臺灣博碩士論文加值系統

第一部份
腸道菌在生長環境中會遇到許多複雜的自然化學訊息 ( physicochemical signals )，如：溫度、pH值、滲透壓、養份和有毒物質，藉由這些環境因素的整合使腸道菌在腸道中找到最適合的生長環境。大腸桿菌可藉由大腸桿菌素的產量增多來增加自己生存和防禦的機會。
大腸桿菌素是由大腸桿菌所產生的一種質體編碼殺菌毒素，其作用範圍為大腸桿菌及相近的腸桿菌科細菌。E7大腸桿菌素操縱子是由三個基因所組成，分別為大腸桿菌素基因 (cea)、免疫蛋白基因 (cei) 及溶菌蛋白基因 (cel)，而這操縱子的表現是受到宿主SOS反應系統的調節。cei基因的產物-免疫蛋白，可以中和其上游 cea基因產物的毒性，以保護宿主；而 cel基因的產物-溶菌蛋白可以將大腸桿菌素和免疫蛋白的複合體運送到菌體外，但當它大量表現時則會造成宿主細胞的溶解而死亡。因此，操縱子基因間的協調表現是很重要的。
對於免疫蛋白 (ImmE7) 最清楚的功能為可以中和大腸桿菌素的毒性。在先前的研究發現，多元轉錄物cea-cei-cel在cei上有一主要的特定點斷裂 (major site-specific cleavage) 的現象，推測此定點斷裂與操縱子的表現調控有關。本論文以探討在cei上特定的斷裂 (site-specific cleavage) 情形會不會受到環境因素的影響和影響的程度為主要的研究方向。
利用反轉錄聚合酵素連鎖反應定量法 (quantitative RT-PCR assay) 對Mitomycin C誘導、增加E7大腸桿菌素操縱子產量、高溫、高pH值等不同環境因素影響 cei上特定斷裂的程度作探討。結果顯示，SOS反應對 cei上特定斷裂現象是一個強烈的訊息。 cei上特定斷裂現象隨著E7大腸桿菌素操縱子產量而變化。溫度和 pH值影響 cei上特定斷裂現象的程度較緩和。這些結果顯示，環境因素影響大腸桿菌素 ColE7 operon上cei特定斷裂的機制有兩個： (一) cei上特定斷裂的情形會隨著E7大腸桿菌素操縱子產量而變化，如：增加E7大腸桿菌素操縱子產量。(二) 當大腸桿菌素ColE7操縱子的表現突然過量時，會引發菌體中某些機制去對應大腸桿菌素操縱子大量表現的強烈反應，如：mitomycin C的誘導, 高溫, 高pH值之下。在未來的工作中，將進一步探討目前已知細菌用來對環境做適應調節的系統與 cei上特定斷裂現象的關係，及所參與的因子和調控的機制。
第二部份
大腸桿菌素E7是去氧核醣核酸酵素型式的細菌毒素。當大腸桿菌遭遇環境壓力時會分泌此毒素殺害其它細菌以增加其在此環境中的競爭力。當大腸桿菌素在細胞中被轉譯時，同時產生免疫蛋白質與毒性區域結合以中和其毒性因而避免此毒素對菌體自身所造成傷害。經轉譯後，大腸桿菌素 (ColE7)與免疫蛋白 (ImmE7) 會以異體複合物的形式分泌到細胞外。大腸桿菌素進入細胞內必需藉由一些外膜接受器 (如：BtuB)。然後，大腸桿菌素進入胞膜間區 (periplasmic space) 是利用Tol系統並有許多不同的胞膜間區或內膜蛋白質的參與其中。最後，當大腸桿菌素到達胞膜間區之後，大腸桿菌素會依據其不同的毒性特質，造成細胞的受損，甚至死亡；本論文以探討大腸桿菌素ColE7的胞膜移位過程還有那些不同的胞膜間區或內膜蛋白質參與為主要的研究方向。
利用transposon mutagenesis法來尋找參與translocation相關的基因，並尋求參與translocation相關的蛋白質。結果，發現有56個被transposon插入genomic DNA的菌株；接著，發現有36個被transposon破壞的菌株是可以抗colicin的毒殺；最後，對插入的基因做定序後確定各突變株只有一個transposon插入到genomic DNA中，得到被插入的基因都不含胞膜蛋白有關的基因，然而這些突變株如何影響ColE7的胞膜位移，這是值得做進一步的探討。
接著，利用反轉錄聚合酵素連鎖反應定量法 ( quantitative RT-PCR assay )來測定抗colicin毒殺的突變菌株中的有關胞膜位移基因 ( 包括btuB, ompC, ompF, pa l) 的表現情形。結果發現在每一個抗colicin毒殺的菌株中btuB, ompC, ompF, pal的表現量和野生型 ( wild type ) 比較有所不同。因此證明所測定的突變基因與大腸桿菌素胞膜位移過程有關。
總合以上的結果，我們已發現一些新型基因與大腸桿菌素胞膜位移過程有關。未來工作中，將測定每一突變菌株對大腸桿菌素的胞膜位移所做成的影響，再從突變菌株定出些基因和蛋白與野生株之異同。最後，再研究彼此間的關係和受那些因子的調控，藉以鉤勒出一個完整的大腸桿菌素ColE7胞膜位移過程的機制。

中文摘要…………………………………………….……………………….…Ⅰ
