請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/63664
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林俊彬 | |
dc.contributor.author | Ya-An Cheng | en |
dc.contributor.author | 鄭雅安 | zh_TW |
dc.date.accessioned | 2021-06-16T17:15:56Z | - |
dc.date.available | 2012-09-17 | |
dc.date.copyright | 2012-09-17 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-08-19 | |
dc.identifier.citation | Actis LA, Rhodes ER, Tomaras AP (2003). Genetic and molecular characterization of a dental pathogen using genome-wide approaches. Adv Dent Res 17:95-99.
Adams DE, Bliska JB, Cozzarelli NR (1992). Cre-lox recombination in Escherichia coli cells. Mechanistic differences from the in vitro reaction. J Mol Biol 226(3):661-673. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W et al. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389-3402. Balashova NV, Park DH, Patel JK, Figurski DH, Kachlany SC (2007). Interaction between leukotoxin and Cu,Zn superoxide dismutase in Aggregatibacter actinomycetemcomitans. Infect Immun 75(9):4490-4497. Bhattacharjee MK, Kachlany SC, Fine DH, Figurski DH (2001). Nonspecific adherence and fibril biogenesis by Actinobacillus actinomycetemcomitans: TadA protein is an ATPase. J Bacteriol 183(20):5927-5936. Braunthal SD, Holt SC, Tanner AC, Socransky SS (1980). Cellular fatty acid composition of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus. J Clin Microbiol 11(6):625-630. Brogan JM, Lally ET, Demuth DR (1996). Construction of pYGK, an Actinobacillus actinomycetemcomitans-Escherichia coli shuttle vector. Gene 169(1):141-142. Bron PA, Grangette C, Mercenier A, de Vos WM, Kleerebezem M (2004). Identification of Lactobacillus plantarum genes that are induced in the gastrointestinal tract of mice. J Bacteriol 186(17):5721-5729. Brondz I, Olsen I (1989). Chemical differences in lipopolysaccharides from Actinobacillus (Haemophilus) actinomycetemcomitans and Haemophilus aphrophilus: clues to differences in periodontopathogenic potential and taxonomic distinction. Infect Immun 57(10):3106-3109. Brondz I, Olsen I (1990). Multivariate analyses of carbohydrate data from lipopolysaccharides of Actinobacillus (Haemophilus) actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus. Int J of Syst Bacteriol 40(4):405-408. Brown LJ, Brunelle JA, Kingman A (1996). Periodontal status in the United States, 1988-1991: prevalence, extent, and demographic variation. J Dent Res 75 (Spec issue):672-683. Calhoon DA, Mayberry WR, Slots J (1981). Cellular fatty acid and soluble protein composition of Actinobacillus actinomycetemcomitans and related organisms. J Clin Microbiol 14(4):376-382. Chelikani P, Fita I, Loewen PC (2004). Diversity of structures and properties among catalases. Cell Mol Life Sci 61(2):192-208. Chen C, Wang T, Chen W (2010). Occurrence of Aggregatibacter actinomycetemcomitans serotypes in subgingival plaque from United States subjects. Mol Oral Microbiol 25(3):207-214. Chien JT, Lin CH, Chen YC, Lay CJ, Wang CL, Tsai CC (2009). Epidural abscess caused by Haemophilus aphrophilus misidentified as Pasteurella species. Internal medicine 48(10):853-858. Di Bonaventura MP, DeSalle R, Pop M, Nagarajan N, Figurski DH, Fine DH et al. (2009). Complete genome sequence of Aggregatibacter (Haemophilus) aphrophilus NJ8700. J Bacteriol 191(14):4693-4694. Diard M, Garry L, Selva M, Mosser T, Denamur E, Matic I (2010). Pathogenicity-Associated Islands in Extraintestinal Pathogenic Escherichia coli Are Fitness Elements Involved in Intestinal Colonization. J Bacteriol 192(19):4885-4893. DiRienzo JM, McKay TL (1994). Identification and characterization of genetic cluster groups of Actinobacillus actinomycetemcomitans isolated from the human oral cavity. J Clin Microbiol 32(1):75-81. DiRienzo JM, Slots J, Sixou M, Sol MA, Harmon R, McKay TL (1994). Specific genetic variants of Actinobacillus actinomycetemcomitans correlate with disease