ABSTRACT CRAIG, KELLY. Examination of Pseudomonas sp. Response to Industrial Processing Stresses (Under the direction of Dr. Amy Grunden). Members of the genus Pseudomonas have drawn interest for their biotechnological and agricultural potential along with their medical importance as plant and animal pathogens. Several species of Pseudomonas have been targeted for their ability to repress microbial plant pathogens, insects, and nematodes. To succeed as viable candidates for agricultural applications, biological control strains need to survive the formulation process, prolonged periods of storage, and challenging environmental conditions. During the formulation process, beneficial bacteria can be dried to halt metabolism and improve shelf-life stability, transportability, and ease of application in the field. A high throughput screening strategy was developed to identify soil-associated microbes capable of surviving drying methods. The microbial diversity of soil bacterial communities was analyzed after exposure to spray drying and oven tray drying. In addition, a Gram-negative bacteria targeted isolation method was established to study the survival capabilities of asporulous Gram-negative bacteria. Bacillus, a Gram-positive spore forming bacterium, has an advantage in surviving and quickly recovering from harsh drying methods such as spray drying, whereas Pseudomonas sp. and other Gram-negative bacteria are capable of surviving milder formulation strategies such as oven tray drying. A diverse set of Pseudomonas species was subjected to heat shock conditions to determine which species are capable of surviving heat shock. A second study assessed the ability of a panel of protectants to improve stress recovery of the Pseudomonas strains. P. thermotolerans and P. aeruginosa were the top two species capable of surviving a 60°C five- minute heat shock. Heat shock surviving strains were enriched with genes encoding for heat shock proteins and universal stress response proteins such as HSP70, GroEL/ES, and clp protease compared to strains that could not survive heat shock. A subset of the Pseudomonas diversity panel was treated with common protectants prior to exposure to heat shock temperatures. Skim milk improved the recovery of the broadest range of Pseudomonas strains subjected to heat shock. These studies evaluated the capabilities of Pseudomonas species to survive formulation related stress, detected phylogenetic and genotypic predictors of heat shock survival, and determined the highest performing heat shock protectants for a diverse set of Pseudomonas species. It can take years of research to develop an efficacious and reliable product with a long shelf life. Developing a better understanding of optimal drying methods and protectants for Pseudomonas species will accelerate the capabilities of these microorganisms as biological control products. © Copyright 2021 by Kelly Craig All Rights Reserved Examination of Pseudomonas sp. Response to Industrial Processing Stresses by Kelly Craig A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science Microbiology Raleigh, North Carolina 2021 APPROVED BY: _______________________________ _______________________________ Dr. Amy Grunden Dr. Jose Bruno-Barcena Committee Chair _______________________________ _______________________________ Dr. Paul Hamilton Dr. James Kremer DEDICATION I would like to dedicate my thesis research to my husband, Chris, and my parents. Thank you for your never-ending support and encouragement. ii BIOGRAPHY Kelly Craig was born in Sayre, PA to parents Deborah and Brian Craig on February 4th, 1992. She was raised in Cary, NC with her three brothers and graduated from Athens Drive High School in 2010. Kelly attended North Carolina State University and received her B.S. in agricultural and environmental technologies in 2015. During her final year at North Carolina State University, Kelly worked at the Plant Disease and Insect Clinic where she discovered her passion for microbiology. After graduating, Kelly was offered a position at an agricultural biotechnology company named AgBiome. Kelly has worked at AgBiome for the past six years focusing on screening for biological control agents against plant pathogens and searching for probiotics to control pathogen outbreaks in newly weened piglets. Kelly joined the microbiology program at North Carolina State University to pursue a master’s degree under the mentorship of Dr. Amy Grunden. Her research focused on the tolerance of Pseudomonas to heat stress and the translation of these adaptations to surviving the industrial formulation process. Kelly lives in Durham, NC with her husband Chris and their two kittens, Sylphrena and Nyx. iii ACKNOWLEDGMENTS First, I would like to thank my committee chair, Dr. Amy Grunden, for her support and guidance throughout my graduate career. You are brilliant, incredibly patient, and I am very grateful for your help over the years. I would also like to thank Dr. Brant Johnson and my committee members, Dr. James Kremer, Dr. Jose Bruno-Barcena, and Dr. Paul Hamilton, for their support and advice. A special thanks to Dr. James Kremer and Dr. Brant Johnson for supporting my graduate efforts at AgBiome. Attending graduate school while remaining full time at AgBiome was not easy and I was only successful because of your encouragement and mentorship. I have had the opportunity to work with phenomenal scientists at AgBiome. This was my first time venturing into formulations and bioinformatics, and I would like to thank Alex Schlesinger, Matthew Biggs, Jake Trimble, Chuck Pepe-Ranne, and Jessica Parks for answering my never-ending questions on experimental design and data analysis. Thank you to everyone in Dr. Grunden’s lab for being so welcoming and supportive: Hannah Wapshott-Stehli, Mara Cuebas-Irizarry, Jabeen Ahmad, Deaja Sanders, Enrique Garcia, Micaela Robson, and Jason Whitham. iv TABLE OF CONTENTS LIST OF TABLES ...................................................................................................................... viii LIST OF FIGURES ...................................................................................................................... ix Chapter 1: Leveraging Pseudomonas Stress Response Mechanisms for Industrial Applications .................................................................................................................................. 1 1.1 Abstract .................................................................................................................................... 2 1.2 Introduction .............................................................................................................................. 3 1.3 Desiccation Stress .................................................................................................................... 7 1.4 Heat Stress ............................................................................................................................. 17 1.5 Cold Stress ............................................................................................................................. 27 1.6 General Stress Response ........................................................................................................ 33 1.7 Conclusions and Future Directions ........................................................................................ 42 1.8 References .............................................................................................................................. 44 Chapter 2: Diversity of Soil Bacterial Communities After Exposure to Formulation Stress ........................................................................................................................................... 62 2.1 Abstract .................................................................................................................................. 63 2.2 Introduction ............................................................................................................................ 64 2.3 Materials and Methods ........................................................................................................... 66 2.4 Results .................................................................................................................................... 74 2.5 Discussion .............................................................................................................................. 87 2.6 Conclusions ............................................................................................................................ 89 2.7 References .............................................................................................................................. 90 Chapter 3: Evaluation of Pseudomonas Heat Shock Survival in Response to Protectant Addition ...................................................................................................................................... 93 3.1 Abstract .................................................................................................................................. 94 3.2 Introduction ............................................................................................................................ 95 3.3 Materials and Methods ........................................................................................................... 97 3.4 Results .................................................................................................................................