英文摘要…………………………………………….…………………………Ⅳ
壹、序論………………………………………………………………….1 ~ 13
第一部份
一、背景介紹………………………………………………………….…1
(一)大腸桿菌素ColE7基因與其操縱子上其它基因的功能……..1
(二)大腸桿菌素的分類………………………………………….…..2
(三)大腸桿菌素的作用機制………………………………………...3
二、大腸桿菌素E7質體之特性………………………………….…..…3
三、大腸桿菌素E7操縱子基因表現之調節…………………………...4
四、大腸桿菌素質體之運用及本實驗室ColE7-K317質體研究之概況…………………………………………………………………….5
五、環境因素對cei上特定的斷裂 (site-specific cleavage) 情形之影響………………………………………………………….……..…..7
六、實驗目的………………………………………………….…..……...8
第二部份
一、大腸桿菌素進入敏感細胞之途徑………………………..…………9
二、目前已知參與大腸桿菌素ColE7的胞膜移位過程所需蛋白之探討………………………………………….....……………….…..…10
三、目前對於大腸桿菌素進入敏感細菌 (sensitive bacteria) 移位過程 (translocation process) 研究之概況…………………...…………...11
四、實驗目的………………………………………………….…..……...12
貳、實驗材料與方法…………………………………………………….14 ~ 46
一、細菌菌株………………………………………………….…..…… 14
二、質體及其建構…………………………………………….…..…… 14
三、化學藥品與酵素………………………………….…………..…… 15
四、一般分生研究的方法…………………………………..….... 17 ~ 26
(一)細菌培養………………………………………………..……... 17
(二)在大腸桿菌中質體DNA之分離與純化……………..……..... 18
(三)大腸桿菌中全部DNA (total genomic DNA) 之分離與純化...20
(四)聚合酵素鏈鎖反應…………………………………………..... 21
(五)限制酵素的切割……………………………..……………..…. 22
(六)CIAP處理…………………………………..…………………..22
(七)Klenow反應處理…………………………………………..…...22
(八)DNA的連接反應………………………………………..…..… 23
(九)洋菜膠體電泳……………………………...……………...……24
(十)從洋菜膠中回收DNA片段及純化…………...…………….... 24
(十一)細菌的轉形作用……………………………………...…….. 25
(十二)選殖菌株之篩選-細菌質體之快速篩選法………………… 27
五、蛋白質電泳……………………………..……………………… 28 ~ 32
(一)SDS-聚丙醯胺膠電泳……….…………………………………28
(二)蛋白質染色……………………………………………………..31
六、西方墨點測試………………………………………………………...32
七、遠西墨點測試………………………………………………………...33
八、Colicin活性測試 (Colicin activity test)……………………………..34
九、RNA的抽取 (RNA isolation)……………………………….……….34
十、反轉錄聚合酵素鏈鎖反應…………………………………..……….35
參、結果……………………………………………………….…………. 37 ~ 50
第一部份
一、建立cei-mRNA上特定斷裂 (site-specific cleavage) 程度的定量方法…….……………………………………………………………… 37
二、Mitomycin C的誘導之下對免疫基因mRNA上特定點斷裂(site-specific cleavage) 現象的影響…………………………..…… 39
三、在沒有DNA損壞的情況下觀察在菌體中total mRNA量對免疫基因mRNA上特定點斷裂 (site-specific cleavage) 現象的影響……….41
四、高溫下對免疫基因mRNA上特定點斷裂 (site-specific cleavage) 現象的影響………………………………………………………….….43
五、高pH值下對免疫基因mRNA上特定點斷裂(site-specific cleavage) 現象的影響……………………………………………………….….44
