Characterisation of the unique Campylobacter jejuni cytochrome P450, CYP172A1. A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences. 2013 Peter C. Elliott Preliminary Pages Table of Contents List of Figures ................................................................................................................................ 7 List of Tables ............................................................................................................................... 10 List of abbreviations .................................................................................................................... 11 Abstract ........................................................................................................................................ 14 Declaration ................................................................................................................................... 15 Copyright ..................................................................................................................................... 15 Acknowledgements ...................................................................................................................... 16 Chapter 1. Introduction ................................................................................................................ 17 1.1.1. The Campylobacter genus and related organisms ......................................................... 17 1.1.2 Campylobacter jejuni and its general features ................................................................ 18 1.1.3. C. jejuni infection sources .............................................................................................. 20 1.2. C. jejuni infection and symptoms ..................................................................................... 21 1.2.1. C. jejuni adhesion to epithelial cells .............................................................................. 21 1.2.3. C. jejuni invasion of epithelial cells ............................................................................... 22 1.3. Antibiotic resistance in Campylobacter species ............................................................... 23 1.4. Surface structures of C. jejuni. .......................................................................................... 25 1.4.1. The Flagellum ................................................................................................................ 25 1.4.2. C. jejuni lipooligosaccharide (LOS) .............................................................................. 28 1.4.3. C. jejuni capsule polysaccharide (CPS) locus ................................................................ 30 1.4.4. C. jejuni CPS structural variability and serotyping ........................................................ 30 1.5. The presence of CYP172A1 in Campylobacter jejuni. ..................................................... 34 1.6. History of the cytochrome P450 ....................................................................................... 36 1.6.1. The prevalence of P450s in nature ................................................................................. 38 1.6.2. Bacterial P450s .............................................................................................................. 40 1.7. P450 Structure ................................................................................................................... 40 1.8. Catalytic cycle of P450s .................................................................................................... 42 1.9. Redox partners .................................................................................................................. 48 1.10. Flavin-containing proteins .............................................................................................. 50 1.10.1. Ferredoxin/Flavodoxin reductases ............................................................................... 52 2 Preliminary Pages 1.10.2. Flavodoxins .................................................................................................................. 53 1.10.3. Iron-sulphur containing proteins .................................................................................. 54 1.11. Inhibitors of P450s .......................................................................................................... 55 1.12. Aims of the project ......................................................................................................... 56 Chapter 2. Materials and Methods ............................................................................................... 58 2.1. Materials ........................................................................................................................... 58 2.2. Growth media ................................................................................................................... 58 2.3. Antibiotics ......................................................................................................................... 61 2.4. Campylobacter jeuni strains ............................................................................................. 62 2.5. Purification of C. jejuni genomic DNA ............................................................................ 63 2.6. Amplification of target DNA by PCR .............................................................................. 63 2.7. Preparation of electrocompetent C. jejuni ........................................................................ 64 2.8. Sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis .... 65 2.9. Western blotting ................................................................................................................ 65 2.10. C. jejuni growth assays ................................................................................................... 67 2.11. C. jejuni autoagglutination assays ................................................................................... 67 2.12. Electron Microscopy (EM) ............................................................................................. 68 2.13. C. jejuni motility Assay .................................................................................................. 68 2.14. Minimum Inhibitory Concentration (MIC) assays for C. jejuni ..................................... 68 2.15. Polymyxin B susceptibility assays .................................................................................. 69 2.16. Disc diffusion assay for C. jejuni .................................................................................... 70 2.17. Tricine-SDS-PAGE for C. jejuni capsule and LOS visualisation. .................................. 70 2.18.1. Fourier Transform Infrared (FT-IR) Spectroscopy ...................................................... 71 2.18.2. Cluster Analysis of FT-IR ............................................................................................ 72 2.19. Surface Enhanced Raman Spectroscopy (SERS)............................................................ 73 2.20. Bacterial cell strains ........................................................................................................ 74 2.21. Restriction enzyme digests ............................................................................................. 74 2.22. E. coli transformations .................................................................................................... 75 2.24. Site Directed Mutagenesis (SDM) .................................................................................. 76 2.25. Preparation of E. coli glycerol stocks ............................................................................. 77 2.26. E. coli expression trials of the cj1411c gene product ..................................................... 77 2.27. CYP172A1 solubility trials ............................................................................................. 78 3 Preliminary Pages 2.28. Temperature trials for CYP172A1 expression in E. coli ................................................ 78 2.28.1 IPTG gene induction trials ............................................................................................ 79 2.28.2. Growth Medium trials for target P450 production ....................................................... 79 2.29. Expression and purification of CYP172A1 .................................................................... 79 2.30. CYP172A1 antibody production ..................................................................................... 80 2.31. UV-Visible spectroscopy ................................................................................................ 81 2.31.1. Dual beam UV-Visible spectroscopy ........................................................................... 81 2.31.2. Fluorescence spectroscopy ........................................................................................... 81 2.32. Pyridine Hemochromogen Assay .................................................................................... 82 2.33. Redox potentiometry ....................................................................................................... 82 2.34. Differential Scanning Calorimetry (DSC) ...................................................................... 84 2.35. Electron Paramagnetic Resonance (EPR) Spectroscopy