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Use of a Novel High-Throughput Genetic Selection to Identify Regulators of Selected Vibrio Cholerae Late Genes; Characterization of Phob As a Regulator of Xds

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Use of a Novel High-Throughput Genetic Selection to Identify Regulators of Selected Vibrio Cholerae Late Genes; Characterization of Phob As a Regulator of Xds Use of a novel high-throughput genetic selection to identify regulators of selected Vibrio cholerae late genes; characterization of PhoB as a regulator of xds A thesis submitted by EMILYKATE MCDONOUGH In partial fulfillment of the requirements for the degree of Doctor of Philosophy In Molecular Microbiology TUFTS UNIVERSITY Sackler School of Graduate Biomedical Sciences August, 2014 Advisor: Andrew Camilli 1 To everyone who inspired me to keep going. 2 ACKNOWLEDGEMENTS First and foremost, thank you Andy for mentoring me with such a kind and enthusiastic nature. Thank you for always being approachable and doing your best to make time for teaching. Thank you especially for the first few years in the lab, when I stumbled around my bench, hardly knowing anything about microbiology; you were always supportive and excited about even my smallest achievements. Thank you for always listening to my ideas and for letting me make my own decisions, even when you completely disagree with my choices. As I move through this world, whether in science or film, I will always be thankful for what I have learned and accomplished in your laboratory. I hope you continue to infect those around you with your passion, optimism and kindness. To my committee, thank you for all of your input throughout the years. You have challenged me to think on my feet, a skill that is very useful in this field. Thank you for all of the guidance, encouragement, and kind criticism. Linc – thank you for your constant words of support. Even when I have felt discouraged by my work, whether you know my feelings or not, you have made a point to tell me otherwise. Also thank you for critiquing my writing so carefully. Michael – thank you for your mentorship. I love talking with you about both science and life – and I wish I’d done it more. You have always cared about me as a person, which has helped me remember that there is more to life than graduate school. Ralph – thank you for your ever-questioning ways; you are a true Obie, whatever that means. Having you on my committee has made me stronger and a more confident scientist. Thank you to the Camilli lab members, both past and present members. The lab has been my home for seven years, and I will think fondly of many memories after I have left. Lobster bay – (Katie, Jason, Neil, Steph) thank you for all the conversations about science and life. Our little mascot is falling to pieces, but I hope his namesake will live on. Pick a good replacement for me! Free high fives. Ayman – thank you for opening my eyes to the world of Illustrator short cuts and teaching me the ins and outs of Word. Also thank you for your excitement about biochemistry and your undying willingness to help out. You have been a good source of moral support, and always provide a good laugh. Anne – as one of the strongest women I know, you have been a role model for me. I hope our paths will cross again soon. Rita – thank you for always listening to my ideas and making me feel like they mattered. Evan – I’m so glad we got to write the book chapter together, thanks for that experience. Also, thanks for making the lab a great place to be. Dave – I’m so grateful that you are in the lab. You have been an amazing resource for and critic of my work. Thanks for contributing so much to my thesis work and for making me a better scientist; you are a great one. Also thanks for all the camera talk. Revati and Heather – thanks for all the lunches and great scientific talks. You guys have been great friends. Ellie – I’m so lucky that you joined our lab. You have been a wonderful friend these past few years. Thanks for coffee, walks, cheering me up, morale support, etc. I’m excited to see where your life takes you – I know you’ll reach all your dreams. 3 Thank you to my friends for all of your undying support, and understanding of my lack of time to come play or visit you in NYC. There’s too many of you to thank individually, but know that you are all in my heart. Kayle. Thank you. Thank you for putting up with all my stress and bursts of frustration. Thank you for providing me with so much love and support. Thank you for our scientific conversations. Thank you for reminding me to breathe and to live. I have grown so much from knowing you. I love you. And finally, to my family, thank you. You have always believed in me and told me to follow my dreams. Popsy and momma, thank you for all the opportunities you have given me – I am so very lucky. I’m sorry I didn’t stay little, but I think this accomplishment is pretty awesome, eh? Hannie – you always say that I’m your role model sister, but you should know that I look up to you every day; you’re so smart, driven, and not afraid to stick your neck out. I admire the woman you’ve become. 