六、環境因素對免疫基因mRNA上特定點斷裂(site-specific cleavage) 現象的影響之比較………………………………………………….….45
第二部份
一、以transposon mutagenesis法來尋找與translocation相關的基因藉此完成一個完整的桿菌素胞膜移位過程的模式…….……………… 46
二、大腸桿菌素ColE7的胞膜移位過程所需基因轉錄表現的定量方法……………………………………………………………….…… 48
三、參與大腸桿菌素ColE7胞膜移位過程所需基因與抗colicin毒殺關係之探討………………………………………………………….….49
肆、討論………………………………………………………...……..…. 51 ~ 64
第一部份
一、影響RNA斷裂現象發生可能的環境因素…….…………………… 51
(一)Mitomycin C的誘導之下的影響……………………………...... 52
(二)增加菌體中大腸桿菌素ColE7操縱子表現量之影響………..... 53
(三)高溫下的影響……………………………………………………..54
(四)高pH值下的影響………………………………………….…..... 55
二、環境因素對細菌生存的影響…………………….………………..…56
三、未來工作……………….………………………………………….….57
第二部份
一、可能參與大腸桿菌素ColE7運送的蛋白質之探討…….………..…59
(一)以transposon mutagenesis法找到之基因可能參與大腸桿菌素運送的機制……………………………..…………………………..…...59
二、可能影響大腸桿菌素ColE7的胞膜移位過程的機制…………...…62
三、未來工作……………….…………………………………………..….64
伍、圖表………………………………………………..…………………... 65 ~ 88
陸、參考文獻…………………………………………………….…….……89 ~ 97

Argast, M., and Boos, W. (1980). Co-regulation in Escherichia coli of a novel transport system for sn-glycerol-3-phosphate and outer membrane protein Ic (e, E) with alkaline phosphatase and phosphate-binding protein. J Bacteriol 143, 142-50.
Babitzke, P., and Kushner, S. R. (1991). The Ams (altered mRNA stability) protein and ribonuclease E are encoded by the same structural gene of Escherichia coli. Proc Natl Acad Sci U S A 88, 1-5.
Bowles, L. K., and Konisky, J. (1981). Cleavage of colicin Ia by the Escherichia coli K-12 outer membrane is not mediated by the colicin Ia receptor. J Bacteriol 145, 668-71.
Chak, K. F., and James, R. (1985). Analysis of the promoters for the two immunity genes present in the ColE3-CA38 plasmid using two new promoter probe vectors. Nucleic Acids Res 13, 2519-31.
Chak, K. F., Kuo, W. S., Lu, F. M., and James, R. (1991). Cloning and characterization of the ColE7 plasmid. J Gen Microbiol 137, 91-100.
Chak, K. F., Safo, M. K., Ku, W. Y., Hsieh, S. Y., and Yuan, H. S. (1996). The crystal structure of the immunity protein of colicin E7 suggests a possible colicin-interacting surface. Proc Natl Acad Sci U S A 93, 6437-42.
