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By Katherine E. L. Cox a Dissertation Submitted to Johns Hopkins University in Conformity with the Requirements for the Degree O

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By Katherine E. L. Cox a Dissertation Submitted to Johns Hopkins University in Conformity with the Requirements for the Degree O EFFECTS OF CONJUGATIVE PLASMIDS ON BACTERIAL HOSTS, AND COEVOLUTION OF HOSTS AND PLASMIDS by Katherine E. L. Cox A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland March, 2017 Abstract As agents of horizontal gene transfer, plasmids play a role in the spread of antibiotic resistance and virulence factors, and can carry genetic material between widely divergent species. Typically, plasmids are considered primarily in terms of the genes they carry and the known functions of those genes, such as providing antibiotic resistance or new metabolic pathways. However, plasmids can have a variety of effects on their hosts beyond simply providing new functionality. Interactions between hosts and plasmids can modify host behavior, for example, increasing biofilm formation, modifying of host gene expression, or influencing virulence. We wished to investigate the mechanisms by which plasmids influence host behavior, as well as the evolution of host/plasmid relationships. In this work, we used multiple approaches to explore host/plasmid interactions. We examined a specific plasmid (R1) in great detail, generating a complete sequence and annotation for this plasmid and providing a brief review of the known gene products. We probed the influence of the plasmid-borne traJ gene (a regulator of plasmid transfer) on the neonatal-meningitis clinical isolate E. coli RS218 by measuring changes in the virulence and gene expression of RS218 carrying either wild-type or disrupted copies of traJ. We then examined the evolution of host/plasmid relationships by introducing plasmids into new hosts and coevolving them for 500 generations. We measured changes in phenotype (fitness) and genotype (using whole-genome-sequencing) in the evolved host/plasmid pairs. The R1 plasmid is overall quite similar to the well-studied F and R100 plasmids, though it has some unique regions that appear to have resulted from movement of mobile ii genetic elements. Our work with RS218 revealed that disruption of the traJ gene appears to modify expression of several S-fimbrial adhesin (sfa) genes. Sfa genes are known virulence factors. Finally, the host/plasmid coevolution studies revealed that the initial mutations involved in adaptation were generally highly reproducible within a given host/plasmid pair, but differed depending on both the host and the plasmid involved. Advisor: Joel Schildbach Reader: Lisa Scheifele iii Acknowledgements I want to thank our collaborator, Kwang Sik Kim, for access to strains and reagents and Donna Pearce for training in cell culture and invasion assays. Thanks also to the labs of Robert Schleif and Beverly Wendland for shared reagents and equipment as well as advice. Thanks to Allison Pinder for answering my questions about sequencing, training me on the BioAnalyzer, and allowing me to use it. Thanks especially to Fred Tan for answering all sorts of questions about sequence analysis and computers, for equipment and reagents, general science advice, and opportunities to both learn and teach bioinformatics. I want to thank my lab mates over the years for their advice and encouragement. Casey Hemmis and Tegan Feehery for showing me around the lab. My classmates for fun times and nerdy conversations, about science and otherwise. Allyson Roberts and Ashley Yuen for helping with fitness assays. James Shepherdson and my husband Daniel for helping me learn a bit about how computers work and find where Java lives. More recently Russell Hughes and Kristie Wrasman, for discussions on science and teaching, and for keeping me company up in UTL before it was cool :). To Beth Buenger, my partner-in-crime, for years of co-studying, co-stressing, co-laughing, co-writing and editing, co-sending-scary-emails, co-avoiding, co-pushing-through, co-planning, co- encouraging and co-sciencing. I want to thank the former and current members of my thesis committee, Trina Schroer, Elijah Roberts, and Nick Ingolia for their time and advice, as well as Lisa Scheifele for serving as a reader. And of course my advisor, Joel Schildbach, for iv everything he’s taught me, for being on my side even when science isn’t cooperating, and for his wholehearted support of my interest in teaching. I want to thank my church, for their prayers and encouragement, and for the reminders that there are more important things in life than the experiment that just failed or the presentation I have to prepare for. Thanks especially to Ronee and Paul Adams, Aaron and Alyssa Groen, Mike and Gail Boyes, and all of their children for welcoming me as family and adopting me for the holidays when I wasn’t able to make it home. I want to thank my family for their encouragement-without-pressure and their understanding of my absences. I especially want to thank my mom, who homeschooled me and taught me that learning is fun, reading is wonderful, and I can learn anything if I work at it. Finally, I want to thank my husband, Daniel Cox, for his unwavering support and patience. He married me right as I joined a lab, and has stuck with me through all the ups and downs of this whole PhD process. Through his shared enthusiasm in figuring out how the world works, faithfulness in doing more than his fair share of making our apartment a home, and most of all, his constant assurance of love regardless of my success and failures, he increased my joy on the good days and made it possible to struggle through bad ones. v Table of Contents Abstract .......................................................................................................................... ii Acknowledgements ....................................................................................................... iv Table of Contents .......................................................................................................... vi List of Tables................................................................................................................. ix List of Figures .................................................................................................................x 1 Chapter 1: Introduction .............................................................................................1 1.1 Overview ...........................................................................................................1 1.2 Introducing Plasmids: ........................................................................................2 1.2.1 Plasmid mobility and conjugation ...............................................................3 1.2.2 Plasmids affect host behavior .....................................................................4 1.2.3 Host range and plasmid stability .................................................................6 1.2.4 Fitness cost of plasmids ..............................................................................7 1.2.5 Evolution of plasmids .................................................................................9 1.3 Experimental Evolution ................................................................................... 11 1.3.1 Richard Lenski: First to intentionally investigate evolution of a host/plasmid relationship ........................................................................................ 13 1.3.2 Julian Adams: Early studies on plasmids in chemostats ............................ 15 1.3.3 Fitness Landscapes ................................................................................... 17 1.3.4 Dahlberg and Chao: Coevolution with conjugative plasmids .................... 18 1.3.5 Dionisio et al.: Adaptation in one host can improve fitness in another host 19 1.3.6 Eva Top: How does the host range of plasmids change? ........................... 21 1.3.7 Why do plasmids exist? ............................................................................ 26 1.3.8 Michael Brockhurst: Why do plasmids persist over time? ......................... 27 1.3.9 Craig MacLean: persistence of non-transmissible plasmids....................... 30 1.3.10 Summary .................................................................................................. 32 1.4 Goals of this work ........................................................................................... 32 2 Chapter 2: Sequence of the R1 plasmid and comparison to F and R100 .................. 34 2.1 Abstract ........................................................................................................... 34 2.2 Introduction ..................................................................................................... 34 2.3 Materials and Methods .................................................................................... 35 2.4 Results and Conclusions .................................................................................. 37 2.4.1 Overview .................................................................................................. 37 vi 2.4.2 Conjugative plasmid backbone ................................................................. 43 2.4.2.1 Plasmid replication region ................................................................. 43 2.4.2.2 tra operon .......................................................................................... 44 2.4.2.3 Leading region .................................................................................. 47 2.4.2.4 Plasmid partitioning systems ............................................................. 55 2.4.2.5
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