Plasmid analysis, comparative genomics and transcriptomics of beer-spoilage lactic acid bacteria emphasizing the role of dissolved carbon dioxide and traditional beer-spoilage markers A Thesis Submitted to the College of Graduate Studies and Research In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In the Department of Health Sciences University of Saskatchewan Saskatoon By Jordyn A. Bergsveinson Copyright Jordyn A. Bergsveinson, December, 2015. All rights reserved. Permission to Use In presenting this thesis in partial fulfilment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor who supervised my thesis work or, in his absence, by the Head of the Graduate Program or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or to make other use of material in this thesis in whole or part should be addressed to: Head of the Health Sciences Graduate Program College of Medicine 107 Wiggins Road University of Saskatchewan Saskatoon, Saskatchewan (S7N 5E5) i ABSTRACT Specific isolates of lactic acid bacteria (LAB) are capable of growing in and spoiling beer, and are the cause of product and process contamination, and financial loss for brewers the world over. To date, our understanding of how these contaminants are able to grow in beer is limited to analysis of hop-tolerance mechanisms, with a limited number of putative hop-tolerance genes having been described. In order to demonstrate that these hop-tolerance genes are incomplete descriptors of overall beer-spoilage ability, the transcriptional activity of these genes in two different beer-spoilage related (BSR) LAB isolates, and the prevalence and sequence conservation of hop-tolerance gene horC in BSR LAB with varying beer-spoilage ability is examined. This analysis is followed by work demonstrating that the total plasmid profile of a beer-spoilage LAB, and not just plasmids harboring hop-tolerance genes, contributes to the isolate’s overall beer-spoilage phenotype and highlights redundancy in potential beer-spoilage mechanisms. The next chapter provides evidence that the presence of dissolved CO2 (dCO2) in beer selects for the ability of LAB to spoil packaged beer, and that tolerance to this stress is not correlated with hop-tolerance, indicating that dCO2 stress is an important part of the total beer environment. This is followed by the presentation and analysis of the genome of the rapid beer- spoiling isolate Lactobacillus brevis BSO 464 and subsequent RNA sequencing for this isolate when grown in degassed and gassed beer so as to elucidate which genes are active when grown in beer, and when grown specifically in the presence of dCO2. Global transcriptome sequencing of this L. brevis isolate and Pediococcus claussenii ATCC BAA-344T when each were grown in growth-limiting concentrations of hops was also performed in order to clarify the hop-specific transcriptional response from that of the response when these isolates grow in the total beer environment. Lastly, comparison is made between available genomes of BSR LAB to reveal that the specific brewery environment a BSR LAB is recovered from, influences genetic variability and that comparison within a given LAB species reveals genetic differences that can be exploited as beer-spoilage genetic markers. This comparative analysis reveals that the total plasmid-coding capacity strongly influences individual BSR LAB beer-spoilage phenotype and the environment they are able to grow in. Overall, beer-spoilage ability is shown to be adaptive and acquired incrementally and not solely as a result of the presence of hop-tolerance genes. ii ACKNOWLEDGEMENTS I would like to extend my gratitude and thanks to my research advisory committee members for their support and input to my work over the past four years and allowing me to learn from their combined expertise. Further, I would thank Dr. Vanessa Pittet and all summer and Honours students that I have had the pleasure to work with, and for their contribution to the data presented here. Finally, I must thank Dr. Barry Ziola for his ability to put up with me, his drive to challenge me, and his invaluable guidance and support in and for all my endeavors. I gratefully acknowledge personal support from the following funding sources. I received Devolved and Non-Devolved Graduate Scholarships from University of Saskatchewan at both the Master’s and PhD level. The American Society of Brewing Chemists provided two Eco-Lab Scholarships and a Roger C. Briess Scholarship. Lastly, travel funds were generously provided to me by the University of Saskatchewan, the College of Graduate Studies and Research, the College of Medicine, and the Barbara Moore Memorial Trust Fund and Dr. Thomas A. Cunningham Memorial Fund, each from the Department of Pathology. iii DEDICATION I dedicate this thesis to both of my parents for all forms of their unconditional support; to all the people whom I have had the opportunity to work with, learn from; and to many an interesting beer drank. iv TABLE OF CONTENTS Permission to Use ....................................................................................................................... i ABSTRACT ............................................................................................................................... ii ACKNOWLEDGEMENTS .................................................................................................... iii DEDICATION.......................................................................................................................... iv TABLE OF CONTENTS……………………………………………………………………..v LIST OF TABLES .................................................................................................................... x LIST OF FIGURES ................................................................................................................. xi LIST OF APPENDICES ....................................................................................................... xiii LIST OF ABBREVIATIONS ............................................................................................... xiv Chapter 1: Introduction, literature review, and objectives ...................................................... 1 1. INTERFACE ......................................................................................................................... 1 1.1. GENERAL OVERVIEW .................................................................................................. 1 1.1.1 Introduction to lactic acid bacteria in beer: “The good, the bad and the ugly” ............. 1 1.1.2. The promise of omics for BSR LAB research .............................................................. 3 1.2. General LAB characteristics ............................................................................................. 4 1.2.1. BSR LAB diversity ....................................................................................................... 5 1.3. Traditional and emerging methods for BSR LAB detection and identification .......... 7 1.3.1. Culture-based Methods ................................................................................................. 7 1.3.2. Molecular Techniques ................................................................................................... 8 1.3.3. Multilocus sequence typing ........................................................................................ 12 1.3.4. Omics .......................................................................................................................... 12 1.4. BSR LAB and the brewing environment ....................................................................... 14 1.4.1. Niche adaptation and horizontal gene transfer............................................................ 14 1.4.2. Origin of BSR LAB .................................................................................................... 16 1.5 Hop tolerance .................................................................................................................... 18 1.5.1. Antimicrobial effect of hops ....................................................................................... 18 1.5.2. Hop-tolerance mechanisms ......................................................................................... 18 1.5.3. Proposed hop-tolerance genes..................................................................................... 19 1.5.4. Hop-tolerance genes and the brewing environment .................................................... 21 1.5.5. Utility of BSR LAB hop-tolerance genes ................................................................... 22 1.6. Stress tolerance and adaptation of BSR LAB ............................................................... 23 1.6.1. Stress tolerance to ethanol and low pH ....................................................................... 24 1.6.2. Stress tolerance