Clavel, T., Lazzaroni, J. C., Vianney, A., and Portalier, R. (1996). Expression of the tolQRA genes of Escherichia coli K-12 is controlled by the RcsC sensor protein involved in capsule synthesis. Mol Microbiol 19, 19-25.
Craig, N. L., and Roberts, J. W. (1980). E. coli recA protein-directed cleavage of phage lambda repressor requires polynucleotide. Nature 283, 26-30.
Davies, J. K., and Reeves, P. (1975). Genetics of resistance to colicins in Escherichia coli K-12: cross-resistance among colicins of group B. J Bacteriol 123, 96-101.
Dela, F., Larsen, J. J., Mikines, K. J., Ploug, T., Petersen, L. N., and Galbo, H. (1995). Insulin-stimulated muscle glucose clearance in patients with NIDDM. Effects of one-legged physical training. Diabetes 44, 1010-20.
Di Masi, D. R., White, J. C., Schnaitman, C. A., and Bradbeer, C. (1973). Transport of vitamin B12 in Escherichia coli: common receptor sites for vitamin B12 and the E colicins on the outer membrane of the cell envelope. J Bacteriol 115, 506-13.
Dorman, C. J. (1991). DNA supercoiling and environmental regulation of gene expression in pathogenic bacteria. Infect Immun 59, 745-9.
Dorman, C. J., Bhriain, N. N., and Higgins, C. F. (1990). DNA supercoiling and environmental regulation of virulence gene expression in Shigella flexneri. Nature 344, 789-92.
Duche, D., Baty, D., Chartier, M., and Letellier, L. (1994). Unfolding of colicin A during its translocation through the Escherichia coli envelope as demonstrated by disulfide bond engineering. J Biol Chem 269, 24820-5.
Edwards, R. A., and Puente, J. L. (1998). Fimbrial expression in enteric bacteria: a critical step in intestinal pathogenesis. Trends Microbiol 6, 282-7.
Eraso, J. M., Chidambaram, M., and Weinstock, G. M. (1996). Increased production of colicin E1 in stationary phase. J Bacteriol 178, 1928-35.
Eraso, J. M., and Weinstock, G. M. (1992). Anaerobic control of colicin E1 production. J Bacteriol 174, 5101-9.
Fields, P. I., Groisman, E. A., and Heffron, F. (1989). A Salmonella locus that controls resistance to microbicidal proteins from phagocytic cells. Science 243, 1059-62.
Goransson, M., Sonden, B., Nilsson, P., Dagberg, B., Forsman, K., Emanuelsson, K., and Uhlin, B. E. (1990). Transcriptional silencing and thermoregulation of gene expression in Escherichia coli. Nature 344, 682-5.
Gottesman, S., and Stout, V. (1991). Regulation of capsular polysaccharide synthesis in Escherichia coli K12. Mol Microbiol 5, 1599-606.
Hardy, K. G. (1975). Colicinogeny and related phenomena. Bacteriol Rev 39, 464-515.
Hasegawa, Y., Yamada, H., and Mizushima, S. (1976). Interactions of outer membrane proteins O-8 and O-9 with peptidoglycan sacculus of Escherichia coli K-12. J Biochem (Tokyo) 80, 1401-9.
Higgins, C. F. (1990). DNA supercoiling, chromatin structure and the regulation of gene expression. Antonie Van Leeuwenhoek 58, 155.
Hsieh, S. Y., Ko, T. P., Tseng, M. Y., Ku, W., Chak, K. F., and Yuan, H. S. (1997). A novel role of ImmE7 in the autoregulatory expression of the ColE7 operon and identification of possible RNase active sites in the crystal structure of dimeric ImmE7. EMBO J 16, 1444-54.
Inokuchi, K., Mutoh, N., Matsuyama, S., and Mizushima, S. (1982). Primary structure of the ompF gene that codes for a major outer membrane protein of Escherichia coli K-12. Nucleic Acids Res 10, 6957-68.
