The Aromatic Amino Acid Responsive TyrR Transcription Factor of Enterobacter cloacae UW5: Its Role in Regulation of Indole-3-Acetic Acid Biosynthesis and the Identification of an Expanded Regulon Using RNA-Sequencing by Thomas J.D. Coulson B.Sc. University of New Brunswick 2008 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate Academic Unit of Biology Supervisor: Cheryl L. Patten, PhD, Department of Biology Examining Board: Rene Malenfant, Ph.D, Biology Shawn MacLellan, Ph.D, Biology Ghislain Deslongchamps, Ph.D, Chemistry External Examiner: Ji Yang, Ph.D, Microbiology and Immunology, University of Melbourne This dissertation is accepted by the Dean of Graduate Studies THE UNIVERSITY OF NEW BRUNSWICK July, 2019 ©Thomas J.D. Coulson, 2019 ABSTRACT The control of transcription is an important process in all living cells. In the bacterial family Enterobacteriaceae, the transcription factor TyrR controls genes for aromatic amino acid uptake and biosynthesis. In this thesis, I explore the control of genes by TyrR in Enterobacter cloacae UW5, a soil bacterium commonly associated with plant roots that confer beneficial effects on its host and is also an inhabitant of human intestinal microflora and an opportunistic pathogen. Chapter 1 provides a general introduction to bacterial activities in the plant rhizosphere and transcriptional regulation, especially by TyrR. In Chapter 2, I investigated the regulation of two divergently transcribed genes, ipdC and akr, by TyrR. The ipdC gene encodes indolepyruvate decarboxylase for the production of the plant growth hormone indole-3-acetic acid, which plays an important role in the plant beneficial behavior of E. cloacae. TyrR is required for activation of ipdC by binding a single DNA element upstream of the promoter. All three aromatic amino acids act as cofactors for TyrR to induce ipdC expression. Expression of akr, encoding a putative aldo-keto reductase, was repressed by TyrR independently of aromatic amino acids and involved TyrR binding an atypical DNA site within the promoter. In Chapter 3, I assembled the E. cloacae UW5 genome sequence, which revealed genes and pathways that contribute to its plant-associated lifestyle and served as a reference for mapping RNA-sequencing data. In Chapter 4, I delineated the TyrR regulon by comparing transcription profiles in wild-type and tyrR mutant strains of E. cloacae generated through RNA-sequencing. Broad changes in gene expression were identified and several new TyrR members confirmed, including dmpM encoding a methyltransferase that is highly upregulated by tyrosine and phenylalanine, and cpxP and cpxR, which encode ii components of the envelope stress response. Additionally, pathways for aromatic metabolism, anaerobic respiration, and motility were altered in the tyrR mutant. Chapter 5 summarizes this research that suggests that the E. cloacae TyrR regulon has expanded from that of E. coli to include genes for survival in the diverse environments that this bacterium inhabits and illustrates the expansion and plasticity of transcription factor regulons. iii DEDICATION I dedicate this thesis to my parents, Dana and Heather Coulson, who have supported me both emotionally and financially, allowing me to pursue my lifelong dream of becoming a scientist. I love you both. iv ACKNOWLEDGEMENTS I would first and foremost like to thank my supervisor Dr. Cheryl Patten. I am eternally grateful to have studied under such an amazing mentor, who has supported my efforts over these many years. It has been wonderful to work, study and play in the lab. I would like to thank my supervisory committee members, Dr. Mike Duffy and Dr. Bryan Crawford for their input, suggestions and many conversations which have helped shape the direction of my work. And Dr. René M. Malenfant for his input and assistance with analysis of my sequencing data. Thank you to my family Dana, Heather, Caroline, Victoria, Charlotte, TJ, Shane, Bell, Mocha and Ru for your love, encouragement and patience. To my friends and fellow grad students who I have had the pleasure of knowing over the years I thank you for the insightful conversations, assistance in the lab and our times spent at the Grad House. And finally a super special thank you goes to Nebai, who has been there for me both professionally and emotionally. I look forward to the many adventures we will continue to have together and promise to take you back to Mount Carleton J v Table of Contents ABSTRACT ........................................................................................................................ ii DEDICATION ................................................................................................................... iv ACKNOWLEDGEMENTS ................................................................................................ v Table of Contents ............................................................................................................... vi List of Tables ..................................................................................................................... xi List of Figures ................................................................................................................... xii Chapter 1 Introduction ........................................................................................................ 1 1.1 The Rhizosphere ....................................................................................................... 1 1.1.1 Plant root exudates ............................................................................................. 3 1.1.2 Rhizobacteria ..................................................................................................... 4 1.1.3 Chemotaxis and motility in the rhizosphere ...................................................... 6 1.1.4 Nutrient utilization by rhizobacteria ................................................................ 11 1.1.5 Mechanisms of plant growth promotion by rhizobacteria ............................... 14 1.2 Transcription in prokaryotes ................................................................................... 22 1.2.1 Transcription factors ........................................................................................ 23 1.3 The TyrR transcription factor ................................................................................. 26 1.3.1 Structure of the TyrR protein ........................................................................... 26 1.3.2 Cofactor-mediated oligomerization of TyrR ................................................... 29 1.3.3 TyrR DNA recognition and binding ................................................................ 31 1.3.4 TyrR repression of transcription ...................................................................... 32 1.3.5 TyrR activation of transcription ....................................................................... 37 vi 1.3.6 PhhR: A TyrR homologue in Pseudomonas .................................................... 39 1.4 Enterobacter cloacae .............................................................................................. 43 1.5 Objectives ............................................................................................................... 46 1.6 Organization of thesis ............................................................................................. 47 1.7 References ............................................................................................................... 49 Chapter 2 The TyrR transcription factor regulates the divergent akr-ipdC operons of Enterobacter cloacae UW5a ............................................................................................. 75 2.1 Abstract ................................................................................................................... 75 2.2 Introduction ............................................................................................................. 76 2.3 Materials and Methods ............................................................................................ 80 2.3.1 Bacterial strains and culture conditions ........................................................... 80 2.3.2 Transcription start site identification ............................................................... 80 2.3.3 Construction of reporter gene fusions and promoter mutagenesis ................... 82 2.3.4 Reporter gene expression assays ...................................................................... 84 2.3.5 Electrophoretic mobility shift assays ............................................................... 85 2.3.6 Statistical analysis ............................................................................................ 87 2.4 Results ..................................................................................................................... 87 2.4.1 Mapping of the akr and ipdC promoters .......................................................... 87 2.4.2 ipdC is positively regulated by TyrR and the aromatic amino acids ............... 89 2.4.3 akr is negatively regulated by TyrR ................................................................. 89 vii 2.4.4 ipdC activation requires the strong TyrR binding site ..................................... 90 2.4.5 Repression of akr is facilitated by the weak TyrR binding site occluding the promoter ...................................................................................................................