泌尿道致病型大腸桿菌為主要造成人類泌尿道感染的致病菌，其中與共生性大腸桿菌的最大差別在於毒性基因的分布；除了基因體的差異，在一些特定DNA序列上的修飾也會造成致病菌致病力的改變，而DNA甲基轉移酶為執行DNA修飾的酵素，因此DNA甲基轉移酶也可能成為毒性因子。目前的單一分子即時偵測定序系統雖然能夠挖掘未知甲基轉移酶，但是無法藉由觀察菌株外觀得知結果。另外，當DNA甲基轉移酶與切割甲基化序列的第IV型限制酶共存於同一宿主，若兩酵素的辨認序列重疊時，則宿主DNA即被前者酵素甲基化後又被後者切割造成死亡，限制菌株水平基因轉移的方向。結合上述，我們發展一套自殺型供體菌株的接合作用，篩選呈現死亡現象(Conflict)的菌株，並分析所擁有的DNA甲基轉移酶對於致病菌與其致病力的相關性。我們以泌尿道致病型大腸桿菌作為篩選對象，將第IV型限制酶 (McrBC) 傳送至83株臨床泌尿道感染分離株以及50株健康病人糞便分離株。我們篩選出包含至少9種胞嘧啶甲基轉移酶的29 株Conflict菌株，並進一步在大腸桿菌K-12菌株證實McrBC皆與9種甲基轉移酶之辨認序列重疊。在泌尿道感染分離株的分布中，Conflict菌株的比例 (27.85%) 顯著高於糞便分離株的比例 (10.2%)，暗示所篩選的甲基轉移酶與泌尿道致病菌致病性有關聯性；然而在線蟲模式中，卻沒有發現Conflict菌株與其致病力高低的相關性。綜合以上結果，我們推測該9種DNA胞嘧啶甲基轉移酶可能參與泌尿道致病菌獲得外來基因的演化過程，而非影響菌株致病力能力的重要因素之一。

Uropathogenic E. coli (UPEC) is a subtype of extraintestinal pathogenic E. coli, and many virulence genes have been identified for the uropathogenicity. In this regard, the gene constitution of the E. coli genome is very important. Because DNA methylation of specific sequence on the genome has been identified to be involved in pathogenicity, DNA methyltransferases (DNMT) can be a virulence factor. The single molecular real time (SMRT) sequencing is available to identify methylated sequence. However, phenotypical approach is also necessary to assign the recognition sequence of DNMT. In this study, we develop a suicide-donor-conjugation method to screen a combination of DNMT and restriction enzyme, which recognize overlapping sequences. A type IV restriction enzyme (McrBC), which specifically recognizes methylated site, was used to carry out the screening in UPEC as a model.
A plasmid expressing McrBC was transferred to 83 of clinically isolated UPEC and 50 of fecal E. coli isolates through the suicide donor conjugation. When a recipient harbors a cytosine-5-DNMT which recognizes a sequence overlapping with McrBC, its genome DNA will be methylated and restricted, and then the cell shows a conflict phenotype (lethal). Twenty-nine strains were identified as shown the conflict from the 133 strains. Frequency of the conflict in the UTI isolates (24/83) showed significantly higher than that in the fecal isolates (5/50), suggested that the DNMTs conflicted with McrBC were associated with uropathogenicity. Nine C5-DNMT genes covering 24 of the 29 strains were identified as determinants of the conflict. Their clone showed the conflict and overlapping of recognition sequence with McrBC in K-12 strain. We also challenged whether the DNMTs associated with higher pathogenicity. Caenorhabditis elegans model was adopted for the survey, however, there are no significant association between the existence of DNMT and pathogenicity level in the model. These results implied that the DNMTs involved in horizontal gene transfer to acquire virulence genes, but did not involved to increase the pathogenicity.

1. Croxen, M.A., et al., Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev, 2013. 26(4): p. 822-80.
2. Croxen, M.A. and B.B. Finlay, Molecular mechanisms of Escherichia coli pathogenicity. Nat Rev Microbiol, 2010. 8(1): p. 26-38.
3. Hudault, S., J. Guignot, and A.L. Servin, Escherichia coli strains colonising the gastrointestinal tract protect germfree mice against Salmonella typhimurium infection. Gut, 2001. 49(1): p. 47-55.
4. Kuhnert, P., J. Nicolet, and J. Frey, Rapid and accurate identification of Escherichia coli K-12 strains. Appl Environ Microbiol, 1995. 61(11): p. 4135-9.
