Characterisation Of The ATP-Binding Cassette Transporters Of Pseudomonas aeruginosa Victoria Grace Pederick Research Centre for Infectious Diseases, Department of Microbiology and Immunology, School of Molecular and Biomedical Sciences, University of Adelaide October 2014 TABLE OF CONTENTS ABSTRACT ............................................................................................................................... V DECLARATION .................................................................................................................... VII COPYRIGHT STATEMENT ................................................................................................ VIII ABBREVIATIONS ................................................................................................................. IX TABLE OF TABLES ............................................................................................................. XII TABLE OF FIGURES ........................................................................................................... XIII ACKNOWLEDGEMENTS .................................................................................................... XV CHAPTER 1: INTRODUCTION ........................................................................................ 1 Pseudomonas aeruginosa ........................................................................................... 1 1.1.1. P. aeruginosa and human disease ......................................................................... 1 1.1.1.1. Cystic fibrosis ................................................................................................ 2 1.1.2. Virulence factors ................................................................................................... 3 1.1.3. Transcriptional regulation ..................................................................................... 4 1.1.3.1. Quorum sensing ............................................................................................. 4 1.1.3.2. Two-component regulatory systems .............................................................. 5 1.1.3.3. Metalloregulatory genes ................................................................................ 5 1.1.4. Biofilm .................................................................................................................. 6 1.1.5. Antibiotic resistance ............................................................................................. 8 1.1.5.1. Low membrane permeability ......................................................................... 8 1.1.5.2. Antibiotic inactivating enzymes .................................................................... 9 1.1.5.3. Target modifications .................................................................................... 10 1.1.5.4. Lifestyle-mediated resistance ...................................................................... 10 1.1.5.5. Antibiotic efflux ........................................................................................... 11 ABC transporters ..................................................................................................... 14 1.2.1. Common architecture .......................................................................................... 15 1.2.1.1. Transmembrane domains ............................................................................. 15 1.2.1.2. Nucleotide-binding domains ........................................................................ 16 1.2.2. ABC exporters .................................................................................................... 16 1.2.2.1. Xenobiotic efflux ......................................................................................... 17 1.2.2.2. Protein secretion .......................................................................................... 19 1.2.2.2.1. Toxins and proteases ............................................................................. 20 1.2.2.2.2. Antimicrobials and antibiotic peptides ................................................. 20 1.2.2.2.3. Heme-binding proteins and siderophores ............................................. 21 1.2.2.3. Glycoconjugate secretion ............................................................................. 22 1.2.3. ABC importers .................................................................................................... 22 1.2.3.1. Classification of SBPs ................................................................................. 23 1.2.3.2. Roles of ABC permeases ............................................................................. 25 1.2.3.3. Metal ion acquisition ................................................................................... 26 1.2.3.3.1. Iron ........................................................................................................ 26 1.2.3.3.2. Zinc ....................................................................................................... 27 1.2.3.3.3. Molybdenum ......................................................................................... 27 ABC transporters in P. aeruginosa ........................................................................ 28 1.3.1. Aims of the study ................................................................................................ 29 CHAPTER 2: METHODS AND MATERIALS .............................................................. 30 Materials ................................................................................................................... 31 DNA manipulation ................................................................................................... 31 2.2.1. Plasmid isolation ................................................................................................. 31 2.2.2. Genomic DNA isolation ..................................................................................... 31 2.2.3. DNA electrophoresis ........................................................................................... 31 2.2.4. Assessment of DNA quantity and quality........................................................... 32 II 2.2.5. DNA sequencing ................................................................................................. 32 2.2.6. Polymerase Chain Reaction (PCR) ..................................................................... 32 Bacterial culturing ................................................................................................... 32 Bacterial strains ....................................................................................................... 38 2.4.1. P. aeruginosa ...................................................................................................... 38 2.4.1.1. Mutagenesis ................................................................................................. 38 2.4.2. E. coli .................................................................................................................. 41 2.4.2.1. Generation of chemically competent cells ................................................... 41 2.4.2.2. Transformation of E. coli ............................................................................. 41 Phenotypic analyses ................................................................................................. 43 2.5.1. Growth kinetics assay ......................................................................................... 43 2.5.2. Biofilm formation ............................................................................................... 43 2.5.3. Whole cell metal accumulation ........................................................................... 44 2.5.4. Minimal inhibitory concentration (MIC) analysis .............................................. 44 2.5.5. Fatty acid analysis ............................................................................................... 45 2.5.6. Bacterial adherence to hydrocarbons (BATH) assay .......................................... 45 2.5.7. Volatile compound (VOC) headspace analysis .................................................. 45 2.5.8. Scanning electron microscopy analysis .............................................................. 46 ZnuA biochemical characterisation ....................................................................... 46 2.6.1. Cloning and expression of ZnuA ........................................................................ 46 2.6.2. Purification of ZnuA ........................................................................................... 47 2.6.3. ZnuA zinc binding assays ................................................................................... 48 2.6.4. PAR assay ........................................................................................................... 48 Bioinformatic analyses ............................................................................................ 48 2.7.1. Identification of transcriptional regulatory sites ................................................. 48 2.7.2. Generation of Weblogos ..................................................................................... 49 Transcriptional analyses ......................................................................................... 49 2.8.1. RNA isolation ..................................................................................................... 49 2.8.2. qRT-PCR ...........................................................................................................