4 ABSTRACT Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, is a natural member of temperate aquatic environments around the world. V. cholerae undergoes adaptive shifts in gene expression throughout the various stages of its life cycle. Our laboratory identified 57 V. cholerae genes that are expressed specifically at or near the end of the infection cycle in the infant mouse model of infection. Many of these ‘late’ genes appear to be involved in preparing the bacteria for the shift from the host small intestine to the aquatic environment. In contrast, these same genes are not required for V. cholerae colonization of the mouse small intestine. These data led us to hypothesize that V. cholerae evolved to preinduce such dissemination genes while still in the host intestinal tract in order to optimize their chances of successfully transitioning to the aquatic environment. To gain support for this hypothesis and to extend our understanding of gene regulation at this critical transition, the goal of my thesis project was to determine what signals V. cholerae senses in the small intestine that alert the organism to the upcoming change in environment. The first half of my thesis describes the implementation and results of a high throughput genetic selection we developed to identify transcriptional regulators of late genes. The selection was designed such that both activators and repressors could be identified, as it is not known if these genes are induced or de- repressed late in infection. We settled on three late genes as query genes for the selection: cdpA (encoding a cytoplasmic cyclic-diguanylate phosphodiesterase), emrD (encoding a efflux pump inner membrane subunit), and xds (encoding a secreted DNA exonuclease). The selection results did not identify any regulators of cdpA. Conversely, pepA (encoding 5 a leucyl amino-peptidase) was identified as a repressor of emrD, and phoB (encoding the phosphate starvation response regulator) was identified as an activator of xds. Additionally, I have provided evidence that while regulation of xds by PhoB is relevant to the host intestinal environment, expression of xds can occur early or late in infection depending on the physiological state of the inoculum used for infection. In the second half of my thesis I have worked towards a deeper understanding of the biological significance of the regulation of xds by PhoB. As Xds is an exonuclease, we hypothesized that PhoB induces xds under phosphate limiting conditions as part of the upregulation of phosphate acquisition genes. Indeed, V. cholerae is known to be able to survive using DNA as a sole source of phosphate, and Xds has been described as important for this ability. In this work I describe the identification and preliminary characterization of two nucleotidases, UshA and CpdB, that contribute to the ability of V. cholerae to utilize nucleotides as a source of phosphate. I have shown that both of ushA and cpdB are induced by phosphate limiting conditions, further supporting the significance of nucleotides as sources of phosphate. 6 TABLE OF CONTENTS ACKNOWLEDGEMENTS.............................................................................................. 3 ABSTRACT....................................................................................................................... 5 TABLE OF CONTENTS.................................................................................................. 7 LIST OF FIGURES ........................................................................................................ 11 LIST OF TABLES .......................................................................................................... 14 CHAPTER I: ................................................................................................................... 15 INTRODUCTION........................................................................................................... 15 I.1. V. CHOLERAE AS A MODEL WATERBORNE PATHOGEN................................................ 16 I.1.1. Importance of waterborne microbial pathogens.............................................. 16 I.1.2. Role of the aquatic reservoir in transmission of waterborne pathogens......... 17 I.1.3. Vibrio cholerae as a model waterborne pathogen........................................... 19 I.2. OVERVIEW OF THE V. CHOLERAE VIRULENCE PROGRAM ........................................... 24 I.2.1. Primary virulence genes and their regulation................................................. 24 I.2.2. Quorum sensing and the regulation of virulence............................................. 26 I.2.3. Cyclic diguanylate and the regulation of virulence......................................... 29 I.2.4. Bistable expression of virulence factors
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