bp 9,673-9,248)、pilC (bp 10,015-11,274) 及 pilD (bp 11,384-12,138)。 第三群為一未知功能的 open reading frame 稱為 orfX (bp 12,152-12,754)。 pil 基因串之胺基酸序列比對分析,結果發現 PilA 具有典型type IV pili 特徵,N-端六個帶正電胺基酸形成 leader peptide,序列為 MKKQNG,其後接著 GFTLIE 為 prepilin leader peptidase 所辨識一序列。 由於 Xv36 pil 基因串基因組合與 Xc17 不一樣。 因此想了解其他茄科斑點病菌小種是否也也有相同 pil 基因組合。 根據 Xv36 已知序列在 pilA 上游處 (pilC 之 5’ 端) 及下游處 (pilB 之 5’ 端) 設計引子,與 56 株 Xv 之染色體進行 PCR,結果發現這 56 株 PCR 之產物大小約 1.3 kb 與 Xv36 一樣。 將 pilA 與 pilB 分別以 insertional mutation 方法構築突變株,進行 phage sensitivity 測試,此兩突變株對 Xv 呈現抗性。 在互補試驗中,將 wild type pilA或 pilB 基因送入突變株則恢復 Xv sensitive 性狀。 改將 Xv36 pilA 送入 Xc17 及 Xv3240,可使此兩菌株對 Xv 由 resistant 變為 sensitive。 這結果表示PilA 為線狀噬菌體感染之所需。 在病原性測試中,pilA或 pilB 的突變株對甜椒的致病性與野生株沒差異。
URI: http://hdl.handle.net/11455/20903
Appears in Collections:分子生物學研究所

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