IDENTIFYING THE ACTIVITIES OF RHIZOSPHERE MICROBIAL COMMUNITIES USING METATRANSCRIPTOMICS By Aaron Garoutte A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Microbiology and Molecular Genetics – Doctor of Philosophy 2016 ABSTRACT IDENTIFYING THE ACTIVITIES OF RHIZOSPHERE MICROBIAL COMMUNITIES USING METATRANSCRIPTOMICS By Aaron Garoutte Soil microbial communities carry out many functions, most of which are beneficial to the planet as well as to humans. Soil microbial communities control the biogeochemical cycling rates of key elements such as carbon, nitrogen, sulfur and phosphorous and can also aid in plant growth and disease defense. Microbial ecologists have studied the functional activities of microbial communities for decades often using laboratory incubations. Metagenomics has allowed the identification of the microbes and the potentially functional genes in an environmental sample, but does not allow an assessment of activity. Direct observation of microbial community activity in the field is the desired strategy to build the foundational knowledge required to assess, predict and potentially manage soil microbial community activity. In this dissertation I combine the use of metagenomics with metatranscriptomics to identify functional activity of the microbial community in the soil and rhizosphere of candidate biofuel crops. First, I assessed the efficiency of a novel method of rRNA removal, called the duplex specific nuclease normalization, to remove the dominating rRNA from samples of total RNA, to allow greater sequence coverage of mRNA. While this method did result in about 17% non-rRNA, it did not provide a major gain in sequencing depth. I also established best practices for computational metatranscriptomic analysis, especially the importance of assembling short reads into longer contigs to improve annotation accuracy. Second, I examined the activity of the rhizosphere microbial community of switchgrass, a candidate biofuel crop, using a combination of metagenomics, metatranscriptomics and metaproteomics. I defined a minimum core of microbial community functions of both metagenomic and metatranscriptomic sequences to focus the analysis on the most common sequences that were expressed. Beyond the expected housekeeping functions, the ecologically important functions related to biogeochemical cycling expressed were glycoside hydrolases, ligninolytic enzymes, ammonia assimilation, phosphate metabolism and functions related to plant-microbe interactions were production of auxin, trehalose and ACC-deaminase. Ecologically important genes had lower abundance than housekeeping functions indicating that ecologically important genes may represent keystone functions. I also examined the effect of two plants, switchgrass and corn, on the presence and activity of microbial community functions at various distances from living roots using metagenomics and metatranscriptomics. The metagenomic data was able to differentiate between microbial communities associated with the two different crops and differentiate communities in direct contact with the roots versus those not in direct contact. The metatranscriptomic data was unable to differentiate between bulk and rhizosphere samples indicating others factors are stronger determinants of community transcription. I show that direct observation of the activity of microbial communities associated with biofuel crops in field collected samples is possible through metatranscriptomics and aided by metagenomics and metaproteomics. These data allow the detection of microbial activities related to biogeochemical cycling and plant microbe interactions as well as reveal differences in genetic potential across different soil treatments. This thesis is dedicated to my parents, Kristin Magnuson and Michael Garoutte, and to my wife Jennifer Garoutte iv ACKNOWLEDGEMENTS They say it takes a whole village to raise a child, I think the same could be said about finishing a PhD. I would like to thank my friends and family (Garoutte and Gable), you have all been a fantastic support! Your interest in my work has always encouraged me. I never tire of the question “So, what is it you do again?” I would like to thank my children, Charlie and Claire. Watching you grow, learn and explore has been an inspiration to me as a scientist and as a father. You give me immeasurable joy and remind me there is a world outside of the lab. I will always be proud of you. I love you both dearly. I would also like to thank my wife, Jenny. You have been my rock. Your unwavering confidence in me and your constant support have enabled me to achieve this goal. I love you more than words can convey. Mom, thank you for all your support over the years, through all the ups and downs of life you have always been there for me. It is only now, as I have my own children, that I am starting to understand the sacrifices you and Dad made for me. Dad, you were taken from us too soon. You will always be my role model and I will always strive to be the great father you were to me. I know you’d be proud of me, you said it so many times that now it is the only thing I hear you say when I remember your voice. I would like to thank a few specific teachers and professors who have helped me throughout my now 22 years of education. First, I’d like to thank Mrs. Christine Arenson, you taught this little dyslexic boy to read and helped me overcome my learning disability. Next, I would like to thank Dr. Richard Cass. You showed me that even though I was a slow reader and not the best writer that I could succeed in a college level English course. Finally, I would like to thank Drs. Greg and Kathy Murray. You both shaped me as an ecologist and v gave me my first research experiences. Your classes were hard but the time I put in was never work. Thank you for investing in me. I would like to thank Dion Antonopoulos for taking a chance and hiring a field ecologist with minimal knowledge of microbiology, and next to nothing in terms of lab skills, to work in your lab. You helped ignite my passion for microbial ecology and set me on the path of working with metatranscriptomics. You taught me everything I know about microbiology and molecular biology lab work. I would also like to thank Sarah O’Brien for sharing with me your passion for soil. The two of you set me on a path to explore soil microbial communities in my PhD work. I would also like to thank the Tiedje lab, all current and past members. I was fortunate to have been able to interact with so many people with such diverse backgrounds. Thank you for your support, encouragement and constructive criticism. You helped to make me a better scientist and more well rounded person. Specifically, I’d like to thank Adina Howe. Adina, you were like my second mentor. You taught me to code, how to use the HPCC, how to interview and train students. You helped improve my writing and you were always up for Indian buffet! Thank you for investing in me. Dr. Tiedje, thank you for taking me on as a student. You had no idea what you were getting into! You have helped me learn how to think about science and how to present my work both in publication and presentation form. You have always had the ability to push me outside of my scientific comfort zone, and your unique perspective has always helped bring my work up to the next level. Thank you. Finally, I would like to thank my current committee members Sheng Yang He, Maren Friesen and Ashley Shade. Each of you has helped me grow as a scientist. I value the vi feedback you have given me and appreciate your time. I would also like to thank a former committee member, Titus Brown. Thank you for supporting me as this hybrid biologist bioinformatician. You have always made me feel like I had a place in your lab. You taught me the value of open science and helped me think critically about the code I write. vii TABLE OF CONTENTS LIST OF TABLES …...…..……………………………………………………………………………………...…………….x LIST OF FIGURES.….....………………………………………………………………………………………………..…xiii CHAPTER 1: Bridging the gap between the lab and the field .…………...…………………...................1 Introduction to soil microbial communities…………………………………………………………..2 Soil microbial community functions …………………..…………………………………………………2 The rhizosphere: a focal point of microbial community activity……………………………..5 Disconnect between laboratory-based studies and field microbial communities ……………………………………………………………………………………………………………………6 “Omics” opens new avenues of research………………………………………………………………..7 Evolution of sequencing technology…………………………………………………………...7 Metagenomics………………………………………………………………….....................7 Metatranscriptomics………………………………………………………………………9 The dark side of high throughput sequencing………………………………..10 Observation of microbial community functions to answer ecological questions…....11 Importance of direct observation of microbial community functional activity………12 Questions addressed in this thesis………………………………………………………………………13 REFERENCES……..………………………………………………………………………………………………16 CHAPTER 2: Methodologies for probing the metatranscriptome of grassland soil..................24 Abstract………………………………………………………………………………………………………………………..25 Introduction…………………………………………………………………………………………………………………26 Methods……………………………………………………………………………………………………………………….29 Metatranscriptome sample collection and library preparation……………………………..31 MG-RAST databases used for annotation of unassembled raw reads…………………….32 Metatranscriptome assembly & annotation…………………………………………………………32 Previous