5. Durfee, T., et al., The complete genome sequence of Escherichia coli DH10B: insights into the biology of a laboratory workhorse. J Bacteriol, 2008. 190(7): p. 2597-606.
6. Kaper, J.B., J.P. Nataro, and H.L. Mobley, Pathogenic Escherichia coli. Nat Rev Microbiol, 2004. 2(2): p. 123-40.
7. Souza, V., et al., Genetic Structure of Natural Populations of Escherichia coli in Wild Hosts on Different Continents. Applied and Environmental Microbiology, 1999. 65(8): p. 3373-3385.
8. Edouard Bingen, B.P., Naïma Brahimi, Stéphanie Mathy, Patricia Desjardins, Jacques Elion and Erick Denamur, Phylogenetic Analysis of Escherichia coli Strains Causing Neonatal Meningitis Suggests Horizontal Gene Transfer from a Predominant Pool of Highly Virulent B2 Group Strains. The Journal of Infectious Diseases, 1998. 177(3): p. 642-650.
9. Boyd, E.F. and D.L. Hartl, Chromosomal Regions Specific to Pathogenic Isolates of Escherichia coli Have a Phylogenetically Clustered Distribution. Journal of Bacteriology, 1998. 180(5): p. 1159-1165.
10. Bonacorsi, S.P.P., et al., Identification of Regions of the Escherichia coli Chromosome Specific for Neonatal Meningitis-Associated Strains. Infection and Immunity, 2000. 68(4): p. 2096-2101.
11. Blattner, F.R., et al., The complete genome sequence of Escherichia coli K-12. Science, 1997. 277(5331): p. 1453.
12. Subashchandrabose, S. and H.L. Mobley, Virulence and Fitness Determinants of Uropathogenic Escherichia coli. Microbiol Spectr, 2015. 3(4).
13. Gao, Q., et al., Roles of iron acquisition systems in virulence of extraintestinal pathogenic Escherichia coli: salmochelin and aerobactin contribute more to virulence than heme in a chicken infection model. BMC Microbiol, 2012. 12: p. 143.
14. Alteri, C.J., et al., Mucosal immunization with iron receptor antigens protects against urinary tract infection. PLoS Pathog, 2009. 5(9): p. e1000586.
15. I Johanson, R.L., C Svanborg, Roles of the pap- and prs-encoded adhesins in Escherichia coli adherence to human uroepithelial cells. Infect Immun., 1992. 60(8): p. 3416–3422.
16. Blum, G., et al., Gene clusters encoding the cytotoxic necrotizing factor type 1, Prs-fimbriae and α-hemolysin form the pathogenicity island II of the uropathogenic Escherichia coli strain J96. FEMS Microbiology Letters, 1995. 126(2): p. 189-195.
17. Welch, R.A., Uropathogenic Escherichia coli-Associated Exotoxins. Microbiol Spectr, 2016. 4(3).
18. Guyer, D.M., et al., Sat, the Secreted Autotransporter Toxin of Uropathogenic Escherichia coli, Is a Vacuolating Cytotoxin for Bladder and Kidney Epithelial Cells. Infection and Immunity, 2002. 70(8): p. 4539-4546.
19. Jones, P.A., Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet, 2012. 13(7): p. 484-92.
20. Kommireddy Vasu, V.N., Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense. Microbiol Mol Biol Rev, 2013. 77(1): p. 53-72.
21. Sanchez-Romero, M.A., I. Cota, and J. Casadesus, DNA methylation in bacteria: from the methyl group to the methylome. Curr Opin Microbiol, 2015. 25: p. 9-16.
22. T A Bickle, D.H.K., Biology of DNA Restriction. Microbiol Rev, 1993. 57(2): p. 434–450.
23. Messer, W. and M. Noyer-Weidner, Timing and targeting: The biological functions of Dam methylation in E. coli. Cell, 1988. 54(6): p. 735-737.
24. Low, D.A., N.J. Weyand, and M.J. Mahan, Roles of DNA adenine methylation in regulating bacterial gene expression and virulence. Infect Immun, 2001. 69(12): p. 7197-7204.
25. Blyn, L.B., B.A. Braaten, and D.A. Low, Regulation of pap pilin phase variation by a mechanism involving differential dam methylation states. The EMBO Journal, 1990. 9(12): p. 4045-4054.
26. Murphy, K.C., et al., Dam methyltransferase is required for stable lysogeny of the Shiga toxin (Stx2)-encoding bacteriophage 933W of enterohemorrhagic Escherichia coli O157:H7. J Bacteriol, 2008. 190(1): p. 438-41.