Inukai, M., Ghrayeb, J., Nakamura, K., and Inouye, M. (1984). Apolipoprotein, an intermediate in the processing of the major lipoprotein of the Escherichia coli outer membrane. J Biol Chem 259, 757-60.
James, R., Kleanthous, C., and Moore, G. R. (1996). The biology of E colicins: paradigms and paradoxes. Microbiology 142, 1569-80.
Konisky, J. (1982). Colicins and other bacteriocins with established modes of action. Annu Rev Microbiol 36, 125-44.
Kuhar, I., and Zgur-Bertok, D. (1999). Transcription regulation of the colicin K cka gene reveals induction of colicin synthesis by differential responses to environmental signals. J Bacteriol 181, 7373-80.
Lacroix, J. M., Loubens, I., Tempete, M., Menichi, B., and Bohin, J. P. (1991). The mdoA locus of Escherichia coli consists of an operon under osmotic control. Mol Microbiol 5, 1745-53.
Liao, C. C., Hsiao, K. C., Liu, Y. W., Leng, P. H., Yuen, H. S., and Chak, K. F. (2001). Processing of DNase Domain during Translocation of Colicin E7 across the Membrane of Escherichia coli. Biochem Biophys Res Commun 284, 556-62.
Little, J. W., and Mount, D. W. (1982). The SOS regulatory system of Escherichia coli. Cell 29, 11-22.
Lu, F. M., and Chak, K. F. (1996). Two overlapping SOS-boxes in ColE operons are responsible for the viability of cells harboring the Col plasmid. Mol Gen Genet 251, 407-11.
Lu, F. M., Yuan, H. S., Hsu, Y. C., Chang, S. J., and Chak, K. F. (1999). Hierarchical order of critical residues on the immunity-determining region of the Im7 protein which confer specific immunity to its cognate colicin. Biochem Biophys Res Commun 264, 69-75.
Lugtenberg, B., Peters, R., Bernheimer, H., and Berendsen, W. (1976). Influence of cultural conditions and mutations on the composition of the outer membrane proteins of Escherichia coli. Mol Gen Genet 147, 251-62.
Maupin-Furlow, J. A., Rosentel, J. K., Lee, J. H., Deppenmeier, U., Gunsalus, R. P., and Shanmugam, K. T. (1995). Genetic analysis of the modABCD (molybdate transport) operon of Escherichia coli. J Bacteriol 177, 4851-6.
McCarthy, J. E., and Gualerzi, C. (1990). Translational control of prokaryotic gene expression. Trends Genet 6, 78-85.
Mekalanos, J. J. (1992). Environmental signals controlling expression of virulence determinants in bacteria. J Bacteriol 174, 1-7.
Melefors, O., and von Gabain, A. (1991). Genetic studies of cleavage-initiated mRNA decay and processing of ribosomal 9S RNA show that the Escherichia coli ams and rne loci are the same. Mol Microbiol 5, 857-64.
Miller, S. I. (1991). PhoP/PhoQ: macrophage-specific modulators of Salmonella virulence? Mol Microbiol 5, 2073-8.
Mizuno, T., Chou, M. Y., and Inouye, M. (1983). A comparative study on the genes for three porins of the Escherichia coli outer membrane. DNA sequence of the osmoregulated ompC gene. J Biol Chem 258, 6932-40.
Mock, M., and Pugsley, A. P. (1982). The BtuB group col plasmids and homology between the colicins they encode. J Bacteriol 150, 1069-76.
Muga, A., Gonzalez-Manas, J. M., Lakey, J. H., Pattus, F., and Surewicz, W. K. (1993). pH-dependent stability and membrane interaction of the pore-forming domain of colicin A. J Biol Chem 268, 1553-7.
Mukhopadhyay, P., Mukhopadhyay, M., and Mills, D. (1990). Construction of a stable shuttle vector for high-frequency transformation in Pseudomonas syringae pv. syringae. J Bacteriol 172, 477-80.