and health in a regional population of families with localized juvenile periodontitis. Infect Immun 62(8):3058-3065. Dongari AI, Miyasaki KT (1991). Sensitivity of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus to oxidative killing. Oral Microbiol Immunol 6(6):363-372. Doolittle WF (1998). You are what you eat: a gene transfer ratchet could account for bacterial genes in eukaryotic nuclear genomes. Trends Genet 14(8):307-311. Duncan MJ (2003). Genomics of oral bacteria. Crit Rev Oral Biol Med 14(3):175-187. Eicher T, Brandstatter L, Pos KM (2009). Structural and functional aspects of the multidrug efflux pump AcrB. Biol Chem 390(8):693-699. Eisen JA (2000). Horizontal gene transfer among microbial genomes: new insights from complete genome analysis. Current Opinion in Genetics & Development 10(6):606-611. Fine DH, Markowitz K, Furgang D, Fairlie K, Ferrandiz J, Nasri C et al. (2007). Aggregatibacter actinomycetemcomitans and its relationship to initiation of localized aggressive periodontitis: longitudinal cohort study of initially healthy adolescents. J Clin Microbiol 45(12):3859-3869. Finlay BB, Falkow S (1997). Common themes in microbial pathogenicity revisited. Microbiol Mol Biol Rev 61(2):136-169. Fives-Taylor PM, Meyer DH, Mintz KP, Brissette C (1999). Virulence factors of Actinobacillus actinomycetemcomitans. Periodontol 2000 20:136-167. Fujise O, Chen W, Rich S, Chen C (2004). Clonal diversity and stability of subgingival Eikenella corrodens. J Clin Microbiol 42(5):2036-2042. Fujise O, Wang Y, Chen W, Chen C (2008). Adherence of Aggregatibacter actinomycetemcomitans via serotype-specific polysaccharide antigens in lipopolysaccharides. Oral Microbiol Immunol 23(3):226-233. Groisman EA, Ochman H (1996). Pathogenicity islands: bacterial evolution in quantum leaps. Cell 87(5):791-794. Guthmiller JM, Kolodrubetz D, Kraig E (1995). Mutational analysis of the putative leukotoxin transport genes in Actinobacillus actinomycetemcomitans. Microb Pathog 18(5):307-321. Gwinn ML, Ramanathan R, Smith HO, Tomb JF (1998). A new transformation-deficient mutant of Haemophilus influenzae Rd with normal DNA uptake. J Bacteriol 180(3):746-748. Hacker J, Blum-Oehler G, Muhldorfer I, Tschape H (1997). Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution. Mol Microbiol 23(6):1089-1097. Hacker J, Kaper JB (2000). Pathogenicity islands and the evolution of microbes. Annu Rev Microbiol 54(641-679. Hacker J, Carniel E (2001). Ecological fitness, genomic islands and bacterial pathogenicity. A Darwinian view of the evolution of microbes. EMBO Rep 2(5):376-381. Hacker J, Hentschel U, Dobrindt U (2003). Prokaryotic chromosomes and disease. Science 301(5634):790-793. Haubek D, Ennibi O-K, Poulsen K, V?th M, Poulsen S, Kilian M (2008). Risk of aggressive periodontitis in adolescent carriers of the JP2 clone of Aggregatibacter (Actinobacillus) actinomycetemcomitans in Morocco: a prospective longitudinal cohort study. The Lancet 371(9608):237-242. Hill CW, Sandt CH, Vlazny DA (1994). Rhs elements of Escherichia coli: a family of genetic composites each encoding a large mosaic protein. Mol Microbiol 12(6):865-871. Holt SC, Tanner AC, Socransky SS (1980). Morphology and ultrastructure of oral strains of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus. Infect Immun 30(2):588-600. Huang ST, Lee HC, Lee NY, Liu KH, Ko WC (2005). Clinical characteristics of invasive Haemophilus aphrophilus infections. J Microbiol Immunol Infect 38(4):271-276. Isaza MP, Duncan MS, Kaplan JB, Kachlany SC (2008). Screen for leukotoxin mutants in Aggregatibacter actinomycetemcomitans: genes of the phosphotransferase system are required for leukotoxin biosynthesis. Infect Immun 76(8):3561-3568. Jung GW, Parkins MD, Church D (2009). Pyogenic ventriculitis complicating Aggregatibacter aphrophilus infective endocarditis: A case report and literature review. The Canadian journal of