Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21589
標題: 供噬菌體 phiL7 感染所需之寄主 Xanthomonas campestris pv. campestris 基因及線狀噬菌體 phiLf
Xanthomonas campestris pv. campestris Genes Required for Phage phiL7 Infection and Determination of Filamentous Phage phiLf pIII Region Responsible for Host Recognition
作者: 洪志勳
Hung, Chih-Hsin
關鍵字: phage;噬菌體;adsorption;Xanthomonas campestris pv. campestris;lipopolysaccharide;phage resistant;host specificity;xanA;siderophore;吸附蛋白;十字花科黑腐病菌;脂醣體;噬菌體抗性;寄主專一性;xanA;嵌鐵蛋白
出版社: 分子生物學研究所
摘要: 
革蘭氏陰性的 Xanthomonas campestris pv. campestris 為十字花科黑腐病菌 (又稱黃原菌)。 其所產生的大量胞外黏多醣 (xanthan gum) 已被廣泛應用於農業、石化業及食品業上,作為穩定劑、增稠劑、乳化劑、及懸浮劑之用。 phiL7 及 phiLf 為專一感染本菌之噬菌體。 phiL7,屬於烈性噬菌體 (virulent phage),基因體含有約 37 kb 之雙股DNA; 頭部為六角體,直徑約 60 nm,具不收縮而微彎的尾巴,約為 10 ´ 180 nm。 phiLf 為一線狀噬菌體,大小約為 1000±200 ´ 8 nm,包含單股環狀,長 6008 nt DNA之基因體。 其繁殖方式並不會將寄主溶裂。
為了解 phiL7 感染寄主的機制,本研究分析兩株以 Tn5 誘變,能抵抗 phiL7 感染之X. campestris pv. campestris突變株。 其中一株在 xanA 基因上被插壞,另一株則插在 tonB 基因上。
xanA 基因所主導之蛋白為一雙功能之酵素 phosphoglucomutase/phosphomannomutase, 為胞外多醣合成路徑上前驅物 Glu-1-P 及 Man-1-P 的合成所需,因而 xanA 突變株較不黏溼。 xanA 突變株在點測試 (spot test) 中需 36 小時後才會出現混濁溶菌斑 (turbid plaques),而與野生株在 1.5 小時後即出現透明溶菌斑 (clear plaques) 之情形不同。 以正常 xanA 基因對此突變株進行互補,即可得到與野生株相同之透明溶菌斑。 以噬菌體吸附的方式證實,xanA 突變株對噬菌體 phiL7 的吸附能力比野生株降低約 1500 倍。 由於以胞外多醣突變株 (在不同基因突變者),當對照組進行測試,發現對 phiL7 皆仍敏感 (sensitive),可見胞外多醣並非 phiL7 的感染所需。 從結果推斷 phiL7 的受體為一複合受體 (complex receptor), LPS 為其成分之一。
tonB 所主導的是內膜蛋白,與鐵離子之吸收及病原性有關, 將其上下游基因加以定序分析後,發現其下游包含三個 ORFs,分別為 exbB,exbD1 及 exbD2。 插入致變之結果發現 exbB,exbD1 亦是 phiL7 的感染所需之基因。 以北方雜交 (Northern hybridization) 分析可偵測到 2.4 kb的轉錄抄本 (transcript),其長度相當於 tonB 、exbB、exbD1 及 exbD2 四個基因之總長。 另外以 primer extension 定出轉錄起點位於 tonB 上游第 79 nt 處。 以啟動子偵測方式 (promoter-proving assay),發現只有在 tonB 上游有啟動子,因此可判斷 tonB-exbB-exbD1-exbD2 為一個操縱子 (operon)。 tonB 、exbB、exbD1 基因應為噬菌體穿入 (penetration) 所需而非吸附所需,乃是基於 1) 突變株在 phiL7 吸附能力上與野生株並無差異;2) 而以電孔法 (electroporation) 將 phiL7 genome DNA 送入該等突變株中,則仍能使之釋放出具有感染性之噬菌體。
本論文第三部分乃研究 phiLf 與 phiXv (X. campestris pv. vesicatoria 之線狀噬菌體) 之 pIII (吸附蛋白) 上與寄主辨認有關之區域。 將 phiXv 的吸附蛋白 pIII 之 N 端 154 個胺基酸,以 phiLf 之 pIII N 端的 164 個胺基酸取代後,此重組噬菌體 (phiXvfIII) 即改變其寄主專一性,變成可感染 X. campestris pv. campestris 而不感染其原來的寄主。 由此可見,pIII 上決定噬菌體寄主專一性的胺基酸位於其 N 端。

The Gram-negative plant pathogenic Xanthomonas campestris pv. campestris is the causal agent of black rot in crucifers. The exopolysaccharide (EPS) has a variety of applications in agriculture, petroleum production, cosmetics, and food industry as a stabilizing, viscosifying, emulsifying, thickening, and suspending agent. phiL7 and phiLf are phages that specifically infect X. campestris pv. campestris. phiL7, a virulent phage with a double-stranded DNA genome of 37 kb, has a hexagonal head that measures 60 nm in diameter and a noncontractile tail that is about 10 180 nm. fLf, a filamentous phage of 1000200 8 nm in size with a single-stranded, circular DNA genome of 6,008 nt, propagates without lysis of the host cells.
