Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22040
標題: Xanthomonas fragariae 細菌素之純化與特性探討
Purification and Characterization of Bacteriocin from Xanthomonas fragariae
作者: 林平國
Lin, Ping-Kuo
關鍵字: bacteriocin;細菌素
出版社: 分子生物學研究所
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摘要: 
細菌素大都是由蛋白類物質所組成,具有抑制親緣性相近菌屬生長的功能,本研究嘗試純化Xanthomonas fragariae (Xf) 細菌素並分析其特性。Xf 是感染草莓的植物病原菌,所產生的細菌素對於許多Xanthomonas菌屬都具有抑菌活性。在研究中將 Xf 菌株培養至穩定生長期時抑菌活性最佳,在純化上以PEG 6000 沉澱胞外蛋白,由Sephacryl S-300 HR分子篩管柱或蔗糖梯度離心進行細菌素蛋白純化,由分子篩管柱純化結果,推測 Xf 細菌素分子量大於290 kD,因此Xf 細菌素屬於大分子量的細菌素蛋白。純化後的Xf細菌素蛋白對溫度最高耐受度為40 °C,且對chymotrypsin及trypsin蛋白酶明顯具有抵抗性,但活性會受到proteinase K的抑制。經由SDS-PAGE分析,發現純化後Xf 細菌素蛋白是由許多蛋白次單元所組合而成,取其中的60 kD、40 kD、36 kD、32 kD及22 kD五個蛋白條帶進行液相層析串聯質譜儀 (LC/MS/MS) 分析比對,發現這些蛋白都含有與噬菌體P2相關的胺基酸序列。藉由穿透式電子顯微鏡觀察Xf 細菌素的構造及型態,顯示此細菌素類似phage tail-like bacteriocin,呈桿狀主要由鞘膜 (sheath)、核心蛋白 (core domain) 及尾絲 (tail fibers) 三大部份所組成,而Xf 細菌素尾端的長度、直徑及寬度都與P2 phage 及P2 phage tail-like bacteriocin接近且外形相似。利用質譜分析所得的胜肽片段設計退化性引子,經 PCR 反應得到之 DNA 片段作為探針,從構築的 Xf 基因庫中篩選出四個質體,分別命名為pBXf-p40、pCXf-p40、pEXf-p40及pXf-p40 。經由序列比對分析後,發現目前篩選出來的片段中含有15個ORFs,相關基因並形成一基因串。由目前定序的結果得知,此基因組是由lysis cassette、主要及次要的結構蛋白所組合而成,這些蛋白經胺基酸序列比對,結果發現 Xf phage tail-like bacteriocin 與 Yersinia pseudotuberculosis IP 32953 的 P2 phage 基因組序列最為相似,其次為與 Yersinia mollaretii ATCC 43969 、 Yersinia frederiksenii ATCC 33641 的 P2 phage 基因組序列。

Bacteriocin are proteins in nature with bactericidal activity towards the same or closely related species of bacteria. This study describes the purification and characterization of a phage tail-like bacteriocin from the plant pathogenic bacterium
Xanthomonas fragariae (Xf). The bacteriocin of Xf has antimicrobial activity toward most of the tested Xanthomonas species. Maximal bacteriocin activity was obtained in the culture broth of early stationary phase of growth. The bacteriocin produced by Xf was purified from culture supernatant by polyethylene glycol 6000 precipitation, Sephacryl S-300 HR gel-filtration chromatography and sucrose density gradient centrifugation. The protein with bacteriocin activity purified from sephacryl S-300 gel filtration chromatography was found to have a molecular weight higher than 290 kD. The purified bacteriocin of Xf was stable at 40℃. The Xf bacteriocin was sensitive to proteolytic enzyme such as trypsin and chymotrypsin, but sensitive to proteinase K digestion. SDS-PAGE analysis indicated that Xf bacteriocin is composed of several protein subunits and exists as a protein complex. The five intense protein bands, 60 kD、40 kD、36 kD、32 kD and 22 kD, were chosen for LC/MS/MS analysis. By searching the MS/MS spectra against the protein database, these protein bands were found to relate to the bacteriophage P2 proteins. In consistent with this, electron microscopy of the purified Xf bacteriocin revealed the presence of a phage tail-like structure with sheath, core domain, and tail fibers. The longitudinal lengths and diameters of Xf bacteriocin were similar to bacteriophage P2 and P2 phage tail-like bacteriocins. By using the identified peptide sequences from LC/MS/MS analysis, degenerate oligonucleotide primers were designed and PCR amplified as probes. Four clones pBXf-p40, pCXf-p40, pEXf-p40, and pXf-p40 were isolated from the constructed Xf genomic library. Sequence analysis revealed that the a total of fifteen ORFs with possible SD sequences can be identified in the cloned fragments. The phage tail-like bacteriocin genes of Xf determined form a gene cluster and consist of lysis cassette, major and minor structural proteins. The amino acid sequence deduced from these ORFs have the highest similarities to P2 bacteriophage proteins of Yersinia pseudotuberculosis IP 32953、Yersinia mollaretii ATCC 43969 and Yersinia frederiksenii ATCC 33641.
URI: http://hdl.handle.net/11455/22040
其他識別: U0005-1808200918570500
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