噬菌體 phiL7 的 p24基因產物之功能探討

羅安琦

doi:10.6845/NCHU.2015.00047

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噬菌體 phiL7 的 p24基因產物之功能探討

Functional investigation of p24 gene of phage phiL7

羅安琦 , Masters　　Advisor：翁淑芬　　

繁體中文 DOI： 10.6845/NCHU.2015.00047

溶裂型噬菌體；轉錄調控因子；抗溶裂效應； phiL7 ； transcription regulator ； anti-lysis effect

Abstract │ Reference (40) │ Times Cited (2) │ Altmetrics

Abstract 〈TOP〉

噬菌體 phiL7為一溶裂型噬菌體，可專一感染革蘭氏陰性植物病原菌Xanthomonas campestris pv. campestris 17 (Xc17)造成宿主細胞破裂死亡。前人的研究顯示，(一) phiL7的 p24 基因可以藉由影響Xc17 RNA polymerase 與 啟動子的結合能力來調節某些噬菌體基因的轉錄表現；(二) 包含p24 基因的Xc17 轉殖株具有抗phiL 7 溶裂的現象；(三) p24基因會受到 p26 基因內的 “TGTCAATA”序列(命名為R2) 調控，此序列與預測的p24基因啟動子之 -35 區域(命名為R1) 完全相同；(四) 當R1與R2 in cis存在時，p24基因抗溶裂的效應即被移除，轉殖株會正常被噬菌體溶裂。本研究目的為進一步了解 P24 如何參與調控噬菌體基因之表現，以及 p24基因的轉錄調控。實驗結果顯示，(ㄧ) 能表現p24基因之Xc17轉殖株不會被phiL7溶裂，但被感染後，菌株仍然可以複製並釋放出少量的噬菌體子代；以p29 溶裂蛋白的抗體進行西方墨點法測試，發現在被感染的轉殖株中無法偵測到溶裂蛋白之表現；(二) 將p35 (主導產生 phiL7 RNA polymerase) 基因與 p24基因共同轉殖表現於 Xc17中，再經以phiL7 感染，發現P35的表現可以消除p24的抗溶裂效應；(三) 由啟動子活性測試的結果發現 P35 可以抑制 phiL7 P56 啟動子的活性，但不影響P35與P15啟動子的活性；(四) P24與 P35共同表現可以活化 P15、P23、P38、P35；(五)為了解R1 與R2的調控作用，構築了包含R1 and/or R2 序列突變的質體，轉殖送入Xc17後，以phiL7感染。發現，點突變後的R1仍具有啟動子活性且不再受 R2 序列影響，而原始或突變的R2序列後仍可以影響且抑制原始之R1序列的效應；(六) 經由生物資訊分析結果發現， Xanthomonas phage ?Xop1 的 ORF24，Xop411 的P25及 Xp10的 P25與 phiL7 的P24具高序列相同度。以phiL7感染表現 Xop411 P25之Xc17 轉殖株，發現Xop411 P25亦能拮抗 phiL7 溶裂宿主。總結上述結果，(一) P24為ㄧ轉錄調節因子，能分別和host RNAP與 phiL7 RNAP 作用；(二) phiL7 RNAP 必須藉由 P24幫助才能調控某些噬菌體基因的轉錄；(三) 有些Xanthomonas phage中也有類似 P24的調控因子。

Parallel Abstract 〈TOP〉

phiL7 is a lytic phage specifically infecting the gram-negative Xanthomonas campestris pv. campestris (Xcc) that causes black rot in crucifers.  Previous studies have shown that 1) p24 gene of phiL7 is able to regulate transcription of several phage genes by altering the binding affinity of the host Xc17 RNA polymerase to the promoters, 2) Xc17 with cloned phiL7 p24 gene, designated Xc17(p24), is not lysed upon phiL7 infection, 3) the expression level of p24 gene is regulated by a “TGTCAATA” sequence (termed R2), which located in p26 gene and is identical in nucleotide sequence to the -35 region of the putative p24 promoter (termed R1), and 4) lysis by phiL7 infection occurs when R1 and R2 are present in cis.  In this study, it was shown that 1) although no lysis was observed for Xc17(p24) upon phiL7 infection, a low titer of phage progeny was still released, 2) in Xc17(p24), expression of lysis protein P29 was repressed as shown in Western blotting, 3) when p35 gene (encoding phiL7 RNA polymerase) was cloned into Xc17(p24), the anti-lysis effect of P24 was abolished, 4) in vivo transcription assay revealed that P35 could repress P56 promoter but not the P35 and P15 promoter, and P35 appeared to interact with P24 to activate P15, P23, P35, and P38 promoter.  To understand the regulation of R1 and R2 sequence, DNA fragment with point mutations in R1 and/or R2 sequence were constructed and transformed into Xc17, followed by phiL7 infection.  Results showed that the promoter activity of R1 sequence was not affected by the mutations, but R1 was no longer regulated by R2.  Data also showed that the wild-type or mutated R2 sequence could still inhibit the wild-type R1 sequence.  Bioinformatic analysis showed that phiL7 P24 has closely related homologues from Xanthomonas phages Xcp1, Xop411, and Xp10.  It was shown that Xc17 with cloned Xop411 P25 was not lysed upon phiL7 infection, suggesting that Xop411 P25 has the analogous function as the phiL7 P24.  In summary, these results suggest that 1) P24 is a transcription regulator that can interact with both phiL7 RNAP and host RNAP, 2) phiL7 RNAP needs P24 to work together to transcribe some phage genes, and 3) other Xanthomonas phages also have a regulator similar to the phiL7 P24.

Reference ( 40 ) 〈TOP〉

李佳霓. 2006. Xanthomonas 溶裂型噬菌體 phiL7與 Xo411之探討. 國立中興大學分子生物學研究所博士論文
連結：
蔡任濠. 2010. 噬菌體 phiL7 溶裂基因串之功能探討. 國立中興大學分子生物學研究所碩士論文
連結：
游佳誠. 2012. 調控噬菌體溶裂效應的 phiL7 p24-p25-p26基因之探討. 國立中興大學分子生物學研究所碩士論文
連結：
簡旭玲. 2007. 十字花科黑腐病菌聚半乳糖醛酸酶基因 pglA 之調控. 國立中興大學分子生物學研究所碩士論文
連結：
Ackermann, H.W. (2001). Frequency of morphological phage descriptions in the year 2000. Brief review. Archives of virology 146, 843-857.
連結：

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