臺灣博碩士論文加值系統

Pectobacterium carotovorum subsp. carotovorum (P.c.c.)為腸道菌科的革蘭氏陰性菌，偏好寄生於植物，常造成十字花科植物根莖部腐爛之疾病，是導致許多植物軟腐病的植物病原細菌。當環境不利於細菌生存時，細菌會分泌一種胞外抗生蛋白 (細菌素)，能夠抑制或殺死與生產菌株有相近親緣關係的菌種。
第三類分泌系統(T3SS)中的可分為刺針型(T3aSS)和鞭毛型(T3bSS)，此兩種機制在結構及功能上是非常密切及相似的，在大部分的結構組成蛋白和功能具有同源性，從本實驗室先前的研究中，發現低分子量的細菌素Carocin，主要是經由第三類分泌系統(T3SS)中的鞭毛系統分泌至細胞外。刺針型(T3aSS)主要能使效應蛋白直接送入宿主細胞，效應蛋白會改變宿主的細胞功能或是細胞膜結構，達到侵入的作用或是共生目的。其鞭毛型(T3bSS)目的是將蛋白從胞內分泌至胞外及在液體中移動。
從本實驗室的研究中得知，在P.c.c.菌種內的Carocin表現相關機制裡，DGC與CRP等調控因子扮演了重要的角色，當調控因子被阻斷掉之後，分別在T3aSS及T3bSS的基因ysaT及flhA等基因均不再表現，進而造成低分子量細菌素無法被分泌至細胞外。此依結果顯示P.c.c.的低分子量細菌素有可能是透過T3aSS及T3bSS兩系統所整合的新形態的第三類分泌系統分泌至胞外。
本實驗成功的將flhA及ysaT基因subcloned至pET32a質體上，並在RT-PCR的實驗中證實了，重組過後的質體是能正常的做轉錄作用。為了讓重組質體能大量表現目標基因的蛋白，本實驗利用IPTG去做誘導表現。由於這兩個蛋白皆為膜蛋白，我們嘗試了許多界面活性劑及各種條件，最後我們得知FlhA蛋白利用1 % Triton X-100才能使之萃取出，YsaT蛋白則是利用1 M NaCl水溶液即可將此蛋白萃取出來。

摘要 i
Abstract ii
目次 iii
表目次 iv
圖目次 v
名詞縮寫表 vi
第一章 緒論 1
1.1 Pectobacterium carotovorum subsp.carotovorum與細菌性軟腐病 1
1.2 細菌素(Bacteriocin) 3
1.3 細菌之分泌系統 6
1.4 第三類分泌系統(Type III secretion system, T3SS) 7
1.5 膜蛋白(Membrane protein) 9
1.6 研究目的 11
第二章 材料與方法 12
2.1 實驗使用之菌株、質體及引子 12
2.2 細菌培養 14
2.3 質體DNA之分離 15
2.4 洋菜膠體(Agarose gel)配置與DNA電泳分析 17
2.5 勝任細胞與轉型作用 18
2.6 DNA片段回收與純化 21
2.7 選殖基因與質體構築 22
2.8 蛋白誘導表現 26
2.9 SDS-PAGE製備與蛋白質電泳分析 27
2.10 西方墨點法(Western blot) 29
2.11 RT-PCR 31
第三章 實驗結果 33
3.1 flhA基因重組載體構築 33
3.2 構築體的mRNA表現 34
3.3 融合蛋白誘導大量表現 35
第四章 討論 36
參考資料 53

參考資料
1.Malcolmson, J.F., A study of Erwinia isolates obtained from soft rots and blackleg of potatoes. Transactions of the British Mycological Society, 1959. 42(2): p. 261-269.
2.Pérombelon, M.C.M., Potato diseases caused by soft rot erwinias: an overview of pathogenesis. Plant Pathology, 2002. 51(1): p. 1-12.
3.LUND, B.M., Formation of Reducing Sugars from Sucrose by Erwinia Species. Microbiology, 1975. 88(2): p. 367-371.
4.Jones, D.R. and W.J. Dowson, On the bacteria responsible for soft rot in stored potatoes, and the reaction of the tuber to invasion by bacterium carotovorum (jones) lehmann & neumann. Annals of Applied Biology, 1950. 37(4): p. 563-569.
5.Chatterjee, A.K. and M.P. Starr, Donor Strains of the Soft-Rot Bacterium Erwinia chrysanthemi and Conjugational Transfer of the Pectolytic Capacity. Journal of Bacteriology, 1977. 132(3): p. 862-869.
6.Chatterjee, A.K., et al., Synthesis and excretion of polygalacturonic acid trans-eliminase in Erwinia, Yersinia, and Klebsiella species. Canadian Journal of Microbiology, 1979. 25(1): p. 94-102.
7.黃㯖昌. 臺灣作物細菌性病害防治要領. 行政院農業委員會臺東區農業改良場.
