FUNCTIONAL ANALYSIS OF THE ROLE OF ALTERNATIVE SIGMA FACTORS IN VIBRIO PARAHAEMOLYTICUS HOST PATHOGEN INTERACTIONS by Brandy L. Haines-Menges A dissertation submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biological Sciences Summer 2015 © 2015 Brandy Haines-Menges All Rights Reserved ProQuest Number: 3730237 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. ProQuest 3730237 Published by ProQuest LLC (2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106 - 1346 FUNCTIONAL ANALYSIS OF THE ROLE OF ALTERNATIVE SIGMA FACTORS IN VIBRIO PARAHAEMOLYTICUS HOST PATHOGEN INTERACTIONS by Brandy L. Haines-Menges Approved: __________________________________________________________ Robin W. Morgan, Ph.D. Chair of the Department of Biological Sciences Approved: __________________________________________________________ George H. Watson, Ph.D. Dean of the College of Arts and Sciences Approved: __________________________________________________________ James G. Richards, Ph.D. Vice Provost for Graduate and Professional Education I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ E. Fidelma Boyd, Ph.D. Professor in charge of dissertation I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Thomas E. Hanson, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Michelle A. Parent, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Daniel Simmons, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Diane Herson, Ph.D. Member of dissertation committee ACKNOWLEDGMENTS When I thought of all the people who deserve an acknowledgement for their guidance and support throughout the Ph.D. process a simple ‘thank you’ seems insufficient to describe the impact they have had on my development as both a scholar and better version of myself. First I have to express sincere gratitude to my advisor and mentor, Dr. E. Fidelma Boyd, for providing the opportunity to work in her lab and for providing this project. She has shown extraordinary patience as I struggled with external factors and finding my scientific passion. It is because of her excellent mentorship and ability to deliver constructive criticism when it was most needed that I have been able to write this dissertation and that I feel prepared to enter the next phase of my career, whatever direction that may take me. Secondly, I would like to thank all of the current and past members of the Boyd lab; I could not have asked for a better group of individuals to work with and develop a friendship with. They are always ready with a listening ear, advice on experiments and constructive criticism when asked while reviewing presentations and manuscripts. I have to particularly thank and acknowledge Dr. W. Brian Whitaker for training me when I first joined the lab and for always answering any questions I ask and for offering advice, even after graduating from the lab. I wish all of you the best of luck in your future endeavors; you deserve all of the successes you achieve. v I would also like to take this time to thank all of the members of my committee for their constant advice and guidance and for pushing me to always become better. Your time and efforts have been much appreciated. Lastly I thank all of the friends and fellow graduate students I have met during my time here. Thank you for your support and guidance and for allowing me to serve as the BGSA president. Thank you for your help in releasing the stresses of graduate school and my personal life throughout the past five years. vi DEDICATIONS I dedicate this dissertation to my husband for all of his love and support throughout my graduate school career. Thank you for appreciating the importance of my own dreams and career aspirations and not allowing yours to overshadow that. The distance between us has not been easy but it has been worthwhile to share this life accomplishment with you. As equal partners, I pledge to you to always be by your side as you continue to fight the traumatic stress left by war and to show the same love and support that you have shown to me. I can’t wait to begin a new journey together. I dedicate this dissertation also to my parents who instilled in me the value of hard work and of obtaining an education. Thank you for your support throughout my entire life and for always pushing me towards higher intellectual pursuits. I dedicate this dissertation to my late grandmother, Nancy Haines, who was always extremely supportive and proud of any accomplishments I have achieved and who showed remarkable strength and courage battling brain cancer. My only wish is that you would have achieved your last wish, to see my college graduation but I know that you were there and at my PhD graduation in spirit and your strength and courage and love will stay with me for the rest of my life. vii TABLE OF CONTENTS LIST OF TABLES ....................................................................................................... xii LIST OF FIGURES ..................................................................................................... xiii ABSTRACT ................................................................................................................. xv Chapter 1 INTRODUCTION .............................................................................................. 1 Vibrio parahaemolyticus .................................................................................... 1 Sigma Factors ..................................................................................................... 5 Function of Alternative Sigma Factors among Vibrio Species ........ 6 Dissertation Work ............................................................................................. 15 2 EVOLUTIONARY ANALYSIS OF SIGMA 70 FAMILY SIGMA FACTORS AMONG VIBRIONACEAE ........................................................... 20 Introduction ...................................................................................................... 20 Materials and Methods ..................................................................................... 23 Housekeeping gene phylogenetic analysis ........................................... 23 Sigma factor sequence acquisition and functional groupings .............. 24 Sigma factor phylogenetic analysis ...................................................... 26 RNA extractions and qPCR .................................................................. 27 RNA-Seq analysis ................................................................................ 28 Results .............................................................................................................. 29 Domains 2 and 4 are the most highly conserved among Sigma 70 Family sigma factors found in V. parahaemolyticus ............................ 29 There is a positive correlation between the total number of sigma factors and genome size ........................................................................ 30 Phylogenetic analysis of all Sigma70 family sigma factors among Vibrionaceae ......................................................................................... 31 RpoD and RpoH are found in single copies in all species analyzed .... 32 Distribution of the second flagella system on chromosome two is clade specific ........................................................................................ 32 viii Several Vibrio species encode a divergent second copy of the RpoS sigma factor .......................................................................................... 33 V. parahaemolyticus possess 5 ECF type sigma factors with varying degrees of conservation ........................................................................ 34 ECF gene neighborhoods found in Vibrio parahaemolyticus are conserved throughout Vibrionaceae ....................................................