infectious diseases & medical microbiology = Journal canadien des maladies infectieuses et de la microbiologie medicale / AMMI Canada 20(3):e107-109. Karched M, Paul-Satyaseela M, Asikainen S (2007). A simple viability-maintaining method produces homogenic cell suspensions of autoaggregating wild-type Actinobacillus actinomycetemcomitans. J Microbiol Meth 68(1):46-51. Kiddy K, Webberley J (1987). Haemophilus aphrophilus as a cause of chronic suppurative pulmonary infection and intra-abdominal abscesses. J Infect 15(2):161-163. Kilian M (1976). A taxonomic study of the genus Haemophilus, with the proposal of a new species. J Gen Microbiol 93(1):9-62. Kinder SA, Badger JL, Bryant GO, Pepe JC, Miller VL (1993). Cloning of the YenI restriction endonuclease and methyltransferase from Yersinia enterocolitica serotype O8 and construction of a transformable R-M+ mutant. Gene 136(1-2):271-275. King EO, Tatum HW (1962). Actinobacillus actinomycetemcomitans and Hemophilus aphrophilus. J Infect Dis 111:85-94. Kittichotirat W, Bumgarner RE, Asikainen S, Chen C (2011). Identification of the pangenome and its components in 14 distinct Aggregatibacter actinomycetemcomitans strains by comparative genomic analysis. PLoS One 6(7):e22420. Kolodrubetz D, Phillips LH, Ezzo PJ, Kraig E (1995). Directed genomic integration in Actinobacillus actinomycetemcomitans: generation of defined leukotoxin-negative mutants. Infect Immun 63(7):2780-2784. Langer SJ, Ghafoori AP, Byrd M, Leinwand L (2002). A genetic screen identifies novel non-compatible loxP sites. Nucleic Acids Res 30(14):3067-3077. Lawrence JG, Ochman H (1997). Amelioration of bacterial genomes: rates of change and exchange. J Mol Evol 44(4):383-397. Lawrence JG, Ochman H (1998). Molecular archaeology of the Escherichia coli genome. Proc Natl Acad Sci U S A 95(16):9413-9417. Lee G, Saito I (1998). Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. Gene 216(1):55-65. Marx CJ, Lidstrom ME (2002). Broad-host-range cre-lox system for antibiotic marker recycling in gram-negative bacteria. Biotechniques 33(5):1062-1067. Mintz KP (2004). Identification of an extracellular matrix protein adhesin, EmaA, which mediates the adhesion of Actinobacillus actinomycetemcomitans to collagen. Microbiol 150(Pt 8):2677-2688. Miyasaki KT, Wilson ME, Reynolds HS, Genco RJ (1984). Resistance of Actinobacillus actinomycetemcomitans and differential susceptibility of oral Haemophilus species to the bactericidal effects of hydrogen peroxide. Infect Immun 46(3):644-648. Miyasaki KT, Wilson ME, Brunetti AJ, Genco RJ (1986a). Oxidative and nonoxidative killing of Actinobacillus actinomycetemcomitans by human neutrophils. Infect Immun 53(1):154-160. Miyasaki KT, Wilson ME, Genco RJ (1986b). Killing of Actinobacillus actinomycetemcomitans by the human neutrophil myeloperoxidase-hydrogen peroxide-chloride system. Infect Immun 53(1):161-165. Mlynarova L, Libantova J, Vrba L, Nap JP (2002). The promiscuity of heterospecific lox sites increases dramatically in the presence of palindromic DNA. Gene 296(1-2):129-137. Musser JM (1996). Molecular population genetic analysis of emerged bacterial pathogens: selected insights. Emerg Infect Dis 2(1):1-17. Nelson KE, Clayton RA, Gill SR, Gwinn ML, Dodson RJ, Haft DH et al. (1999). Evidence for lateral gene transfer between Archaea and bacteria from genome sequence of Thermotoga maritima. Nature 399(6734):323-329. Norskov-Lauritsen N, Kilian M (2006). Reclassification of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis as Aggregatibacter actinomycetemcomitans gen. nov., comb. nov., Aggregatibacter aphrophilus comb. nov. and Aggregatibacter segnis comb. nov., and emended description of Aggregatibacter aphrophilus to include V factor-dependent and V factor-independent isolates. Int J Syst Evol Microbiol 56(Pt 9):2135-2146. Ochman H, Lawrence JG, Groisman EA (2000). Lateral gene transfer and the nature of