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Mechanisms of Polar Growth in the Alphaproteobacterial Order

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Mechanisms of Polar Growth in the Alphaproteobacterial Order MECHANISMS OF POLAR GROWTH IN THE ALPHAPROTEOBACTERIAL ORDER RHIZOBIALES A Dissertation Presented to The Faculty of the Graduate School At the University of Missouri In Partial Fulfillment Of the Requirements for the Degree Doctor of Philosophy By MICHELLE A. WILLIAMS Dr. Pamela J.B. Brown, Dissertation Supervisor DECEMBER 2019 The undersigned, appointed by the dean of the Graduate School, have examined the dissertation entitled MECHANISMS OF POLAR GROWTH IN THE ALPHAPROTEOBACTERIAL ORDER RHIZOBIALES Presented by MICHELLE A. WILLIAMS A candidate for the degree of Doctor of Philosophy And hereby certify that, in their opinion, it is worthy of acceptance. Dr. Pamela J.B. Brown Dr. David J. Schulz Dr. Elizabeth King Dr. Antje Heese ACKNOWLEDGEMENTS First, I would like to extend my sincerest appreciation to my advisor Pam Brown. You are the person who has truly been my encouragement and support through the ups and downs of the last five years. You have gone above and beyond to be a great mentor and friend. Thank you for all the Saturday meetings at Starbucks and the chats about career advice. It is a privilege to be one of the first members of your lab. Through your amazing example, I have grown so much as a critical thinker, designer of experiments and as a mentor to others. To my committee members, Dr. Libby King, Dr. David Schulz, and Dr. Antje Heese, thank you so much for the guidance and support you have provided me on my project and future career. Special thanks to Dr. George Smith, and Dr. Linda Chapman for always attending lab meeting and providing feedback on talks, posters and everything in between. Your mentorship has helped me grow as a scientist. Thank you to the Life Sciences Fellowship Program at the University of Mizzou for providing me with four years of funded research. Especially to Debbie Allen and Mark Hannink, who run the program, and have believed in and supported me. I would like to thank the undergraduate students Andrew Yowell, Jenn Amstutz, Colleen Kennedy, and Grey Gereau for their contributions to this work, and for being amazing mentees. ii To my wonderful lab mates and friends; Wanda Figueroa-Cuilan, Hedieh Attai, Matt Howell, Jeremy Daniel, Jacob Bouchier, and Gustavo Santiago thank you all for the support and encouragement over the years! For always making me laugh, challenging me to think critically, providing feedback on my talks and posters, and going on numerous coffee runs! Special thank you to Matt, Wanda and Amelia Randich for thoughtful reading of this dissertation. To Hedieh and Wanda, we will always be the lab triplets! Thank you for keeping me sane through the long nights in lab, I love you both. Finally, to my family and friends outside of the lab. Without your support I would not be here. To my parents Mary and Wally and my sister Rachelle, thank you for visiting me (and cleaning my house) whenever my life would get a little hectic. I would especially like to thank my grandfather Al Taber, who inspired me to become a scientist. You were always experimenting and inventing, and you encouraged me to do the same. I miss you every day. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ......................................................................................ii LIST OF FIGURES ............................................................................................. viii LIST OF TABLES .................................................................................................xi ABSTRACT ......................................................................................................... xii Chapter 1. Diversity of Growth Patterns in the Alphaproteobacteria Abstract .......................................................................................................... 2 Ecological Diversity of Alphaproteobacteria ................................................... 2 Methods to Visualize Bacteria Growth Patterning .......................................... 5 Growth Patterns During Elongation ................................................................ 7 Growth Patterns During Division .................................................................. 10 Growth Patterns of Budding Bacteria in the Rhizobiales .............................. 11 Growth Patterns of Stalked Budding Bacteria .............................................. 12 Growth Patterns of Prosthecate Budding Bacteria ...................................... 15 Unipolar Elongation of Rod-shaped Bacteria ............................................... 17 Diverse Growth Patterns of Stalked Bacteria in the Caulobacterales .......... 21 Stalked Budding Bacteria within the Caulobacterales .................................. 21 Caulobacterales with Non-reproductive Stalks ............................................. 24 Cell growth in the Rhodospirillales ............................................................... 28 Cell growth in the Rhodobacterales ............................................................. 31 Cell growth in the Rickettsiales .................................................................... 33 Conservation of Core Elongation Machinery in the Alphaproteobacteria ..... 36 Survey of Peptidoglycan Biosynthesis Machinery ........................................ 37 Regulation of Peptidoglycan Biosynthesis Machinery .................................. 41 Concluding remarks ..................................................................................... 44 References .................................................................................................. 45 2. Short-Stalked Prosthecomicrobium hirschii Cells have a Caulobacter-Like Cell Cycle Abstract ........................................................................................................... 56 Introduction ..................................................................................................... 56 iv Materials and Methods .................................................................................... 61 Strain and Culture Conditions ...................................................................... 61 Materials ...................................................................................................... 62 Scanning electron microscopy ..................................................................... 62 Transmission electron microscopy ............................................................... 62 Light microscopy and FDAA staining ........................................................... 63 Initial attachment and polar adhesin detection ............................................. 63 Biofilm formation and LIVE/DEAD staining .................................................. 64 Microfluidic device fabrication ...................................................................... 65 Device operation .......................................................................................... 66 Cell culture, seeding and synchronization .................................................... 67 P. hirschii genome sequencing, assembly, and annotation ......................... 68 Ortholog identification .................................................................................. 69 Genome-wide motiff searches ..................................................................... 70 Phylogenetic tree construction ..................................................................... 70 Results ............................................................................................................ 71 Short-stalked P. hirschii cells are motile....................................................... 71 Short-stalked P. hirschii cells produce a polar polysaccharide ..................... 74 Short-stalked P. hirschii cells can be synchronized ..................................... 76 Conservation of cell cycle regulators and binding sites ................................ 79 Discussion ....................................................................................................... 85 References ...................................................................................................... 92 Supplementary Material .................................................................................. 97 3. Cell Wall Synthesis and Remodeling Enzymes Required for Polar Growth in the Rhizobiales Abstract ......................................................................................................... 108 Introduction ................................................................................................... 109 Materials and Methods .................................................................................. 112 Bacterial strains, plasmids, and growth conditions ..................................... 112 Construction of strains and plasmids ......................................................... 113 Construction of deletion/depletion plasmids and strains ............................ 113 v Phase and fluorescence microscopy ......................................................... 114 Quantification of cell length distributions .................................................... 115 Quantification of cell morphologies , FDAA and FDAAD labeling patterns 115 Peptidoglycan compositional analysis ........................................................ 115 Results .......................................................................................................... 119 Cell wall synthesis enzymes play a key role in A. tumefaciens growth and division ......................................................................................................
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