Proquest Dissertations

THE INTEGRON AND ITS GENE CASSETTES: MOLECULAR ECOLOGY OF A MOBILE GENE POOL by Jeremy Edmund Koenig Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Dalhousie University Halifax, Nova Scotia June 2009 © Copyright by Jeremy Edmund Koenig, 2009 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre refinance ISBN: 978-0-494-56421-9 Our file Notre reference ISBN: 978-0-494-56421-9 NOTICE: AVIS: The author has granted a non L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent §tre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. ••I Canada DALHOUSIE UNIVERSITY To comply with the Canadian Privacy Act the National Library of Canada has requested that the following pages be removed from this copy of the thesis: Preliminary Pages Examiners Signature Page (pii) Dalhousie Library Copyright Agreement (piii) Appendices - Copyright Releases (if applicable) "...if you wish, as I do, to build a society in which individuals cooperate generously and unselfishly towards a common good, you can expect little help from biological nature. Let us try to teach generosity and altruism, because we are born selfish. Let us understand what our own selfish genes are up to, because we may then at least have a chance to upset their designs, something that no other species has ever aspired to do." -Richard Dawkins, The Selfish Gene, 1976 IV TABLE OF CONTENTS LIST OF FIGURES ix LIST OF TABLES xi ABSTRACT xii LIST OF ABBREVIATIONS USED xiii ACKNOWLEDGEMENTS , xiv CHAPTER 1 1 Introduction 1 A Brief History of Lateral Gene Transfer 2 Extraordinary Instances of LGT. 2 LGT within and between the Archaea and the Bacteria 4 Bacterial Homologous and Site-Specific Recombination 6 Homologous Recombination 6 Site-specific Recombination 8 LGT and the Integron 9 Integron Integrase IntI: A Site-specific Recombinase 11 An Evolutionary Account of Integrons: The End of a Class System 14 Integrons Encoded in Mobile Elements 19 Chromosomally Bound Integrons 21 Integron Gene cassettes: Function Encoded in a Mobile Gene Pool 23 General Rationale 24 CHAPTER 2 26 Population Dynamics of Coral-associated Vibrio-Integrons in the Great Barrier Reef: Antimicrobial-Resistance Connections between Coral and Human Microbiomes 26 Rationale ....26 Introduction 26 Materials and Methods 31 Sample Collection and Cultivation 31 Gene Amplification, Cloning and Sequencing 31 Colony Screening with intl PCR 32 Fosmid Library Construction, Screening and Sequencing of Vibrio Cultivars 32 v Housekeeping-gene Sequence Acquisition from Vibrio Cultivars and Genomes 33 Taxonomic Assignment of Vibrio Cultivars by recA Phylogeny 33 Vibrio Concatenated Gene Phylogeny 34 Integron Annotation 34 Cassette Ecology 37 OTU Clustering 38 Statistical Support for attC Clustering 38 Cassette Phylogeny 39 Vibrio Cassette Dendrogram Assembly 39 Results and Discussion 40 Pocillopora damicornis Mucus Contains a Variety o/Vibrio species, some of these Implicated in Pathogenesis 40 The Evolutionary Rate of Integron Cassette Arrays is High in Natural Vibrio Populations 43 Gene Cassette Ecology and Transfer in Vibrio Coral Mucus Cultivars , 45 The Function Most Prevalent in Coral-associated Integron Gene Cassettes is Putative Resistance to Antimicrobials 56 Integron Gene Cassette Connections Between Vibrio Cultivars, Coral and Human Pathogens 60 Concluding Remarks 63 CHAPTER 3 65 Preliminary Evidence for Integron Gene Cassette Ecotype 65 Rationale 65 Introduction 66 Materials and Methods 68 Environmental Samples and Cassette PCR 68 Sequence Analysis and Authentification 70 Cassette Ecology 71 Sequence Annotation 71 SignalP Analysis 73 Phylogenetic Analysis 73 Non-synonymous Versus Synonymous Rates 73 Results and Discussion 74 How Big is the Integron Gene Cassette Metagenome?. 74 VI Functional Diversity Among Cassettes 79 Cellular Targeting of Gene Cassette Products 83 Taxonomic Affiliation of Cassette-encoded Proteins 85 Novel Functions in the Gene Cassette Metagenome 87 Origins of Gene Cassettes 88 Is there Such a Thing as a Cassette Ecotype?. 