Mukhopadhyay, P., Williams, J., and Mills, D. (1988). Molecular analysis of a pathogenicity locus in Pseudomonas syringae pv. syringae. J Bacteriol 170, 5479-88.
Nikaido, H., and Vaara, M. (1985). Molecular basis of bacterial outer membrane permeability. Microbiol Rev 49, 1-32.
Overbeeke, N., Bergmans, H., van Mansfeld, F., and Lugtenberg, B. (1983). Complete nucleotide sequence of phoE, the structural gene for the phosphate limitation inducible outer membrane pore protein of Escherichia coli K12. J Mol Biol 163, 513-32.
Parsot, C., Taxman, E., and Mekalanos, J. J. (1991). ToxR regulates the production of lipoproteins and the expression of serum resistance in Vibrio cholerae. Proc Natl Acad Sci U S A 88, 1641-5.
Pugsley, A. P., and Schwartz, M. (1984). Colicin E2 release: lysis, leakage or secretion? Possible role of a phospholipase. EMBO J 3, 2393-7.
Rech, S., Deppenmeier, U., and Gunsalus, R. P. (1995). Regulation of the molybdate transport operon, modABCD, of Escherichia coli in response to molybdate availability. J Bacteriol 177, 1023-9.
Rigal, A., Bouveret, E., Lloubes, R., Lazdunski, C., and Benedetti, H. (1997). The TolB protein interacts with the porins of Escherichia coli. J Bacteriol 179, 7274-9.
Rosenshine, I., Donnenberg, M. S., Kaper, J. B., and Finlay, B. B. (1992). Signal transduction between enteropathogenic Escherichia coli (EPEC) and epithelial cells: EPEC induces tyrosine phosphorylation of host cell proteins to initiate cytoskeletal rearrangement and bacterial uptake. EMBO J 11, 3551-60.
Russell, J. E., Morales, J., and Liebhaber, S. A. (1997). The role of mRNA stability in the control of globin gene expression. Prog Nucleic Acid Res Mol Biol 57, 249-87.
Santero, E., Hoover, T., Keener, J., and Kustu, S. (1989). In vitro activity of the nitrogen fixation regulatory protein NIFA. Proc Natl Acad Sci U S A 86, 7346-50.
Slilaty, S. N., and Little, J. W. (1987). Lysine-156 and serine-119 are required for LexA repressor cleavage: a possible mechanism. Proc Natl Acad Sci U S A 84, 3987-91.
Soong, B. W., Hsieh, S. Y., and Chak, K. F. (1994). Mapping of transcriptional start sites of the cea and cei genes of the ColE7 operon. Mol Gen Genet 243, 477-81.
Soong, B. W., Lu, F. M., and Chak, K. F. (1992). Characterization of the cea gene of the ColE7 plasmid. Mol Gen Genet 233, 177-83.
Taylor, R. K., Hall, M. N., Enquist, L., and Silhavy, T. J. (1981). Identification of OmpR: a positive regulatory protein controlling expression of the major outer membrane matrix porin proteins of Escherichia coli K-12. J Bacteriol 147, 255-8.
Tommassen, J., and Lugtenberg, B. (1980). Outer membrane protein e of Escherichia coli K-12 is co-regulated with alkaline phosphatase. J Bacteriol 143, 151-7.
Vianney, A., Muller, M. M., Clavel, T., Lazzaroni, J. C., Portalier, R., and Webster, R. E. (1996). Characterization of the tol-pal region of Escherichia coli K-12: translational control of tolR expression by TolQ and identification of a new open reading frame downstream of pal encoding a periplasmic protein. J Bacteriol 178, 4031-8.
Walker, G. C. (1984). Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev 48, 60-93.
Watson, R., Rowsome, W., Tsao, J., and Visentin, L. P. (1981). Identification and characterization of Col plasmids from classical colicin E-producing strains. J Bacteriol 147, 569-77.
Witkin, E. M. (1976). Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol Rev 40, 869-907.
Yanisch-Perron, C., Vieira, J., and Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-19.