UC San Diego UC San Diego Electronic Theses and Dissertations
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UC San Diego UC San Diego Electronic Theses and Dissertations Title Defining the role of bacteriophage in mucosal immunity Permalink https://escholarship.org/uc/item/5px2x5b7 Author Benler, Sean Publication Date 2018 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA SAN DIEGO SAN DIEGO STATE UNIVERSITY DEFINING THE ROLE OF BACTERIOPHAGE IN MUCOSAL IMMUNITY A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biology by Sean Benler Committee in charge: University of California San Diego Professor Partho Ghosh Professor Joseph Pogliano San Diego State University Professor Forest Rohwer, Chair Professor Anca Segall Professor Roland Wolkowicz 2018 The Dissertation of Sean Benler is approved, and it is acceptable in quality and form for publication on microfilm and electronically: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ Chair University of California San Diego San Diego State University 2018 iii Dedication To everyone who pushed me a little further. iv Epigraph And then they ask you, ‘what is the significance of DNA’? -Francis Crick v Table of contents Dedication .................................................................................................................................. iv Epigraph ..................................................................................................................................... v Table of contents ....................................................................................................................... vi List of figures ............................................................................................................................ ix List of tables ............................................................................................................................... x Acknowledgements ................................................................................................................... xi Vita ........................................................................................................................................... xii Abstract of the dissertation ........................................................................................................ iii Chapter 1: Introduction ............................................................................................................... 1 Bacteriophage Adherence to Mucus (BAM): How ecological and evolutionary dynamics create a mucosal immune system ........................................................................................... 1 Mucus is a rapidly changing environment. ......................................................................... 1 Lysogeny and superinfection-exclusion in viruses. ............................................................ 3 Directed evolution by diversity-generating retroelements ................................................. 6 Genome packaging in T4-like bacteriophages: corking the champagne bottle ...................... 9 Acknowledgements .............................................................................................................. 10 References ............................................................................................................................ 11 Chapter 2: gp4 is a nuclease required for morphogenesis of T4-like bacteriophages .............. 17 Abstract ................................................................................................................................. 17 Introduction .......................................................................................................................... 17 Materials and Methods ......................................................................................................... 19 Phage propagation and sequencing .................................................................................. 19 Genome assembly and annotation .................................................................................... 20 Phage proteomic tree ........................................................................................................ 21 Identification of YC in metagenomes ............................................................................... 21 Cloning of wild-type gene 4 ............................................................................................. 22 Isolation and cloning of mutations in gp4 ........................................................................ 22 Protein expression and purification .................................................................................. 22 In vitro nuclease assays .................................................................................................... 23 In vivo complementation assay ......................................................................................... 23 vi Transmission electron microscopy ................................................................................... 24 Results .................................................................................................................................. 24 Bacteriophage YC is a novel member of T4-like bacteriophages .................................... 24 Gene 4 is a putative nuclease found in T4-like phages .................................................... 26 Complementation of amber mutant T4 by gp4 in trans .................................................... 29 Discussion ............................................................................................................................. 31 Acknowledgements .............................................................................................................. 32 Appendix .............................................................................................................................. 33 References ............................................................................................................................ 45 Chapter 3: A diversity-generating retroelement encoded by a globally ubiquitous Bacteroides phage ......................................................................................................................................... 50 Abstract ................................................................................................................................. 50 Introduction .......................................................................................................................... 50 Methods ................................................................................................................................ 52 Identification of DGRs ..................................................................................................... 52 Viral read mapping to predicted prophage-containing regions ........................................ 52 Prophage genome annotation ........................................................................................... 53 Metagenome read mapping to Hankyphage ..................................................................... 54 Phage induction, PCR and transmission electron microscopy ......................................... 54 Results .................................................................................................................................. 55 DGRs found in three temperate phages ............................................................................ 55 DGRs encoded by novel temperate phages ...................................................................... 55 Bacteroides dorei is lysogenized by a DGR-containing temperate phage ....................... 59 Hankyphage exhibits broad-host range and global distribution ....................................... 60 Function of hypervariable proteins ................................................................................... 63 Discussion ............................................................................................................................. 64 Acknowledgements .............................................................................................................. 66 Appendix .............................................................................................................................. 67 References ............................................................................................................................ 87 Chapter 4: Experimental approaches to measure bacteriophage adherence to mucus: challenges and prospects .......................................................................................................... 91 vii Specific Aim 1: Determine the effect of Ig-like domains on phage hunting strategies in a mucosal environment. ........................................................................................................... 91 Protocols ........................................................................................................................... 92 Results .............................................................................................................................