8.Itoh, Y., K. Izaki, and H. Takahashi, Purification and characterization of a bacteriocin from erwinia carotovora.The Journal of General and Applied Microbiology, 1978. 24(1): p. 27-39.
9.Gratia, A., Sur un remarquable exemple d'antagonisme entre deux souches de coilbacille. CR Seances Soc. Biol. Fil., 1925. 93: p. 1040-1041.
10.Gratia, A. and P. Fredericq, *Diversite des souches antibiotiques de b-coli et etendue variable de leur champ daction. Comptes rendus des seances de la societe de biologie et de ses filiales, 1946. 140(11): p. 1032-1033.
11.Lewus, C.B., A. Kaiser, and T.J. Montville, Inhibition of food-borne bacterial pathogens by bacteriocins from lactic acid bacteria isolated from meat. Applied and Environmental Microbiology, 1991. 57(6): p. 1683-1688.
12.Jabeen, N., et al., Isolation, identification and bacteriocin production by indigenous diseased plant and soil associated bacteria. Pakistan J. Biol. Sci, 2004. 7: p. 1893-1897.
13.Strauch, E., et al., Characterization of enterocoliticin, a phage tail-like bacteriocin, and its effect on pathogenic Yersinia enterocolitica strains. Applied and Environmental Microbiology, 2001. 67(12): p. 5634-5642.
14.S?rensen, K.I., et al., A food-grade cloning system for industrial strains of Lactococcus lactis. Applied and Environmental Microbiology, 2000. 66(4): p. 1253-1258.
15.Diez-Gonzalez, F., Applications of bacteriocins in livestock. Current issues in intestinal microbiology, 2007. 8(1): p. 15.
16.Chen, H. and D. Hoover, Bacteriocins and their food applications. Comprehensive reviews in food science and food safety, 2003. 2(3): p. 82-100.
17.Reeves, P., The bacteriocins. Bacteriological reviews, 1965. 29(1): p. 24.
18.Gobbetti, M., et al., Purification and characterization of a proteinaceous compound from Pseudomonas fluorescens ATCC 948 with inhibitory activity against some Gram-positive and Gram-negative bacteria of dairy interest. Le Lait, 1997. 77(2): p. 267-278.
19.Griffiths, G., et al., Vibriobactin, a siderophore from Vibrio cholerae. Journal of Biological Chemistry, 1984. 259(1): p. 383-385.
20.Oudega, B., J. Van der Molen, and F. De Graaf, In vitro binding of cloacin DF13 to its purified outer membrane receptor protein and effect of peptidoglycan on bacteriocin-receptor interaction. Journal of Bacteriology, 1979. 140(3): p. 964-970.
21.Konisky, J., Colicins and other bacteriocins with established modes of action. Annual Reviews in Microbiology, 1982. 36(1): p. 125-144.
22.Tagg, J.R., A.S. Dajani, and L.W. Wannamaker, Bacteriocins of gram-positive bacteria. Bacteriological reviews, 1976. 40(3): p. 722.
23.Dale, C., et al., The insect endosymbiont Sodalis glossinidius utilizes a type III secretion system for cell invasion. Proceedings of the National Academy of Sciences, 2001. 98(4): p. 1883-1888.
24.Bradley, D.E., Ultrastructure of bacteriophage and bacteriocins. Bacteriological reviews, 1967. 31(4): p. 230.
25.Nguyen, A.H., et al., A simple purification method and morphology and component analyses for carotovoricin Er, a phage-tail-like bacteriocin from the plant pathogen Erwinia carotovora Er. Bioscience, biotechnology, and biochemistry, 1999. 63(8): p. 1360-1369.
26.Chuang, D.-y., Y.-c. Chien, and H.-P. Wu, Cloning and expression of the Erwinia carotovora subsp. carotovora gene encoding the low-molecular-weight bacteriocin carocin S1. Journal of Bacteriology, 2007. 189(2): p. 620-626.
27.Dean, M., Y. Hamon, and G. Chimini, The human ATP-binding cassette (ABC) transporter superfamily. Journal of lipid research, 2001. 42(7): p. 1007-1017.
28.Sandkvist, M., Type II secretion and pathogenesis. Infection and immunity, 2001. 69(6): p. 3523-3535.
29.Voulhoux, R., et al., Involvement of the twin‐arginine translocation system in protein secretion via the type II pathway. The EMBO journal, 2001. 20(23): p. 6735-6741.
30.Sandkvist, M., Biology of type II secretion. Molecular microbiology, 2001. 40(2): p. 271-283.
31.Christie, P.J. and J.P. Vogel, Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells. Trends in microbiology, 2000. 8(8): p. 354-360.
32.Henderson, I.R., et al., Type V protein secretion pathway: the autotransporter story. Microbiology and molecular biology reviews, 2004. 68(4): p. 692-744.
