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Helicobacter Pylori: Comparative Genomics and Structure-Function Analysis of the Flagellum Biogenesis Protein HP0958

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Helicobacter Pylori: Comparative Genomics and Structure-Function Analysis of the Flagellum Biogenesis Protein HP0958 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
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