DNA on the move: Investigation into two mobile genetic elements in Vibrio species

..............................................................................................................................xviii Chapter 1: Introduction ...........................................................................................................1 1.1: Preface ..........................................................................................................................1 1.2: Vibrio species: An overview ........................................................................................1 1.2.1: The diverse roles of vibrios...................................................................................2 1.2.1.1: Mutual symbiotic relationships ......................................................................3 1.2.1.2: Pathogenic relationships.................................................................................4 1.2.1.2.1: Human pathogens....................................................................................5 1.2.1.2.2: Marine animal pathogens ........................................................................9 1.3: Lateral gene transfer: a major mechanism for bacterial evolution.............................10 1.3.1: Transformation ....................................................................................................12 1.3.2: Conjugation .........................................................................................................12 1.3.3: Transduction........................................................................................................13 1.3.3.1: Lysogenic conversion...................................................................................14 1.3.4: Integration/maintenance of DNA sequences.......................................................15 1.4: Mobile genetic elements: a source of DNA ...............................................................17 1.4.1: Plasmids ..............................................................................................................17 1.4.2: Transposons.........................................................................................................18 iv 1.4.3: Bacteriophage......................................................................................................20 1.4.4: Genomic islands ..................................................................................................23 1.4.4.1: Genomic islands and the evolution of pathogenic/pandemic V. cholerae ...25 1.4.5: Integrative conjugative elements: the tip of the ICEberg....................................26 1.5: LGT: a major mechanism for bacterial evolution ......................................................27 1.5.1: V. cholerae: a paradigm for the importance of LGT in adaptation and evolution .......................................................................................................................................28 1.6: The integron/gene cassette system .............................................................................31 1.6.1: A big black box in our understanding of gene cassette-associated function(s) ..35 1.7: Project aims ................................................................................................................41 1.7.1: How does the integron/gene cassette system impact on cell physiology? ..........41 1.7.2: How does a novel genomic island impact on cell survival and overall fitness? .41 Chapter 2: Materials and Methods ........................................................................................43 2.1: Bacterial strains and growth conditions .....................................................................43 2.2: List of primers ............................................................................................................51 2.3: DNA methods.............................................................................................................54 2.3.1: PCR and agarose gel electrophoresis ..................................................................54 2.3.2: Genomic DNA extraction ...................................................................................55 2.3.2.1: Crude extraction for PCR.............................................................................55 2.3.2.2: XS buffer method .........................................................................................56 2.3.3: Plasmid extraction ...............................................................................................56 2.3.4: Fosmid library construction, transposon mutagenesis and screening of RME ...57 2.3.5: DNA and whole genome sequencing ..................................................................58 2.3.6: Extraction, purification and quantification of DNA samples from agarose gels 58 2.3.7: Preparation of competent cells ............................................................................58