bacterial innovation. Nature 405(6784):299-304. Oscarsson J, Karched M, Thay B, Chen C, Asikainen S (2008). Proinflammatory effect in whole blood by free soluble bacterial components released from planktonic and biofilm cells. BMC Microbiol 8:206. Pallen MJ, Wren BW (2007). Bacterial pathogenomics. Nature 449(7164):835-842. Palmeros B, Wild J, Szybalski W, Le Borgne S, Hernandez-Chavez G, Gosset G et al. (2000). A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria. Gene 247(1-2):255-264. Pasqualini L, Mencacci A, Scarponi AM, Leli C, Fabbriciani G, Callarelli L et al. (2008). Cervical spondylodiscitis with spinal epidural abscess caused by Aggregatibacter aphrophilus. J Med Microbiol 57(Pt 5):652-655. Paster BJ, Olsen I, Aas JA, Dewhirst FE (2006). The breadth of bacterial diversity in the human periodontal pocket and other oral sites. Periodontol 2000 42:80-87. Perera IC, Lee YH, Wilkinson SP, Grove A (2009). Mechanism for attenuation of DNA binding by MarR family transcriptional regulators by small molecule ligands. J Mol Biol 390(5):1019-1029. Quenee L, Lamotte D, Polack B (2005). Combined sacB-based negative selection and cre-lox antibiotic marker recycling for efficient gene deletion in Pseudomonas aeruginosa. Biotechniques 38(1):63-67. Rylev M, Kilian M (2008). Prevalence and distribution of principal periodontal pathogens worldwide. J Clin Periodontol 35:346-361. Sambrook J, Fritsch EF, Maniatis T, editors (1989). Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory Press. Schmidt H, Hensel M (2004). Pathogenicity islands in bacterial pathogenesis. Clin Microbiol Rev 17(1):14-56. Selander RK, Musser JM, Caugant DA, Gilmore MN, Whittam TS (1987). Population genetics of pathogenic bacteria. Microbiol Pathog 3:1-7. Shao H, James D, Lamont RJ, Demuth DR (2007). Differential interaction of Aggregatibacter (Actinobacillus) actinomycetemcomitans LsrB and RbsB proteins with autoinducer 2. J Bacteriol 189(15):5559-5565. Slots J (1982). Salient Biochemical Characters of Actinobacillus actinomycetemcomitans. Arch Microbiol 131(1):60-67. Slots J (1999). Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in periodontal disease: introduction. Periodontol 2000 20:7-13. Sreenivasan PK, Fives-Taylor P (1994). Isolation and characterization of deletion derivatives of pDL282, an Actinobacillus actinomycetemcomitans/Escherichia coli shuttle plasmid. Plasmid 31(2):207-214. Swick MC, Morgan-Linnell SK, Carlson KM, Zechiedrich L (2011). Expression of Multidrug Efflux Pump Genes acrAB-tolC, mdfA, and norE in Escherichia coli Clinical Isolates as a Function of Fluoroquinolone and Multidrug Resistance. Antimicrob Agents Chemother 55(2):921-924. Tempro PJ, Slots J (1986). Selective medium for the isolation of Haemophilus aphrophilus from the human periodontium and other oral sites and the low proportion of the organism in the oral flora. J Clin Microbiol 23(4):777-782. Thomson VJ, Bhattacharjee MK, Fine DH, Derbyshire KM, Figurski DH (1999). Direct selection of IS903 transposon insertions by use of a broad-host-range vector: isolation of catalase-deficient mutants of Actinobacillus actinomycetemcomitans. J Bacteriol 181(23):7298-7307. Todar K (2009). Todar's online text book of bacteriology. In: K Todar editor: University of Wisconsin. Wang Y, Goodman SD, Redfield RJ, Chen C (2002). Natural transformation and DNA uptake signal sequences in Actinobacillus actinomycetemcomitans. J Bacteriol 184(13):3442-3449. Wang Y, Shi W, Chen W, Chen C (2003). Type IV pilus gene homologs pilABCD are required for natural transformation in Actinobacillus actinomycetemcomitans. Gene 312:249-255. Wang Y, Chen C (2005). Mutation analysis of the flp operon in Actinobacillus actinomycetemcomitans. Gene 351:61-71. Wang Y, Liu A, Chen C (2005). Genetic basis for conversion of rough-to-smooth colony morphology in Actinobacillus actinomycetemcomitans. Infect Immun 73(6):3749-3753. Wang YD, Zhao S, Hill CW (1998). Rhs elements comprise three subfamilies which diverged prior to acquisition by Escherichia coli. J Bacteriol 180(16):4102-4110. Wilkinson SP, Grove A (2006). Ligand-responsive transcriptional regulation by members of the MarR family of winged helix proteins. Curr Issues Mol Biol 8(1):51-62. Wolf J, Curtis N (2008). Brain abscess secondary to dental braces. Pediatr Infect Dis J 27(1):84-85. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/63664 | - |