27. Cohen, N.R., et al., A role for the bacterial GATC methylome in antibiotic stress survival. Nature Genetics, 2016. 48: p. 581.
28. Militello, K.T., et al., Conservation of Dcm-mediated cytosine DNA methylation in Escherichia coli. FEMS Microbiol Lett, 2012. 328(1): p. 78-85.
29. Flusberg, B.A., et al., Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat Methods, 2010. 7(6): p. 461-5.
30. A Janulaitis, M.P., Z Maneliene, S Klimasauskas, V Butkus, Purification and properties of the Eco57I restriction endonuclease and methylase--prototypes of a new class (type IV). Nucleic Acids Res., 1992. 20(22): p. 6043–6049.
31. Loenen, W.A. and E.A. Raleigh, The other face of restriction: modification-dependent enzymes. Nucleic Acids Res, 2014. 42(1): p. 56-69.
32. Liu, G., et al., Cleavage of phosphorothioated DNA and methylated DNA by the type IV restriction endonuclease ScoMcrA. PLoS Genet, 2010. 6(12): p. e1001253.
33. Tesfazgi Mebrhatu, M., et al., Evidence for an evolutionary antagonism between Mrr and Type III modification systems. Nucleic Acids Res, 2011. 39(14): p. 5991-6001.
34. Ellen Sutherland, L.C., Elisabeth A. Raleigh, McrBC a Multisubunit GTP-dependent Restriction Endonuclease. Journal of Molecular Biology, 1992. 225(2): p. 327-348.
35. Mulligan, E.A. and J.J. Dunn, Cloning, purification and initial characterization of E. coli McrA, a putative 5-methylcytosine-specific nuclease. Protein Expr Purif, 2008. 62(1): p. 98-103.
36. P A Waite-Rees, C.J.K., L S Moran, B E Slatko, L J Hornstra, J S Benner, Characterization and expression of the Escherichia coli Mrr restriction system. J Bacteriol, 1991. 173(16): p. 5207–5219.
37. Fukuda, E., et al., Correction: Cell death upon epigenetic genome methylation: a novel function of methyl-specific deoxyribonucleases. Genome Biology, 2011. 12(11): p. 412.
38. Chen, I., P.J. Christie, and D. Dubnau, The ins and outs of DNA transfer in bacteria. Science, 2005. 310(5753): p. 1456-60.
39. Skurray, N.A.F.a.K.I.-I.a.R.A., Structure and Function of the F Factor and Mechanism of Conjugation. 1999.
40. Springer, B., et al., Mechanisms of streptomycin resistance: selection of mutations in the 16S rRNA gene conferring resistance. Antimicrob Agents Chemother, 2001. 45(10): p. 2877-84.
41. Tan, M.-W., S. Mahajan-Miklos, and F.M. Ausubel, Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis. Proceedings of the National Academy of Sciences of the United States of America, 1999. 96(2): p. 715-720.
42. Yuen, G.J. and F.M. Ausubel, Both live and dead Enterococci activate Caenorhabditis elegans host defense via immune and stress pathways. Virulence, 2018. 9(1): p. 683-699.
43. Diard, M., et al., Caenorhabditis elegans as a simple model to study phenotypic and genetic virulence determinants of extraintestinal pathogenic Escherichia coli. Microbes Infect, 2007. 9(2): p. 214-23.
44. Alexandra Merkx-Jacques, A.C., Roland Brousseauc, Luke Masson, Alberto Mazza, Yuan-Ching Tien, Edward Topp, Evaluating the pathogenic potential of environmental Escherichia coli by using the Caenorhabditis elegans infection model. Appl. Environ. Microbiol., 2013. 79(7): p. 2435-2445.
45. Byerly, L., R.C. Cassada, and R.L. Russell, The life cycle of the nematode Caenorhabditis elegans: I. Wild-type growth and reproduction. Developmental Biology, 1976. 51(1): p. 23-33.
46. Cassada, R.C. and R.L. Russell, The dauerlarva, a post-embryonic developmental variant of the nematode Caenorhabditis elegans. Developmental Biology, 1975. 46(2): p. 326-342.
47. Chin-Chung Tseng, J.-J.H., Wen-Chien Ko, Jing-Jou Yan, Jiunn-Jong Wu, Decreased Predominance of papG Class II Allele In Escherichia Coli Strains Isolated From Adults With Acute Pyelonephritis and Urinary Tract Abnormalities. The Journal of Urology, 2001. 166(5): p. 1643-1646.
