UCC Library and UCC researchers have made this item openly available. Please let us know how this has helped you. Thanks! Title Helicobacter pylori: comparative genomics and structure-function analysis of the flagellum biogenesis protein HP0958 Author(s) de Lacy Clancy, Ceara A. Publication date 2014 Original citation de Lacy Clancy, C. A. 2014. Helicobacter pylori: comparative genomics and structure-function analysis of the flagellum biogenesis protein HP0958. PhD Thesis, University College Cork. Type of publication Doctoral thesis Rights © 2014, Ceara A. De Lacy Clancy. http://creativecommons.org/licenses/by-nc-nd/3.0/ Item downloaded http://hdl.handle.net/10468/1684 from Downloaded on 2021-10-10T14:33:51Z Helicobacter pylori: Comparative genomics and structure-function analysis of the flagellum biogenesis protein HP0958 A Thesis Presented in Partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy by Ceara de Lacy Clancy, B.Sc. School of Microbiology National University of Ireland, Cork Supervisor: Prof. Paul W. O’Toole Head of School of Microbiology: Prof. Gerald Fitzgerald February 2014 Table of Contents Table of Contents Abstract ........................................................................................................................ i Chapter 1 Literature Review....................................................................................................... 1 1 Helicobacter pylori .................................................................................................. 2 1.1 Discovery of H. pylori.............................................................................. 2 1.2 Morphology and Physiology .................................................................... 5 1.3 Genetics .................................................................................................... 7 2 Pathogenesis of Helicobacter pylori ...................................................................... 10 2.1 Epidemiology ......................................................................................... 10 2.2 Transmission .......................................................................................... 11 2.3 Infection and Inflammation .................................................................... 12 2.4 Virulence Factors ................................................................................... 14 2.5 Disease ................................................................................................... 20 2.6 Diagnostics ............................................................................................. 21 2.7 Treatment ............................................................................................... 22 3 Bacterial Secretion Systems .................................................................................. 24 3.1 Type I Secretion System ........................................................................ 26 3.2 Type II Secretion System ....................................................................... 26 3.3 Type III Secretion System ...................................................................... 27 3.4 Type IV Secretion System ..................................................................... 29 3.5 Type V Secretion System ....................................................................... 30 3.6 Type VI Secretion System ..................................................................... 31 3.7 Type VII Secretion System .................................................................... 32 4 Composition and Organization of the Bacterial Flagellum ................................... 33 4.1 Morphology of the flagellum in Enterobacteriaceae............................. 33 4.2 Flagellum Assembly in Enterobacteriaceae .......................................... 37 4.3 Regulation of Flagellar Assembly .......................................................... 39 4.4 Flagellar chaperones in Enterobacteriaceae .......................................... 40 4.5 Regulation of flagellum biogenesis in H. pylori .................................... 41 4.6 HP0958 ................................................................................................... 44 5 Aims of this Study ................................................................................................. 48 6 References ............................................................................................................. 50 Chapter 2 Genome Sequences and Comparative Genomics of Two Motile Helicobacter pylori Strains ....................................................................................... 83 Published Genome Announcement: Draft Genome Sequences of Helicobacter pylori Strains 17874 and P79............. 84 1 Abstract .................................................................................................................. 85 2 Genome Announcement ........................................................................................ 85 3 Nucleotide Sequence Accession Numbers ............................................................ 86 4 Acknowledgements ............................................................................................... 86 5 References ............................................................................................................. 87 Expanded Thesis Analysis: Comparative Genomics of Two Motile Helicobacter pylori Strains and Core Genome Analysis of 43 Strains ...................................................................... 88 1 Abstract .................................................................................................................. 89 Table of Contents 2 Introduction ........................................................................................................... 90 3 Methods ................................................................................................................. 92 3.1 Bacterial Strains and Culture Conditions ............................................... 92 3.2 Genomic DNA Extraction ...................................................................... 92 3.3 Genome Sequencing and Annotation ..................................................... 92 3.4 Genome Comparisons ............................................................................ 93 4 Results and Discussion .......................................................................................... 94 4.1 General Genome Features ...................................................................... 94 4.2 Plasticity Zones ...................................................................................... 95 4.3 Motility Genes, Virulence Factors and OMPs ....................................... 96 4.4 Homopolymeric Repeats ........................................................................ 98 4.5 Natural Competence ............................................................................... 99 4.6 The Core Genome of H. pylori ............................................................ 101 4.7 Unique Genes ....................................................................................... 103 5 Conclusions ......................................................................................................... 105 6 Tables and Figures ............................................................................................... 106 7 References ........................................................................................................... 138 Chapter 3 Structure-function analysis of the H. pylori flagellum biogenesis protein HP0958 .................................................................................................................... 145 1 Abstract ................................................................................................................ 146 2 Introduction ......................................................................................................... 147 3 Methods ............................................................................................................... 149 3.1 Bacterial Strains and Culture Conditions ............................................. 149 3.2 Molecular Cloning ............................................................................... 149 3.3 Site-Directed Mutagenesis ................................................................... 150 3.4 Allelic Exchange Mutagenesis ............................................................. 150 3.5 Natural Transformation of H. pylori .................................................... 151 3.6 Motility Assay ...................................................................................... 151 3.7 Electron Microscopy ............................................................................ 152 3.8 Preparation of Whole Cell Fractions .................................................... 152 3.9 Protein Electrophoresis and Western Blot ........................................... 152 3.10 Quantitative Analysis of Transcription by Real-Time PCR ................ 153 3.11 Protein Over-Expression and Purification ........................................... 153 3.12 GST Pull-Down Assay ......................................................................... 154 3.13 Yeast Two-Hybrid Assay ..................................................................... 154 3.14 Plate X-Gal Assay ................................................................................ 155 3.15