Ethylene Response and Phytohormone-Mediated Regulation of Gene Expression in Komagataeibacter xylinus ATCC 53582 by Richard Vincent Augimeri A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Masters of Science In The Faculty of Science Applied Bioscience University of Ontario Institute of Technology April 2016 © Richard Vincent Augimeri, 2016 ABSTRACT Komagataeibacter xylinus ATCC 53582 is a fruit-associated, cellulose-producing bacterium that responds to and synthesizes phytohormones. This thesis elaborates on the ecophysiology of K. xylinus. Responses to indole-3-acetic acid (IAA), abscisic acid (ABA) and ethylene, produced in situ from ethephon, were of particular focus. The effect of these phytohormones on K. xylinus cellulose production and expression of cellulose biosynthesis-related genes (bcsA, bcsB, bcsC, bcsD, cmcAx, ccpAx and bglAx) were determined using pellicle assays and reverse transcription quantitative polymerase chain reaction (RT-qPCR), respectively. Ethylene enhanced cellulose yield by upregulating bcsA and bcsB expression, while IAA decreased cellulose yield by downregulating bcsA. Differential gene expression within the bacterial cellulose synthesis (bcs) operon is reported and a phytohormone-regulated CRP/FNR transcription factor was identified that may influence K. xylinus cellulose biosynthesis. Based on evidence provided in this thesis, the classification of K. xylinus as a saprophyte and its potential to accelerate fruit ripening in nature is proposed. ii Keywords Komagataeibacter xylinus ATCC 53582 Bacterial cellulose bcs operon Ethylene Ethephon Indole-3-acetic acid Abscisic acid CRP/FNR Plant-microbe interaction Fruit-bacteria interaction Fruit ripening Saprophyte Ecophysiology Biofilm Pellicle RT-qPCR iii ACKNOWLEDGEMENTS I am profoundly indebted to my supervisor and mentor, Dr. Janice Strap, for her ample amounts of time spent assisting me with experiments and for her continuous support and guidance throughout this research project. Without her constant motivation, understanding and wise words, this work would not have been possible. I would also like to thank my advisory committee for their constructive comments, suggestions and motivation. In particular, I would like to extend my gratitude to Dr. Dario Bonetta for teaching me the ways of a plant scientist, providing Arabidopsis thaliana seeds, assisting me with gas chromatography and for helpful discussions. Many thanks go out to all of my lab mates over the past few years, but especially to Andrew Varley for enlightening conservations and for collaborating with me on our review paper. I also want to thank Dr. Liliana Trevani and Dr. Jean-Paul Desaulniers for letting me use their UV-visible spectrophotometers, Dr. Julia Green-Johnson for providing centrifuges, microscopes and the qPCR thermocycler, and Dr. Franco Gaspari and Simone Quaranta for providing and teaching me how to use the FT-IR spectrometer. A big thank you goes out to my girlfriend for her ongoing love, encouragement and motivation. Lastly, I would like to thank my parents for their unconditional love and support throughout my years as a university student. I am forever grateful for my experience in the Molecular Microbial Biochemistry Laboratory and look forward to sharing the knowledge I have obtained during my studies in my future endeavors. iv TABLE OF CONTENTS 1 INTRODUCTION ..................................................................................................1 Cellulose: Structure and uses ............................................................................1 Cellulose biosynthesis: Plants vs. bacteria ........................................................2 1.2.1 Cellulose synthesis complexes ....................................................................2 1.2.2 Bacterial cellulose synthesis complex subunits ...........................................6 1.2.3 Cellulose biosynthesis .............................................................................. 10 1.2.4 Cellulose crystallization............................................................................ 11 1.2.5 Plant vs. bacterial cellulose ....................................................................... 12 Microbial biofilm formation ........................................................................... 13 Environmental diversity of BC producers ....................................................... 17 1.4.1 Insect-bacteria interactions of BC-producing acetic acid bacteria .............. 18 1.4.2 Plant-bacteria interactions of BC producers .............................................. 20 1.4.2.1 Persistence of pathogenic Enterobacteriaceae on fresh produce ........ 20 1.4.2.2 Fruit-bacteria interactions of Komagataeibacter xylinus .................... 22 1.4.2.2.1 Phytohormone-mediated fruit-microbe interactions of K. xylinus ... 25 Ethylene: Response and biosynthesis in plants and bacteria ............................ 33 Purpose, hypotheses, and rationale of thesis research ...................................... 37 2 METHODOLOGY ............................................................................................... 39 Chemicals and growth medium ...................................................................... 39 Bacteria and starter culture growth conditions ................................................ 39 Periplasmic protein isolation and analysis....................................................... 40 Prediction of disordered protein domains ........................................................ 41 Triple response assay...................................................................................... 42 Gas chromatography (GC) .............................................................................. 43 Time-course pH analysis of ethephon-exposed cultures .................................. 44 Minimum inhibitory concentration (MIC) assay ............................................. 47 Growth kinetics .............................................................................................. 47 Pellicle assays and analysis ............................................................................ 48 Colony morphology........................................................................................ 50 Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) ...... 50 2.12.1 Growth conditions .................................................................................... 50 v 2.12.2 RNA purification, quality control and first-strand cDNA synthesis ........... 51 2.12.3 Bioinformatic identification of crp/fnrKx ................................................... 52 2.12.4 Primer Design........................................................................................... 53 2.12.5 RT-qPCR ................................................................................................. 56 2.12.6 Analysis and selection of reference genes ................................................. 57 2.12.7 RT-qPCR quality control, data analysis and statistics................................ 60 3 RESULTS ............................................................................................................. 61 IAA and ABA influence the periplasmic protein profiles of K. xylinus ........... 61 The MtfB protein from K. xylinus has predicted disordered regions ................ 66 K. xylinus produces low levels of endogenous ethylene .................................. 66 K. xylinus has proteins similar to other ethylene-forming enzymes ................. 67 Ethylene is produced from ethephon decomposition in SH medium (pH 7)..... 67 K. xylinus culture pH allows for ethephon decomposition ............................... 71 K. xylinus cultures have an increased final pH in the presence of ethylene ...... 71 Ethephon is relatively non-toxic to K. xylinus ................................................. 74 Ethylene does not affect the growth of K. xylinus in agitated broth cultures .... 75 Ethylene increases BC yield and decreases K. xylinus pellicle hydration due to an increase in crystallinity .......................................................................................... 77 Ethylene increases K. xylinus BC production on solid medium ....................... 83 The genome of K. xylinus contains potential ethylene-receptor genes ............. 85 RT-qPCR ....................................................................................................... 86 3.13.1 Steady-state mRNA levels are affected by IAA and ABA in K. xylinus..... 86 3.13.1.1 IAA and ABA induce differential steady-state expression of bcs operon genes that depends on hormone concentration and bacterial growth phase ........... 86 3.13.1.2 IAA and ABA affect the steady-state expression levels of bglAx, but do not affect expression of cmcAx and ccpAx ........................................................... 89 3.13.1.3 IAA and ABA upregulate the steady-state levels of crp/fnrKx during the exponential growth phase of K. xylinus ............................................................... 91 3.13.1.4 The steady-state expression levels of oprB are downregulated by IAA and upregulated by ABA during the early stages of K. xylinus growth ................. 91 3.13.2 Phytohormones influence the active mRNA levels of K. xylinus genes ..... 94 3.13.2.1 Ethylene and IAA cause differential expression of bcs operon genes . 94 3.13.2.2 Ethylene and ABA affect the expression of genes flanking the bcs operon 95 vi 3.13.2.3