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標題: The Xanthomonas campestris pv. campestris genes required for phages fLf and fL7 infection at early steps
噬菌體fLf與fL7感染寄主Xanthomonas campestris pv. campestris 初期 所需之寄主基因
作者: 楊奇凡
Yang, Chi-fan
關鍵字: phage;噬菌體;infection;Xanthomonas campestris pv. campestris;感染;十字花科黑腐病菌
出版社: 分子生物研究所
Xanthomonas campestris pv. campestris (簡稱 Xc) 是十字花科植物
黑腐病之病原菌, 屬於革蘭氏陰性菌, 菌體呈短直桿狀, 具單極生鞭毛。
由於會產生黃色素及胞外黏多醣, 菌落外觀呈黃色黏濕狀。 苯實驗室曾
分離出兩種能專一感染十字花科黑腐病菌 Xanthomonas campestris pv.
campestris 的噬菌體: 烈噬性噬菌體fL7 (virulent phage) 與線狀噬菌
體 fLf (filamentous phage)。為了研究 fLf 感染寄主之機制, 本實驗
係利用mini-Tn5 mutagenesis 方法, 期望藉由 phage fLf-resistant 突
變株的構築與研究, 找出 fLf 感染所需之寄主結構或運輸系統。 結果篩
選出五株不會產生溶菌斑 (plaque) 的突變株, 從中選取一個突變株
FR26 作進一步分析。 當 fLf 以 MOI=0.0001 自然感染突變株 FR26 時,
菌的生長並不受影響, phage titer 也無明顯增加, 顯示 FR26 並不會被
fLf 感染。 但是 fLf 的 RF DNA 以電孔法 (electroporation) 送入
FR26 菌體後, FR26 能夠持續地製造並釋放 fLf, 故推論突變株 FR26之
所以不受 fLf 感染, 係由於噬菌體感染侵入菌體時所需之寄主構造受到
破壞。 接著將相當於 FR26 中 miniTn5Tc 插入點附近之染色體片段選殖
出, 已完成定序的 4.8 kb 部份含有 4 個可能的 open reading frame
(ORF): ORF268, ORF398, ORF223, 和 ORF253。 利用 marker exchange
(或稱 gene replacement) 分析, 顯示ORF268, ORF398, 與 ORF223 均與
fLf 的感染有關。 將這些 ORF 的核酸序列轉譯成氨基酸序列後, 以電腦
比對分析, 顯示可能分別為 lipoprotein, outer membrane /
periplasmic protein, 及 inner membrane protein。 但是目前尚無法
將 fLf-resistant 突變株互補回 wild type (fLf-sensitive) 特性。而
在此三個與 fLf 的感染有關之 ORF 下游的 ORF253, 則發現是與 fL7 感
染有關, 分析其蛋白產物可能位於 inner membrane。 將 ORF253 以 E.
coli S30 表現系統所產生之蛋白產物, 其大小亦與預測相符。 ORF253
與 E. coli 及 Pseudomonas aeruginosa 的 TolQ 分別有 30% 及 34.3%
identity。 並且, 將帶有此 ORF253 之重組質體 pTDX 經電孔法送入
fL7-resistant 突變株後, 可將之完全互補回 fL7-sensitive。
The gram-negative bacterium Xanthomonas campestris pv.
campestris is the phytopathogen causing black rot in cruciferous
plants. This bacterium, with yellow pigment, short rod shape,
and one polar flagellum, can synthesize great amounts of
exopolysaccharide, xanthan gum, rendering the colonies yellow
and mucoid. Two bacteriophages that specifically infect
Xanthomonas campestris pv. campestris have been isolated:
virulent phage fL7 and filamentous phage fLf. In order to
investigate the mechanism involved in fLf infection, efforts
were made to search the host structure forming a transport
system required for fLf infection. By mini-Tn5 mutagenesis, five
phage fLf-resistant mutants that prevented plaque formation was
constructed. FR26 was chosen from these mutants for further
studies. At multiplicity of infection of 0.0001, the growth of
FR26 was not affected, and no increase in phage titer was
observed. These results suggest that FR26 was resistant to fLf.
Electroporation of the fLf RF DNA into FR26 resulted in release
of phage progeny efficiently, indicating that the phage fLf-
resistance phenotype of FR26 is due to the blocking at the early
step of fLf infection.The chromosomal fragment of Xc11P20H (6.8
kb) containing the regionof the mini-Tn5Tc insertion in FR26 was
cloned. Sequence analysis on the 4.8-kb region reveled four open
reading frames: ORF268, ORF398, ORF223, and ORF253, encoding
three proteins which were predicted to be lipoprotein, outer
membrane/periplasmic protein, and inner membrane protein,
respectively, according to the analysis of the deduced amino
acid sequence by computer program PSORT. Analysis with marker
exchage (or gene replacement) indicated that ORF268, ORF398, and
ORF223 are involved in fLf infection. However, fLf-sensitive
phenotype was still not restored to FR26 by these ORFs.
Downstream to these open reading frames involved in fLf
infection, an ORF (ORF253), whose product was predicted to be
located in inner membrane, was found to be required for fL7
infection. Expression of ORF253 in vitro using E. coli S30
transcription/translation system produced a protein of 25 kDa
which is similar to the MW deduced from nucleotide sequence.
ORF253 showed 30% and 34.3% identities in amino acid sequence to
E. coli TolQ and Pseudomonas aeruginosa TolQ, respectively.
Phage fL7-sensitive phenotype was restored by pTDX, containing
ORF253, after being electroporated into the fL7-resistant mutant
Appears in Collections:分子生物學研究所

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