Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2021 Light as a global regulator of gene expression and an anticipatory signal for environmental water loss in Pseudomonas syringae Bridget M. Hatfield Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Recommended Citation Hatfield, Bridget M., "Light as a global egulatr or of gene expression and an anticipatory signal for environmental water loss in Pseudomonas syringae" (2021). Graduate Theses and Dissertations. 18506. https://lib.dr.iastate.edu/etd/18506 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Light as a global regulator of gene expression and an anticipatory signal for environmental water loss in Pseudomonas syringae by Bridget M. Hatfield A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Genetics Program of Study Committee: Gwyn A. Beattie, Major Professor Mark L. Gleason Justin W. Walley Larry J. Halverson Kyaw J. Aung The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this dissertation. The Graduate College will ensure this dissertation is globally accessible and will not permit alterations after a degree is conferred. Iowa State University Ames, Iowa 2021 Copyright © Bridget M. Hatfield, 2021. All rights reserved. ii DEDICATION To my husband, Kirk, your belief in me, encouragement, and love kept me going. To my daughter, Maeve, your never-ending joy always brightens my day. To my family, you have taught me to fight for my dreams and because of you I know that through hard work and persistence I can accomplish anything I set my mind to. iii TABLE OF CONTENTS Page ACKNOWLEDGMENTS ............................................................................................................. vi ABSTRACT .................................................................................................................................. vii CHAPTER 1. GENERAL INTRODUCTION ................................................................................1 Dissertation Organization .......................................................................................................... 1 Literature Review ...................................................................................................................... 1 Photosensory proteins are used to sense light ...................................................................... 2 Diverse photosensory proteins beyond those in Pss B728a ................................................. 3 Photosensory proteins in plants ............................................................................................ 4 LOV proteins in microorganisms ......................................................................................... 4 Phytochromes in microorganisms ........................................................................................ 6 Photosensory proteins in Pseudomonas syringae ................................................................ 7 P. syringae can survive in a wide range of environments .................................................... 8 Pss B728a as a plant pathogen ............................................................................................. 9 Pss B728a strategies for coping with fluctuating water availability on leaves .................... 9 Goal of the thesis ................................................................................................................ 11 References ............................................................................................................................... 11 CHAPTER 2. A BACTERIOPHYTOCHROME ORCHESTRATES TRANSCRIPTIONAL REPROGRAMMING IN RESPONSE TO MULTIPLE LIGHT WAVELENGTHS IN PSEUDOMONAS SYRINGAE B728a ............................................................................................17 Abstract .................................................................................................................................... 17 Introduction ............................................................................................................................. 18 Materials and Methods ............................................................................................................ 20 Bacterial strains and growth conditions. ............................................................................ 20 Construction of a chloramphenicol resistant B728a strain. ................................................ 22 Construction of pET21a-bphOP1-His6X. ........................................................................... 22 Lighting conditions............................................................................................................. 22 Purification of BphP1 protein............................................................................................. 23 Spectroscopic evaluation of BphP1 protein. ...................................................................... 23 Exposure of cells to light for RNAseq analysis. ................................................................ 23 RNA extraction................................................................................................................... 24 Mapping the reads. ............................................................................................................. 25 RNAseq data analysis. ........................................................................................................ 25 Characterization of algD expression at various light intensities. ....................................... 26 Conjugation assays. ............................................................................................................ 26 Statistical analyses. ............................................................................................................. 27 Results ..................................................................................................................................... 27 Purified BphP1 responds to far-red, blue, and red light in vitro ........................................ 27 Light is a global signal for B728a gene expression............................................................ 28 BphP1 regulates the majority of the light-responsive genes in B728a ............................... 31 iv Over-representation analyses identify functional groups that are strongly regulated by light ..................................................................................................................................... 34 Genes involved in osmotolerance are prominent among light- and BphP1-induced genes ................................................................................................................................... 37 The light-dependent BphP1 regulon overlaps with the osmotic stress-dependent AlgU regulon ...................................................................................................................... 40 BphP1 disproportionately induces genes that encode membrane-localized proteins......... 41 algD expression is sensitive to both quality and quantity of light ..................................... 42 BphP1 regulates conjugation in a light and wavelength specific manner .......................... 43 Discussion ................................................................................................................................ 47 Acknowledgements ................................................................................................................. 53 References ............................................................................................................................... 54 Supplementary Data ................................................................................................................ 59 CHAPTER 3. PLANT-ASSOCIATED PSEUDOMONADS USE LIGHT AS AN ANTICIPATORY CUE TO ENHANCE OSMOTOLERANCE ..................................................62 Abstract .................................................................................................................................... 62 Introduction ............................................................................................................................. 63 Methods ................................................................................................................................... 67 Bacterial strains and growth conditions ............................................................................. 67 Light exposure setup .......................................................................................................... 68 Assessment of light impacts on bacterial osmotolerance based on optical density measurements ..................................................................................................................... 68 Assessment of light impacts on bacterial osmotolerance based on cell viability ............... 69 Environmental monitoring preceding and following dawn