To understand phage-host interactions, two phiL7-resistant mutants isolated from strain X. campestris pv. campestris 17 by Tn5 mutagenesis were studied. One was mutated in xanA gene and the other in tonB gene.
The xanA gene has previously been shown to code for the bifunctional phosphoglucomutase/phosphomannomutase required for the synthesis of precursors, Glu-1-P and Man-1-P, for the biosynthesis of both exopolysaccharide and lipopolysaccharide (LPS). Therefore, the xanA mutant is non-mucoid and rough in morphology. In spot tests with phiL7, lysis zones can be formed in 1.5 hr on the lawn of the wild-type cells. In contrast, it took 36 hr for turbid lysis zones to appear on the lawns of the xanA mutant cells. The ability to form the wild-type clear lysis zones was restored by complementation with the wild-type xanA gene. The adsorption efficiency of xanA mutant is decreased by 1,500-fold compared with that of the wild-type cells. EPS mutants are still sensitive to phiL7, indicating that EPS is not required for phiL7 infection. These data together suggest LPS is one of the components forming the complex receptors for fL7.
The tonB is the gene previously shown to code for an inner membrane protein required for iron uptake and pathogenesis. Sequencing of the DNA fragment containing tonB, revealed three downstream genes, exbB, exbD1 and exbD2. Insertional mutation showed that exbB and exbD1 are also required for phiL7 infection. Northern hybridization detected a transcript of 2.4 kb, equivalent to the sum of the four genes, with a transcription initiation site located 79 nt upstream of tonB as determined by primer extension. Promoter-proving assay showed only one promoter located upstream of tonB. These data together indicate that they are organized into an operon, tonB-exbB-exbD1-exbD2. It is suggested that the tonB, exbB, and exbD1 genes are required for penetration instead of adsorption, based on the observations that 1) no differences in the efficiency of phiL7 adsorption were found between the wild-type and mutant cells, and 2) the mutant cells were able to release infective phage particles upon electroporation with the phiL7 genomic DNA.
The third part of this study was to determine the region of pIII (the adsorption protein) of phiLf and phiXv (a filamentous phage of X. campestris pv. vesicatoria) that is responsible for host recognition. For this purpose, the N-terminal 154 amino acid residues of the phiXv pIII were exchanged with the N-terminal 164 residues of phiLf pIII. The resultant recombinant phage, phiXvfIII, is able to infect X. campestris pv. campestris, but not X. campestris pv. vesicatoria. These data indicate that the region controlling host recognition locates in the N-terminal third of the pIIIs.
URI: http://hdl.handle.net/11455/21589
Appears in Collections:分子生物學研究所

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