48. Mao, B.H., et al., Identification of Escherichia coli genes associated with urinary tract infections. J Clin Microbiol, 2012. 50(2): p. 449-56.
49. Olivier Clermont, S.B., Edouard Bingen, Rapid and Simple Determination of the Escherichia coli Phylogenetic Group. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2000. 66(10): p. 4555–4558.
50. Nipic, D., et al., Escherichia coli uropathogenic-specific protein, Usp, is a bacteriocin-like genotoxin. J Infect Dis, 2013. 208(10): p. 1545-52.
51. Chapman, T.A., et al., Comparison of virulence gene profiles of Escherichia coli strains isolated from healthy and diarrheic swine. Appl Environ Microbiol, 2006. 72(7): p. 4782-95.
52. James R. Johnson, A.L.S., Extended Virulence Genotypes of Escherichia coli Strains from Patients with Urosepsis in Relation to Phylogeny and Host Compromise. The Journal of Infectious Diseases, 2000. 181(1): p. 261-272.
53. Li, L.C. and R. Dahiya, MethPrimer: designing primers for methylation PCRs. Bioinformatics, 2002. 18(11): p. 1427-1431.
54. Sulston, J. and J. Hodgkin, Methods. 1988. Vol. 17. 1988.
55. Stiernagle, T., Maintenance of C. elegans. WormBook, 2006: p. 1-11.
56. Johnson, J.R., et al., Bacterial characteristics in relation to clinical source of Escherichia coli isolates from women with acute cystitis or pyelonephritis and uninfected women. J Clin Microbiol, 2005. 43(12): p. 6064-72.
57. Duriez, P., et al., Commensal Escherichia coli isolates are phylogenetically distributed among geographically distinct human populations. Microbiology, 2001. 147(6): p. 1671-1676.
58. Merlin, C., et al., Why Is Carbonic Anhydrase Essential to Escherichia coli? Journal of Bacteriology, 2003. 185(21): p. 6415-6424.
59. Masayuki HASHIMOTO, J.-i.K., Indispensability of the Escherichia coli carbonic anhydrases YadF and CynT in cell proliferation at a low CO2 partial pressure. Biosci. Biotechnol. Biochem, 2003. 67(4): p. 919-922.
60. János Pósfai, A.S.B., Gyorgy Pósfai', and Richard J. Roberts, Predictive motifs derived from cytosine methyltransferases. Nucleic Acids Res, 1989. 17: p. 2421-2435.
61. Page, R.D.M., TreeView: An application to display phylogenetic trees on personal computers. Bioinformatics and Computational Biology, 1996. 12(4): p. 357-358.
62. Anastasi, E.M., et al., Pathogenic Escherichia coli found in sewage treatment plants and environmental waters. Appl Environ Microbiol, 2012. 78(16): p. 5536-41.
63. Bergeron, C.R., et al., Chicken as reservoir for extraintestinal pathogenic Escherichia coli in humans, Canada. Emerg Infect Dis, 2012. 18(3): p. 415-21.
64. Xia, X., et al., Escherichia coli from retail meats carry genes associated with uropathogenic Escherichia coli, but are weakly invasive in human bladder cell culture. J Appl Microbiol, 2011. 110(5): p. 1166-76.
65. Tenaillon, O., et al., The population genetics of commensal Escherichia coli. Nat Rev Microbiol, 2010. 8(3): p. 207-17.
66. Chiou, C.S., et al., Identification of prophage gene z2389 in Escherichia coli EDL933 encoding a DNA cytosine methyltransferase for full protection of NotI sites. Int J Med Microbiol, 2010. 300(5): p. 296-303.
67. Lee KF, K.K., Shaw PC, A bacterial methyltransferase M.EcoHK31I requires two proteins for in vitro methylation. Nucleic Acids Res. , 1995. 23(1): p. 103-108.
68. Aaron D Hernday, B.A.B., David A Low, The Mechanism by which DNA Adenine Methylase and PapI Activate the Pap Epigenetic Switch. Molecular Cell, 2003. 12(4): p. 947-957.
69. Oza, J.P., J.B. Yeh, and N.O. Reich, DNA methylation modulates Salmonella enterica serovar Typhimurium virulence in Caenorhabditis elegans. FEMS Microbiol Lett, 2005. 245(1): p. 53-9.
70. Jonsson, M., Investigation of in vivo effects by E. coli virulence factors using wild type and mutant Caenorhabditis elegans as an invertebrate animal infection model system.