耐輻射奇異球菌PprI蛋白質對大腸桿菌基因調控之蛋白質體研究

Proteomic study of gene regulation by Deira-PprI in Escherichia coli.

蔡淑雅(Tsai shu-ya)

指導教授 ： 張文章

國立臺灣大學/生命科學院/生化科學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.01700

摘要

耐輻射奇異球菌是目前最被廣泛研究的一株耐輻射細菌，以其特殊的耐輻射能力著稱。耐輻射奇異球菌能承受高達5kGy的輻射劑量，且不造成其存活率上的影響。因為地球上從來沒有一處環境擁有如此高的輻射劑量，因此很難以天擇說來解釋耐輻射奇異球菌之耐輻射能力的獲得。有人提出耐輻射能力可能是耐輻射奇異球菌為了適應其他環境逆境，例如:乾燥環境，所意外獲得的能力。因為根據研究，乾燥環境所引發的DNA損傷，包括:DNA 雙股斷裂ヽDNA單股斷裂ヽ及DNA交叉連結(crosslinks) ，與輻射線照射下產生的DNA損傷是相同的。

耐輻射奇異球菌在相同輻射劑量處理下產生DNA雙股斷裂的速率與大腸桿菌是相同的。所以耐輻射奇異球菌並非透過保護DNA免於輻射線傷害，有效的DNA修補機制才是其耐輻射的原因。

近年來文獻資料顯示，PprI被發現是耐輻射奇異球菌DNA修補及保護機置相關的一個蛋白質。將PprI蛋白質表現在大腸桿菌中可以增加大腸桿菌耐γ-輻射線的能力。

為了驗證PprI蛋白質是否真的能增加其他菌種的耐輻射能力。我們將PprI蛋白質大量表現於大腸桿菌BL21菌株體內，並施以1.3 kGy X光照射或五天的乾燥處理。實驗結果卻不同於以往的研究報告，我們發現在有PprI蛋白質表現的大腸桿菌在1.3kGyX光照射及五天的乾燥實驗中降低了存活率。另外，在一些清除自由基的酵素活性研究上，例如:SOD及catalse，我們也發現在PprI蛋白質表現下，大腸桿菌的SOD和catalase的酵素活性有減低的趨勢。二維電泳分析實驗及質譜分析結果顯示，PprI表現下的大腸桿菌體內，有18個蛋白質的表現量受到PprI的影響，其中有13個蛋白質表現量大於1.5倍以上; 5個蛋白質表現量下降至0.6倍以下，這些蛋白質的功能包括:轉錄ヽ轉譯ヽ訊息傳遞ヽ碳水化合物的代謝ヽ後修飾作用ヽ胺基酸的運送子…等等。

PprI表現下的大腸桿菌體內，有一些與胺基酸的運送和ATP生成相關的蛋白質表現量的下降，以及SOD與catalase活性的降低，可能是大腸桿菌抗輻射能力降低的原因。

關鍵字

耐輻射奇異球菌 ； 耐輻射性

並列摘要

The polyextremophile bacterium Deinococcus radiodurans is most famous for its extraordinary resistance to ionizing radiation. Exponential-phase cultures of D. radiodurans can survive exposure to γ-irradiation at dose as high as 5 kGy without loss of viability or evidence of DNA damage. It is hard to rationalize the extreme radioresistance of D. radiodurans in terms of natural selection, because there has been no high radiation natural environment on earth that would exert selection pressures on microbes to evolve into such a high radioresistant strain. It has been proposed that the radioresistance may be a coincidence with the adaptation to desiccation. The process of desiccation may introduce a substantial number of DNA double-strand breaks, single-strand breaks, and DNA crosslinks that are also observed following exposure to ionizing radiation.

Recent evidences have shown that measurable double-strand breaks form at the same rate in E. coli and D .radiodurans if cultures are irradiated under identical conditions. So the radioresistance of D. radiodurans can not result from prevention of DNA damage but from efficient DNA damage repair system.

PprI, a newly identified gene switch, is responsible for DNA damage repair and protection pathways in response to radiation stress in D. radiodurans. Expression of D. radiodurans PprI (Deira-PprI) can also enhance the resistance of E. coli to radioresistance.

To evaluate whether PprI also induces radioresistance in other organisms, Deira- PprI was overexpressed in E. coli BL21 (DE3) followed by a high dose x-ray or desiccation treatment. The complemented E. coli strain decreased the radioresistance to 1.3 kGy x-ray treatment or 5-day desiccation. Overexpression of PprI also showed a decrease of SOD and catalase activity in E. coli. By using two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometry, we found that 18 proteins’ expression levels were affected by the overexpression of PprI. The expression levels of 13 proteins showed >1.5- fold increase, other 5 proteins dropped to <60 % of the control. These proteins exhibited various cellular functions, including transcription, translation, signal transduction, carbohydrate metabolism, post-translational modification, amino acid transport etc.

Decrease of expression level of proteins which function as amino acid transporter or participate in carbohydrate metabolism and the decrease of enzyme activities of SOD and catalase could be the reason why PprI could not enhance the radioresistance of E. coli.

並列關鍵字

Deinococcus radiodurans ； PprI ； radioresistance

參考文獻

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