Species wide analysis of Escherichia coli identifies a negative regulatory input that controls flhDC transcription

i Dedication . ii Acknowledgements . iii Table of Figures . viii Table of Tables . xi List of Abbreviations . xii Chapter One: Introduction . 1 1.1. Escherichia coli basic characteristics . 2 1.2. Strain diversity of the species E. coli . 2 1.2.1. E. coli and its pangenome . 2 1.2. 2. Early molecular approaches for bacterial classification . 7 1.2. 3. Multilocus sequence typing . 11 1.2. 4. Model strains of E. coli . 12 1.3. Pathogenicity and virulence factors of E. coli pathovars . 15 1.3.1. Enteropathogenic E. coli (EPEC) . 15 1.3.2. Enterohaemorrhagic E. coli (EHEC) . 16 1.3.3. Enterotoxigenic E. coli (ETEC) . 16 1.3.4. Enteroinvasive E. coli (EIEC) . 17 1.3.5. Enteroaggregative E. coli (EAEC) . 20 1.3.6. Diffusely adherent E. coli (DAEC) . 22 1.3.7. Adherent invasive E. coli (AIEC) . 22 1.3.8. Neonatal meningitis E. coli (NMEC) . 23 1.3.9. Uropathogenic E. coli (UPEC) . 23 1.4. Key methods in bacterial recombineering . 28 1.4. 1. Datsenko and Wanner . 28 1.4. 2. Blank method . 30 1.4. 3. CRISPR-Cas9 system . 33 1.4. 4. Bioluminescence and fluorescent reporters. 36 1.5. Movement in bacteria . 38 1.5. 1. Flagella independent motility . 39 1.5. 2. Flagella dependent motility . 40 1.6. The structure and assembly of the flagellum . 40 1.6. 1. The basal body . 41 1.6. 2. The motor . 42 v 1.6. 3. The flagellar protein secretion apparatus . 44 1.6. 4. The hook . 47 1.6. 5. The filament . 47 1.7. The regulation of flagellar genes . 48 1.7. 1. The flagellar master reulator: FlhD4C2 . 51 1.7. 2. Transcription of flhDC . 52 1.7. 3. IS elements . 56 1.7. 4. Degradation of FlhD4C2 . 61 1.7. 5. Other aspects of regulation in the flagellar system . 62 1.7. 6. FlgM-σ28 regulatory checkpoint . 64 1.8. Biofilm formation: The other life style . 68 Chapter Two: Aims of Study . 75 2.1. Aims of study. 76 Chapter Three: Materials and Methods . 79 3.1. Bacterial Strains Activation and Growth Conditions . 80 3.1.1. Standard growth conditions . 80 3.1.2. Growth curve in minimal media . 80 3.2. Antibiotic concentrations . 80 3.3. Motility assay. 81 3.4. Polymerase chain reaction . 81 3.5. Agarose gel DNA electrophoresis . 82 3.6. DNA Sequencing . 82 3.7. Isolation of plasmid DNA . 82 3.7. 1. Crude mini method . 82 3.7. 2. Miniprep kit purification of DNA . 83 3.7. 3. Midiprep kit purification of DNA . 83 3.8. Isolation of bacterial genomic DNA . 83 3.8. 1. Colony-PCR . 83 3.8. 2. Culture-PCR . 83 3.8. 3. Bacterial genomic DNA isolation kit . 84 3.9. DNA extraction from a gel . 84 3.10. Purification of PCR products . 84 3.11. Concentrating DNA by ethanol precipitation . 84 3.12. Molecular cloning . 85 3.12.1. Restriction Digest . 85 3.12. 2. DNA ligation . 85 vi 3.12. 3. Gibson assembly . 86 3.13. DNA Transformation . 86 3.13.1. Preparation of competent E. coli cells for heat-shock . 87 3.13.2. Chemical transformation of DNA (heat shock) . 87 3.13.3. Preparation of competent E. coli cells for electroporation . 88 3.13.4. Transforming DNA by electroporation . 88 3.14. DNA recombination . 89 3.14. 1. Datsenko and Wanner method . 89 3.14. 2. Blank method . 90 3.14. 3. CRISPR-Cas9 method . 91 3.15. Measuring bioluminescence activity . 91 3.16. Fluorescence microscopy . 92 3.17. Image analysis . 92 3.18. Multilocus sequence typing (MLST) . 93 3.19. MLST alignment . 94 3.20. Biofilm formation . 94 3.21. Statistical analysis . 95 Chapter Four: Species Wide Analysis of Escherichia coli flhDC Promoter Activity and Regulation . 97 4.