33.Cianfanelli, F.R., L. Monlezun, and S.J. Coulthurst, Aim, load, fire: the type VI secretion system, a bacterial nanoweapon. Trends in microbiology, 2016. 24(1): p. 51-62.
34.Coulthurst, S.J., The Type VI secretion system–a widespread and versatile cell targeting system. Research in Microbiology, 2013. 164(6): p. 640-654.
35.Salmond, G.P. and P.J. Reeves, Membrance traffic wardens and protein secretion in Gram-negative bacteria. Trends in biochemical sciences, 1993. 18(1): p. 7-12.
36.Galán, J.E. and A. Collmer, Type III secretion machines: bacterial devices for protein delivery into host cells. Science, 1999. 284(5418): p. 1322-1328.
37.Desvaux, M., et al., Type III secretion: what's in a name? Trends in microbiology, 2006. 14(4): p. 157-160.
38.Tampakaki, A., et al., Conserved features of type III secretion. Cellular microbiology, 2004. 6(9): p. 805-816.
39.Cornelis, G.R., The type III secretion injectisome. Nature Reviews Microbiology, 2006. 4(11): p. 811-825.
40.Erhardt, M., K. Namba, and K.T. Hughes, Bacterial nanomachines: the flagellum and type III injectisome. Cold Spring Harbor perspectives in biology, 2010. 2(11): p. a000299.
41.Macnab, R.M., How bacteria assemble flagella. Annual Reviews in Microbiology, 2003. 57(1): p. 77-100.
42.Minamino, T. and R.M. Macnab, Components of the Salmonella flagellar export apparatus and classification of export substrates. Journal of Bacteriology, 1999. 181(5): p. 1388-1394.
43.Minamino, T., K. Imada, and K. Namba, Mechanisms of type III protein export for bacterial flagellar assembly. Molecular BioSystems, 2008. 4(11): p. 1105-1115.
44.Manson, M.D., et al., A protonmotive force drives bacterial flagella. Proceedings of the National Academy of Sciences, 1977. 74(7): p. 3060-3064.
45.Matsuura, S., J.-i. Shioi, and Y. Imae, Motility in Bacillus subtilis driven by an artificial protonmotive force. FEBS letters, 1977. 82(2): p. 187-190.
46.Andreeva, A., et al., SCOP2 prototype: a new approach to protein structure mining. Nucleic Acids Research, 2014. 42(D1): p. D310-D314.
47.Overington, J.P., B. Al-Lazikani, and A.L. Hopkins, How many drug targets are there? Nat Rev Drug Discov, 2006. 5(12): p. 993-996.
48.Almén, M.S., et al., Mapping the human membrane proteome: a majority of the human membrane proteins can be classified according to function and evolutionary origin. BMC biology, 2009. 7(1): p. 50.
49.Lin, Y., et al., The substitution of Arg149 with Cys fixes the melibiose transporter in an inward-open conformation. Biochimica et Biophysica Acta (BBA)-Biomembranes, 2013. 1828(8): p. 1690-1699.
50.Liszewski, K., Dissecting the structure of membrane proteins. 2015.
51.Johnson, J.E. and R.B. Cornell, Amphitropic proteins: regulation by reversible membrane interactions. Molecular membrane biology, 1999. 16(3): p. 217-235.
52.Alenghat, F.J. and D.E. Golan, Membrane protein dynamics and functional implications in mammalian cells. Current topics in membranes, 2013. 72: p. 89.
53.Ghosh, M., et al., Properties of the Group IV phospholipase A2 family. Progress in Lipid Research, 2006. 45(6): p. 487-510.
54.Chan, Y.-c., H.-P. Wu, and D.-y. Chuang, Extracellular secretion of Carocin S1 in Pectobacterium carotovorum subsp. carotovorum occurs via the type III secretion system integral to the bacterial flagellum. BMC microbiology, 2009. 9(1): p. 181.
55.陳坤弘, Pectobacterium carotovorum subsp. carotovorum胞外分泌系統的結構蛋白與調控因子之關係探討. 2016.
56.Chiu, J., et al., Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h. Nucleic Acids Research, 2004. 32(21): p. e174-e174.
57.McMurry, J.L., et al., Analysis of the Cytoplasmic Domains of Salmonella FlhA and Interactions with Components of the Flagellar Export Machinery. Journal of Bacteriology, 2004. 186(22): p. 7586-7592.
58.Minamino, T., T. Iino, and K. Kutuskake, Molecular characterization of the Salmonella typhimurium flhB operon and its protein products. Journal of Bacteriology, 1994. 176(24): p. 7630-7637.
59.詹永傑, Erwinia carotovora subsp. carotovora低分子量細菌素分泌蛋白基因的選殖與分析.國立中興大學化學所, 2005.
60.Kaur, S. and S. Kaur, Bacteriocins as Potential Anticancer Agents. Frontiers in Pharmacology, 2015. 6: p. 272.