University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School 3-26-2014 Sigma Factor N: A Novel Regulator of Acid Resistance and Locus of Enterocyte Effacement in Escherichia coli O157:H7 Avishek Mitra University of South Florida, [email protected] Follow this and additional works at: https://scholarcommons.usf.edu/etd Part of the Molecular Biology Commons Scholar Commons Citation Mitra, Avishek, "Sigma Factor N: A Novel Regulator of Acid Resistance and Locus of Enterocyte Effacement in Escherichia coli O157:H7" (2014). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/5077 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. Sigma Factor N: A Novel Regulator of Acid Resistance and Locus of Enterocyte Effacement in Escherichia coli O157:H7 by Avishek Mitra A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Cell, Microbiology and Molecular Biology College of Arts and Sciences University of South Florida Major Professor: Dr. James Riordan, Ph.D. Dr. Lindsey Shaw, Ph.D. Dr. Katie Scott, Ph.D. Dr. Stanley Stevens, Ph.D. Date of Approval: March 26, 2014 Keywords: rpoN, ntrC, rtcR, GDAR, LEE, EHEC Copyright © 2014, Avishek Mitra Dedication I dedicate this work to my parents. Regardless of all the things that we have fought about in the past, they have been the one constant in my life and have been there for me through thick and thin. I wouldn’t be who I am or where I am, without their love and support. I want to thank Edin, Mike and Vedad, my three friends who have I have known for over ten years and have become my brothers from other mothers. I feel lucky to have you all in my life and I cannot express enough in words what my parents and my friends mean to me. Acknowledgements I cannot express enough gratitude for Dr. Riordan, whose brilliance, selflessness and patience has allowed me to become the researcher I am today. I can truly say that I have cherished every moment of being under your mentorship and doing research in the lab for you. Those first two months cleaning and setting up the empty lab have been some of the happiest memories that I will forever cherish. I will always remember everything that you have taught me and how much you have inspired me to do research. I only hope that in the future I can do for other graduate students, what you have done for me. I want to thank Dr. Shaw from the bottom of my heart for being the first person who inspired me to pursue further studies in microbiology. At a time during my undergrad, when I was completely unsure about my future, your lectures in molecular microbiology gave me the first hope as to what I could work towards in my future. You were the first person to give me the opportunity to do research in your lab and I am forever grateful to you for that. I would like to thank Dr. Stevens and Dr. Scott, for being such great advisors for my degree and for giving me some amazingly insightful leads for my work over the last five years. I cannot thank you all enough for mentoring me. To Pamela, Jason, Nada and Katie, you have all been such great lab mates. I have such great memories with all of you and I wish you all the best in your future. I am definitely going to miss pranking Jason and spoiling movie endings for him. Thank you all for being such great people. Table of Contents List of Figures .............................................................................................................................................. iv List of Table ................................................................................................................................................. vi Abstract ....................................................................................................................................................... vii Chapter One: Regulation of Sigma Factor N and its Contribution to Bacterial Virulence and Pathogenesis ........................................................................................................................................... 1 1.1 Bacterial RNA Polymerase ........................................................................................................ 1 1.2 Bacterial Sigma Factors and Their Role in Transcription .......................................................... 3 1.2.1 The transcription and sigma factor cycle ................................................................... 4 1.2.2 The σ70 family: structure and functions ..................................................................... 4 1.3 The σ54 Family ........................................................................................................................... 6 1.3.1 Key components and structure of the σ54 protein ....................................................... 8 1.3.2 Key components and structure of bacterial enhancer binding proteins ................... 11 1.3.3 bEBPs lacking regulatory and DNA binding domains ............................................ 16 1.4 Factors That Influence σ54-Dependent Transcription ............................................................... 17 1.4.1 The effects of ppGpp and its cofactor DksA on σ54-dependent transcription .......... 18 1.4.2 Regulation of σ54-dependent transcription by integration host factor ...................... 21 1.4.3 Other factors that affect σ54 association with RNAP ............................................... 23 1.5 Role of σ54 in Bacterial Pathogenesis and Stress Fitness ......................................................... 24 1.5.1 Borrelia burgdorferi ................................................................................................ 25 1.5.2 Escherichia coli O157:H7 ........................................................................................ 26 1.5.3 Pseudomonas aeruginosa ........................................................................................ 28 1.5.4 Pseudomonas syringae............................................................................................. 29 1.5.5 Salmonella typhi ...................................................................................................... 30 1.5.6 Other species ............................................................................................................ 31 Concluding Remarks ...................................................................................................................... 32 Chapter Two: Sigma Factor N, Liaison to an NtrC and RpoS Dependent Regulatory Pathway Controlling Acid Resistance and the LEE in Enterohemorrhagic Escherichia coli ............................. 34 2.1 Abstract .................................................................................................................................... 34 2.2 Introduction .............................................................................................................................. 35 2.1 Results ...................................................................................................................................... 38 2.2.1 σN-σS directed regulation of glutamate-dependent acid resistance and the locus of enterocyte effacement ......................................................................................... 38 2.2.2 Effect of rpoN mutation on rpoS mRNA and σS stability in EHEC ........................ 42 2.2.3 Role for core RNA polymerase and σN–dependent transcription in the σS stability, GDAR and LEE expression phenotype of TW14359ΔrpoN ............................. 44 2.2.4 Sensitivity of σN-dependent GDAR and LEE expression to protease inhibition ..... 47 2.2.5 Identification of the enhancer-binding protein required for σN-directed regulation of GDAR and the LEE ..................................................................................... 49 i 2.3 Discussion ................................................................................................................................ 51 2.4 Materials and Methods ............................................................................................................. 56 2.4.1 Bacterial strains and culture conditions ................................................................... 56 2.4.2 Directed gene deletion and site-specific mutation ................................................... 57 2.4.3 Tests for acid resistance ........................................................................................... 57 2.4.4 Quantitative real-time PCR (qRT-PCR) .................................................................. 58 2.4.5 Protein extraction, SDS-PAGE and western immunoblots ...................................... 58 2.4.6 σS and rpoS mRNA stability .................................................................................... 59 2.4.7 lacZ transcriptional fusions and β-galactosidase assay ............................................ 60 2.4.8 Serine protease inhibition ........................................................................................ 61 Chapter Three: Essential Pathway Components and Stimuli for NtrC/σN-Dependent Regulation of Acid Resistance and the Locus of Enterocyte Effacement (LEE) in Enterohemorrhagic Escherichia coli (EHEC) .....................................................................................................................