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標題: 調控噬菌體溶裂效應的 phiL7 p24-p25-p26基因之探討
Functional investigation of phiL7 p24-p25-p26 genes in lysis effect
作者: 游佳誠
Yu, Chia-Cheng
Contributors: 翁淑芬 教授
分子生物學研究所
關鍵字: 溶裂型噬菌體;轉錄調節子
lytic phage;transcriptional regulator
日期: 2012
Issue Date: 2013-11-07 12:20:48 (UTC+8)
Publisher: 分子生物學研究所
摘要: 革蘭氏陰性嗜氧菌Xanthomonas campestris pv. campestris (簡稱Xcc)為十字花科蔬菜黑腐病之病原菌。先前研究已知結論;(1) 噬菌體 phiL7為專一感染 Xcc 的溶裂型噬菌體,屬於 Siphoviridae 科,(2) 序列分析顯示 phiL7具有雙股線型 DNA,內含44,080 bp,60個ORFs。(3) phiL7的 p24~p26為極早期轉錄之基因,經由生物資訊分析比對,發現其它 Xanthomonas phage 具有與 P24相似的蛋白,但功能註解為 hypothetical protein;而 p25與p26未能比對到相似的基因。 p27~p29.1為溶裂基因串,(4) p27、p28、p29與29.1產物分別具有類似 holin、lysozyme、Rz 與Rz1 功能,(5) Xcc 如果存在包含完整 p24基因的質體時,菌體不會被 phiL7溶裂。本研究目的為探討噬菌體 phiL7早期基因 p24~p26在溶裂過程中所扮演的角色。首先構築包含 p24~p26不同基因組合與長短之重組質體,發現包含完整 p24 基因的轉殖株,會抑制 phiL7溶裂菌體,但包含 p24~ p26片段的 Xcc菌體則仍然會被噬菌體溶裂。為了解 p24與 p26 的產物為蛋白質或 RNA,分別在目標基因的 coding region 中製造 nonsense mutation。p24的 nonsense mutation會失去抑制菌體被溶裂的功能;但p26的 nonsense mutation 並不會影響其效應,顯示 p24產物具有蛋白質層面的影響,而 p26則可能為 RNA 層面的效應。針對 p24的功能探討,得到以下結論:(1) p24產物不會影響噬菌體 phiL7 吸附感染宿主的能力,也不會拮抗溶裂蛋白的功能,(2) p24產物可能藉由降低 Xc17 RNA polymerase 與啟動子 P15區域的鍵結能力,達到抑制 P15啟動子的活性;而 p24則可能藉由增加 Xc17 RNA polymerase 與 P38啟動子區域的鍵結能力,活化 P38啟動子的活性,(3) Quantitative RT-PCR 結果顯示 p24可降低 phiL7 晚期基因p15、p19與p28 mRNA之表現量。針對 p26的功能探討,得到以下結論:(1) p24-p25-p26基因以 in cis 形式存在於 Xc17時,該菌體會被 phiL7溶裂;但以 in trans 形式存在時,菌體不會被 phiL7溶裂,(2) In cis形式存在時,p24、p25與 p26 之 mRNA 表現量都相對降低,(3) 將 p26 基因由 3’→5’ 逐步刪除,發現p26第29 ~ 36 bp具有一 “TGTCAATA” 序列,與推測的 p24 基因啟動子之 -35 區域序列完全相同,將 p26的這8個核苷酸突變後,存在包含 p24-p25-p26之菌體就無法被 phiL7溶裂。由目前獲得之實驗結果推測p24為一轉錄調節基因,屬於極早期基因,其所轉錄之產物具有活化某些基因表現,以及抑制某些晚期基因表現之功能。p26會影響 p24基因之表現量,而 p24基因表現量的高低影響菌體是否被溶裂的重要關鍵之一。
The gram-negative plant pathogenic Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in cruciferous plants. Previous studies have shown that 1) phiL7, belonging to Siphoviridae, is a lytic phage that specifically infects Xcc, 2) the genome of phiL7 has a linear double-stranded DNA containing 44,080 bp with 60 ORFs, 3) p24-p26 of phiL7 are immediate-early genes, whose encoded proteins share no similarity with those in database, 4) P27, P28, P29, and P29.1 of phiL7 are similar to holin, lysozyme, Rz and Rz1 from phages, respectively, and 5) cells of Xc17 carrying a plasmid with cloned p24 gene is resistant to lysis by infected phiL7. The purpose of this study was to explore the roles of phiL7 p24-p26 in the lysis of Xc17 cells upon phiL7 infection. Recombinant plasmids containing combinations of complete or truncated genes p24, p25 and p26 were constructed and introduced into Xc17. It was found that the construct with p24 gene alone inhibits the lysis by phiL7 but not the clone containing p24-p25-p26. To understand the functions, nonsense mutation in the coding regions of p24 and p26 were constructed. Results obtained by testing with these constructs show that non-sense mutation in p24 causes the loss of its inhibitory effect on phiL7 lysis, but not the mutation in p26, indicating that the p24 product exerts its effect at protein level, while p26 may exert the effect at RNA level. Further studies reveal that the p24 product 1) neither affects the adsorption ability of phiL7 nor antagonizes the phage lysis, 2) likely represses promoter P15 by lowering the binding ability of Xc17 RNA polymerase to the promoter region and activates P38 promoter by enhancing the enzyme binding to the promoter, and 3) possibly reduces mRNA levels of phiL7 late genes p15, p19 and p28, as shown by quantitative RT-PCR. Study on p26 function shows that 1) lysis by phiL7 infection occurs when p24-p25-p26 genes are present in cis but not in trans, 2) p24, p25 and p26 mRNA levels are lower when p24-p25-p26 is present in cis, (3) nested deletions of p26 from 3''→5'' showed that "TGTCAATA" sequence at 29-36 nt of p26 is identical to that of -35 block in p24 promoter; mutation of this block abolishes lysis by phiL7 infection. These results together suggest p24 to be a transcriptional regulator whose product can activate or repress the expression of some genes. The level of p24, the factor determining whether the host cell is lysed upon phiL7 infection, in turn is regulated by p26.
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