dc.description.abstract | Horizontal gene transfer (HGT) is a process by which bacteria acquire genes from organisms of related or distant taxa. HGT is now recognized as a major driving force in the evolution of bacterial pathogens. Through this process, bacteria may accumulate blocks of DNA such as genomic islands (GEIs) that encode fitness or virulence factors. Aggregatibacter actinomycetemcomitans and Aggregatibacter aphrophilus are Gram-negative facultative oral bacteria. These two species share a high degree of genetic relatedness and identical oral ecological niches. However, the former is considered a major periodontal pathogen, while the latter is a commensal bacterium found frequently in healthy individuals. Moreover, A. actinomycetemcomitans has also been known to exhibit variable virulence potentials among strains. It was postulated that variation in virulence potentials between these Aggregatibacter spp., and among A. actinomycetemcomitans strains could be explained by different genomic contents due to HGT of individual virulence genes or GEI that carry virulence genes. Presumably HGT acquired genes will function in closely related bacterial host species such as A. actinomycetemcomitans and A. aphrophilus. Based on the hypothesis, we made the following predictions: (1) there are genomic islands unique to some strains of A. actinomycetemcomitans. The deletion of some of the islands may lead to reduced virulence potentials of the strains. (2) There are virulence genes presenting in A. actinomycetemcomitans but not in A. aphrophilus. The A. actinomycetemcomitans-specific virulence genes could be transferred and function in A. aphrophilus. Two aims were set to examine the hypothesis.
First, two known virulence determinants and seven strain-specific GEIs were deleted by the markless Cre/loxP system in natural transformable A. actinomycetemcomitans strain D7S-1. Subsequently the wildtype and mutants were tested for their growth rates and biofilm formation. The results indicated the deletion of the strain-specific 285-island (encoding proteins related to cross-membrane translocations and the multidrug efflux pump), significantly increased the growth rate of D7S-1. No other phenotypes were detected in other mutants. Also, two identified strain-specific genomic islands AAI-1 and AAI-2 in A. actinomycetemcomitans strain HK 1651 were deleted by the markless Cre/loxP system via conjugation. The functional annotations of AAI-1 are hypothetical proteins, putative adhesion and putative transposase, and AAI-2 encodes hypothetical proteins and Rhs family protein. The insertional inactivation of comM byAAI-2 may relate to the loss of natural competence of strain HK1651. While it was recognized that AAI-1 and AAI-2 were acquired by HGT, the growth curve study of the knockout mutants of AAI-1and AAI-2 did not reveal a notable variation in their phenotype of growth rate. In the second aim, the simulation of the acquisition of specific virulence determinants between Aggregatibacter species was developed by transferring the kat gene, an A. actinomycetemcomitans specific virulence factor, to A. aphrophilus strain NJ8700. The protective function against the bactericidal effect of hydrogen peroxide was carried out in the wild type A. actinomycetemcomitans D7S-1, rescued D7S-1 kat-deleted mutant and mutant A. aphrophilus expressing kat, while wild type NJ8700 and kat-deleted mutants