90 Concluding Remarks 93 CHAPTER 4 97 A Different Pool of Integron Gene Cassettes 97 Rationale 97 Introduction 97 Materials and Methods 102 Culture Independent Sample Collection 102 intl Gene Amplification from Cultured Organisms 102 PCR Amplification 102 Long-walk PCR Amplification 103 Clone Library Construction and DNA Sequencing 103 Sequence Analysis and Authentification 103 Compilation of Statistics on Diversity 104 Phylogeny 104 Results and Discussion 105 Bacterial and Archaeal Diversity in the Sydney Tar Ponds 105 Intl Diversity in the Sydney Tar Ponds 107 Gene Cassette Diversity in the Sydney Tar Ponds 110 Long-walk PCR of Cassette-encoded EC 4.1.1.44 119 Taxonomic Assignment of Gene Cassettes 119 Culture Dependent Analysis of Integrons 122 Concluding Remarks 125 CHAPTER 5 128 Conclusion 128 Rationale 128 Materials and Methods 128 Cassette Clustering 128 vii Cassette Phytogeny 129 Population Dynamics of Coral-associated Vibrio-Integrons in the Great Barrier Reef: Antimicrobial-Resistance Connections between Coral and Human Microbiomes 130 Preliminary Evidence for Integron Gene Cassette Ecotype 132 A Different Pool of Integron Gene Cassettes 134 Integron Gene Cassettes: A Global Perspective 135 Integrons and their Gene Cassettes: A Mere Snapshot of a Dynamic Evolutionary Process 140 Final Thoughts 142 References 144 APPENDIX 160 Vlll LIST OF FIGURES Figure 1.1 General Structure of the Integron/Gene-cassette System. 10 Figure 1.2 attC Recombination Sites and Intl. 12 Figure 1.3 Intl-mediated Site-specific Recombination of Gene cassettes. 13 Figure 1.4 RpoB Versus IntI Phylogeny for Bacteria Encoding Integrons. 15 Figure 2.1 attC String-search-scoring Method. 36 Figure 2.2 Taxonomic Distribution of recA Nucleotide Sequences 41 Amplified from Cultivars Isolated from Coral Mucus Samples. Figure 2.3 Integron Gene Cassette Array Dynamics of Vibrio Isolates 44 Cultured from Coral Mucus. Figure 2.4 Phylogenetic Analysis of Integron Gene Cassettes Sequenced 47 from Vibrio Coral Mucus Isolates. Figure 2.5 Network Clusters Based on the Integron attC Recombination 48 Sequences Encoded by Marine Vibrio spp. Figure 2.6 Pairwise Comparison of the Genetic Identity Versus Integron 49 Gene Cassette Array Conservation in Vibrio Isolates. Figure 2.7 Phylogeny of Vibrio Housekeeping-genes Versus Shared 51 Cassettes. Figure 2.8 Taxonomical Assignment of Integron Gene Cassettes Encoded 54 by Vibrio Isolated from Coral Mucus. Figure 2.9 Amino Acid Alignment of Cassette-encoded Aminoglycosides. 58 Figure 2.10 Network of Furthest Neighbour Coral-pathogen-encoded Cassettes with Amino Acid BSR Values > 0.70. 62 Figure 3.1 Comparison of Halifax Metagenome and Vibrio cholera Gene 72 Cassette Orientations. Figure 3.2 Rarefaction Curves of Integron Gene Cassettes Amplified 75 From Marine Sediment. Figure 3.3 attC Sites Found in PCR Amplicons Containing Two Cassettes. 78 IX Figure 3.4 Proportion of Cassettes that Encode Signal Peptides. 84 Figure 3.5 Taxonomic Distribution of the Highest BLASTx Scores of 86 Gene Cassette-encoded Proteins. Figure 3.6 Integron Gene Cassette Recruitment. 91 Figure 3.7 Distribution of Cassette-types Shared between the Four 92 Marine Sediment Sample-sites. Figure 4.1 Timeline of Events Related to Steel Production in Sydney 101 Cape Breton, Nova Scotia, Canada. Figure 4.2 Integron Integrase Diversity in the Sydney Tar Ponds. 108 Figure 4.3 Metabolic Atlas of Integron Gene Cassettes from the Cassette 114 Metagenome and those Amplified from the Sydney Tar Ponds. Figure 4.4 Phylogenetic Analysis of Cassette-encoded Homologs to 117 Contaminant-degrading Proteins Amplified from the Sydney Tar Ponds. Figure 4.5 Partial Integron Gene Cassette Array Obtained by Long-walk 120 PCR on DNA Extracted from the Sydney Tar Ponds. Figure 4.6 Nucleotide Alignment of Divergent attC Recombination 121 Sequences Encoded in Multiple-cassette-amplicons.

Load more