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

• Main Authors: Tsai shu-ya, 蔡淑雅
• Other Authors: 張文章
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/71028343720119415799

碩士 === 國立臺灣大學 === 生化科學研究所 === 94 === 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.