of D7S-1 were sensitive to hydrogen peroxide treatment. The results supported that the lack of species barrier in the transfer and expression of genes between these two species. In conclusion, the strain specific GEI may play a role in the phenotype variations of A. actinomycetemcomitans. The natural transformation characteristic of A. actinomycetemcomitans and Cre/loxP recombination system proved to be an efficient and reliable approach in the construction of A. actinomycetemcomitans mutants with gene(s) deletion. In addition, Kat gene is able to be transferred form A. actinomycetemcomitans to A. aphrophilus, suggested that differential acquisition of genes for virulence determinants could occur during the evolution of the Aggregatibacter species. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T17:15:56Z (GMT). No. of bitstreams: 1 ntu-101-D92422004-1.pdf: 4862797 bytes, checksum: 019c23e326a678b87ce35eaa3675f194 (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 口試委員會審定書口試委員會審定書……………………………………………… i
謝誌……………………………………………………………………………………ii 中文摘要………………………………………….…………………………………..iii Abstract…….……………………………………………………………..…………..v Chapter 1. General Introduction ……………………………………………………..1 Chapter 2. Deletions of two strain-specific genomic islands in A. actinomycetemcomitans strain HK1651 and growth curve study 2.1 Introduction………………………………………………………………….....6 2.2 Materials and Methods 2.2.1 Bacterial strains and plasmids……………………………………………..8 2.2.2 DNA manipulations………………………………………………………..8 2.2.3 Site-specific deletion with the loxP /Cre system……………….………….9 2.2.4 Growth assay.……………………………………………….…………….11 2.3 Results 2.3.1 Construction of mutants with AAI-I/AAI-2 deletion……………..………12 2.3.2 Growth assay..……………………………………………….……………12 2.4 Discussion…………………………………………………………………......13 Chapter 3. Mutations of strain-specific genomic islands in A. actinomycetemcomitans strain D7S and phenotype study 3.1 Introduction……………………………………………………...………….....18 3.2 Materials and Methods 3.2.1 Bacterial strains and plasmids…………………………………………….19 3.2.2 DNA manipulations……………………………………………………….20 3.2.3 Construction of vectors containing the wild-type loxP-Spe-loxP cassette and its variants……………………………………………………………20 3.2.4 Site-specific gene deletion with the loxP /Cre system……...…………….21 3.2.5 Restoration of the deleted genes………………………………………….23 3.2.6 Determination of growth doubling time………………………………….23 3.2.7 Biofilm formation assay………………………………………………….24 3.3 Results 3.3.1 Construction of mutants …………………………...……………..………25 3.3.2 Growth and biofilm formation……………………………….…………...26 3.4 Discussion…………………………………………………………………......27 Chapter 4. Deletion, restoration and transfer of kat gene in Aggregatibacter species 4.1 Introduction……………………………….…………………….……………..31 4.2 Materials and Methods 4.2.1 Bacterial strains and plasmids…………………………………………….32 4.2.2 DNA manipulations……………………………………………………….33 4.2.3 Natural transformation assay…………………..………………………….33 4.2.4 Site-specific kat gene deletion and restoration with the loxP /Cre system……………………………………………………………….……34 4.2.5 kat gene in A. actinomycetemcomitans strain D7S-1 transferring to A. aphrophilus strain NJ8700…………………………….………………….36 4.2.6 The H2O2 killing assay…………………..…………………………….….36 4.3 Results 4.3.1 Natural competence of A. aphrophilus……...……...……………..………37 4.3.2 Construction of mutants………..…………………………….…………...37 4.3.3 The H2O2 killing assay…………………..…………………………….….38 4.4 Discussion………………….………………………………………………….38 Summary statements…..……………………………………………………..……..41 References………………………….………………………………………………..42 Figures……………………………………………………………………………….49 Tables………………………………………………………………………………...74 Appendix……………………………………………………………………….........91 List of Figures Figure 1. Comparison between strain HK1651 and strain ANH9381 at the AAI-1 insertion site………………………………………………………….……49 Figure 2. Comparison between strain HK1651 and strain D7S at the AAI-2 insertion site………………………………………………………….