Shiga toxin encoding bacteriophages and horizontal gene transfer in Escherichia coli O157 Eby Mazini Sim A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy July 2016 Certificate of Authorship/ Originality Certificate of Authorship/Originality I certify that the work presented in this thesis has not previously been submitted for a degree, nor has it been submitted as part of the requirements for a degree except as fully acknowledged within the text. I also certify that the written preparation of the thesis, and all experimental work associated with it, has been carried out solely by me, unless otherwise indicated. Finally, I certify that all information sources and literature used are acknowledged in the text. ____________________________ Eby Mazini Sim i Acknowledgements Acknowledgements My journey as a PhD student, starting from The University of Queensland, Brisbane and the subsequent relocation to The University of Technology, Sydney, has been challenging, exciting and immensely rewarding. However, this thesis would not have been possible without the support and guidance from a number of amazing people along the way. First and foremost, I would like to acknowledge my principal supervisor Dr. Nico Petty. Nico not only supervised the work presented in this thesis and provided feedback on this thesis, she also guided and encouraged me through these years. Her passion for research is truly inspiring. I would also like to acknowledge my co-supervisor Dr. Kari Gobius. In spite of his responsibilities at the CSIRO, Kari always made time to mentor me. Both of my supervisors contributed enormously to my development as a scientist and I couldn’t have asked for a better supervisory team for my PhD journey. A huge thank you to all past and present members of the Petty lab and colleagues in the CSIRO, who have made this journey an amazing and productive one: Glen Mellor, Robert Barlow, Lesley Duffy, Kate McMillian, Salma Lahjar, Tuan Nyguen, Neralle Fegan, Scott Chandry, Leanne Haggerty, Alejandro Marquez, Lucy Martindale and Dylan Mansfield. To my family, especially my Mum, thank you for your words of support over all these years. I am particularly grateful for your encouragement of my PhD work as well as my aspirations. To Melissa Hamwood, thank you for being there for me in both the good times and the hard times and for supporting me through this journey. Lastly, I would like to acknowledge the financial assistance from the UQ International Scholarship awarded during my stay at UQ, the UTS International Research Scholarship awarded during my stay at UTS as well as funding from the CSIRO. ii Content page Content page Certificate of Authorship/Originality ......................................................................... i Acknowledgements ..................................................................................................... ii Content page ............................................................................................................... iii List of Figures ............................................................................................................ vii List of Tables ............................................................................................................... ix Abbreviations ............................................................................................................... x Research outputs ....................................................................................................... xii Publications ............................................................................................................. xii Conference proceedings .......................................................................................... xii Book Chapter .......................................................................................................... xii Abstract ..................................................................................................................... xiii 1. Introduction ............................................................................................................ 1 1.1. Bacteriophages .................................................................................................. 1 1.1.1 The structure of tailed phages ..................................................................... 2 1.1.2. Phage genomes ........................................................................................... 4 1.1.3. Phage life cycle .......................................................................................... 5 1.1.3.1 Lytic life cycle ..................................................................................... 6 1.1.3.2. Lysogenic life cycle ............................................................................ 7 1.1.3.3. Pseudolysogeny.................................................................................. 9 1.1.3.4. Satellite phages- a subset of temperate phages ................................. 10 1.1.4 Coevolution of phages and bacteria .......................................................... 10 1.1.4.1. Inhibition of phage adsorption .......................................................... 10 1.1.4.2. Preventing DNA entry ...................................................................... 11 1.1.4.3. CRISPR-Cas system ......................................................................... 11 1.1.4.4. Restriction-modification systems ...................................................... 12 1.1.4.5. Abortive infection ............................................................................. 12 1.1.5. Phages and horizontal gene transfer ........................................................ 13 1.1.5.1 Transduction ...................................................................................... 14 1.1.5.1.1. Generalised transduction ................................................................ 14 1.1.5.1.2. Specialised transduction ................................................................. 15 1.1.5.2. Lysogenic conversion ....................................................................... 16 1.2. Escherichia coli .............................................................................................. 17 1.3. Escherichia coli O157 ..................................................................................... 18 1.3.1. Transmission of STEC O157 ................................................................... 19 1.3.2. Geographical variation in STEC O157 related disease ............................ 20 1.3.3. Diversity of STEC O157 .......................................................................... 21 1.3.4. Virulence factors of STEC O157 strains ................................................. 23 1.3.4.1. The STEC O157 specific plasmid pO157 ......................................... 23 1.3.4.2 Locus of Enterocyte Effacement pathogenicity island ...................... 24 1.3.4.3. Shiga toxin ........................................................................................ 25 1.4. Shiga toxin-encoding phages .......................................................................... 27 1.4.1. Stx phage genomes .................................................................................. 27 1.4.2. Horizontal gene transfer and Stx phages ................................................ 32 iii Content page 1.4.3. Induction of Stx phages ........................................................................... 33 1.4.4. Persistence in the environment ................................................................ 33 1.4.5. Stx phage host range ................................................................................ 33 1.4.6. The impact of Stx prophages on their bacterial host ................................ 36 1.4.7. Insertion sites of Stx phages .................................................................... 37 1.5. Australian STEC O157 and Stx phages .......................................................... 38 1.6. Objectives of this study ................................................................................... 39 2. Materials and methods ........................................................................................ 40 2.1. Media, supplements and solutions .................................................................. 40 2.2. Bacterial strains, phages and culture conditions ............................................. 41 2.3. Bacterial phenotype test .................................................................................. 44 2.3.1. Differentiation of STEC O157 from other bacteria ................................. 44 2.3.2. Stx production .......................................................................................... 44 2.4. Phage methods ................................................................................................ 44 2.4.1. Isolation of Stx phages ............................................................................. 44 2.4.2. Phage concentration and purification ....................................................... 45 2.4.2.1. Isopycnic ultracentrifugation .............................................................. 45 2.4.2.1. Ultrafiltration ...................................................................................... 45 2.4.3. Phage quantification ................................................................................. 46 2.4.3.1. Double agar overlay