………...……50 Figure 3. The structures of suicide plasmids pk-loxT1 and pEP-lox1……………....51 Figure 4. The strategy of creating the deletion mutant HK (AAI-1) ………..……....52 Figure 5. The strategy of creating the deletion mutant HK (AAI-2) ………………..53 Figure 6. The steps of performing growth assay for wildtype and mutant HK1651...54 Figure 7A. Mutant verifications by PCR…………………………………………….55 B. Gel picture of PCR verifications for deletion mutants…………………..56 Figure 8. The sequencing confirmation of AAI-1 deletion in HK1651S-F………….57 Figure 9. The sequencing confirmation of AAI-2 deletion in HK1651S-F………….58 Figure 10 A. The growth curve plotted by OD600 versus day B. The growth curve plotted by log (CFU/ml) versus day………………..59 Figure 11 A. The growth curve plotted by OD600 versus hour B. The growth curve plotted by log(CFU/ml) versus hour…………….....60 Figure 12. The natural transformation protocol for D7S-1…………………………..61 Figure 13A. Deletion of the 285-island of D7S-1…………………………………..62 B. Restoration of the 285-island of D7S-1…………………………...…..63 Figure 14 A. Mutant verifications by PCR…………………………….…………….64 B. Gel picture of PCR verifications for deletion mutants…….…………..65 Figure 15. Microscopic images of biofilm formation of D7S-1……………………..66 Figure 16. Genetic comparison of catalase in A. actinomycetemcomitans genome and the comparable region in A. aphrophilus genome……..……..………….67 Figure 17. Steps for construction of kat-deleted mutants of A. actinomycetemcomitans strain D7S-1…………….………………………..………………………68 Figure 18. Steps for construction of kat-restored mutants for D7S-kat-………..…....69 Figure 19. The catalase bubbling reaction…………………………………………...70 Figure 20. Steps for construction of the plasmids carrying kat gene, served as donor DNA in A. aphrophilus transformation………………………...………..71 Figure 21. Steps for construction of kat-expressed mutants of A. aphrophilus……...72 Figure 22. Sequencing confirmation of A. aphrophilus and A. actinomycetemcomitans mutants…………………………………………………………………...73 List of Tables Table 1. All bacterial strains used in this work………………………………………74 Table 2.All plasmids used in this work………………………………………………76 Table 3. List of primers used for AAI-1 and AAI-2 deletions in HK1651S-F……….77 Table 4. Doubling times of A. actinomycetemcomitans strain HK1651 wildtype and mutants………………………………………………………...……………78 Table 5. List of primers for D7S-1 genes/GEIs mutations……………………..……79 Table 6. Doubling times of A. actinomycetemcomitans strain D7S-1 wildtype and mutants………………………………………………………...……………83 Table 7. Comparison of biofilm formation of A. actinomycetemcomitans strain D7S-1 wildtype and mutants………………………………………………….……84 Table 8. List of primers used for kat gene deletion and restoration in A. actinomycetemcomitans strain D7S-1……………………………………....85 Table 9. List of primers used for kat gene transfer to A. aphrophilus NJ8700 from D7S-1………………………………………………………...……………..86 Table 10. General taxonomy and differences between A. aphrophilus and A. actinomycetemcomitans…………………………………………………..87 Table 11. H2O2 bactericidal assay……………………………………………..……..90 | |
dc.language.iso | en | |
dc.title | Aggregatibacter菌種水平轉移基因分析 | zh_TW |
dc.title | Genetic analysis of horizontally transferred genes in Aggregatibacter species | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 鄧麗珍,傅鍔,藍萬烘,林弘萍,鄭景暉 | |
dc.subject.keyword | A. actinomycetemcomitans,Aggregatibacter species,水平基因轉移,基因組島,毒性因子, | zh_TW |
dc.subject.keyword | A. actinomycetemcomitans,Aggregatibacter species,genomic island,horizontal gene transfer,virulence factor, | en |
dc.relation.page | 90 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-08-19 | |
dc.contributor.author-college | 牙醫專業學院 | zh_TW |
dc.contributor.author-dept | 臨床牙醫學研究所 | zh_TW |
顯示於系所單位: | 臨床牙醫學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-101-1.pdf 目前未授權公開取用 | 4.75 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。