Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Evolution of the Spherical Cell Shape in Bacteria A thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Albany New Zealand. Paul Richard Jesena Yulo 2019 Abstract Cell shape is an important feature of bacterial cells. It is involved in critical aspects of bacterial cell biology such as motility, growth, and the evasion of predators. Despite this, how cell shape has evolved in bacteria is unclear. For most rod-shaped bacteria, the maintenance of cell shape depends primarily on the bacterial actin-like protein, MreB. In this study, we show that the deletion of MreB from the rod-shaped model organism Pseudomonas fluorescens SBW25 results in the formation of aberrant spherical cells that have increased size and reduced fitness. This new MreB-null strain (ΔmreB) is susceptible to mechanical damage and grows poorly due to cell division defects. Furthermore, synthesized peptidoglycan (PG) chains were shorter and cell wall assembly was disorganised in this strain. A 1,000-generation evolution experiment comprised of multiple independent lineages produced spherical cells that have a reduced cell size and improved fitness. Mutations in the PG synthesis protein PBP1a were found across multiple lineages. Genetic reconstructions demonstrated that these mutations have a loss-of-function effect that reduced PG cross-linking and restored the ordered assembly of the cell wall, thereby reducing cell size and improving fitness in MreB-null cells. In one lineage, a five-gene deletion that included the gene coding for the outer membrane channel OprD was found to be beneficial. This deletion reduced cell size, improved fitness, and restored orderly cell wall construction. The mechanism responsible for this is unknown, but it may be related to modifications in septum localisation via the Min system. Finally, we show using phylogenetic analysis that PBP loss is a general trend in bacteria that evolved to become spherical, hinting at a plausible strategy for the evolution of the spherical cell shape from rod-shaped progenitors. i Acknowledgements First and foremost, I would like to thank my advisor, Dr. Heather Hendrickson. I am honoured to be her first Ph.D. student, and greatly appreciate all the time, energy, and guidance she has given me over several years of mentorship. I am deeply indebted for the training I have received terms of doing good science and in being an effective communicator, but most especially for the kind and ceaseless support she continued to provide when the journey became overwhelming. I would like to thank my co-supervisors, Dr. Austen Ganley who helped me find my footing as a provisional Ph.D. student, and Dr. Olin Silander for his critical advice and practical approach that helped bring focus to my project. I am thankful to my collaborators for all the amazing work that they have done: to Dr. Monica Gerth for creating the nucleus on which this project is based; to Dr. Yunhao Liu and Dr. Xue-Xian Zhang for creating the reconstruction strains that have been the pillars of my characterisation work; to Dr. Akhilesh Yadav and Dr. Felipe Cava for sharing their resources and expertise in PG analysis; to Dr. Ashar Malik and Dr. Jane Allison for helping me with protein modelling and bioinformatics analysis; and to Dr. Peter Lind for helping me learn flow cytometry. I am grateful to my institute, the Institute of Natural and Mathematical Sciences, for providing the scholarship that has made my project possible. And to my lab mates, of past and present, for their camaraderie, constructive insights, and meaningful discussions. To my family – I thank my parents, my mother Selena and my father Joe, for their unwavering support. Finally, I dedicate this work to my wife, Owee, for always being my constant supporter and inspiration. ii Table of Contents Abstract .......................................................................................................................................................... i Acknowledgements ....................................................................................................................................... ii List of Figures ................................................................................................................................................ v List of Tables .............................................................................................................................................. viii Chapter 1 Introduction .............................................................................................................................. 1 1.1. Evidence for the existence of rods first, then spheres ..................................................................... 2 1.2. Cell shape has a selective value ........................................................................................................ 4 1.3. The bacterial cell envelope ............................................................................................................... 7 1.3.1. The cytoplasmic or inner membrane .................................................................................... 7 1.3.2. The outer membrane ............................................................................................................ 8 1.3.3. PG cell wall ............................................................................................................................ 8 1.4. PG synthesis .................................................................................................................................... 11 1.5. Bacterial cytoskeleton ..................................................................................................................... 15 1.5.1. Tubulin-like proteins ........................................................................................................... 15 1.5.2. IF-like proteins .................................................................................................................... 16 1.5.3. Actin-like proteins ............................................................................................................... 17 1.6. MreB – the major rod-shape determining protein ......................................................................... 17 1.6.1. MreB coordinates PG synthesis .......................................................................................... 18 1.6.2. MreB can influence the cell division machinery ................................................................. 21 1.6.3. Loss of MreB or MreB-like proteins lead to shape defects and cell death ......................... 22 1.6.4. MreB loss may be an early first step to evolving spherical bacterial cells .......................... 23 1.7. Rationale and objectives of the study............................................................................................. 24 Chapter 2 Materials and Methods ........................................................................................................... 26 2.1. Media, bacterial strains, and growth conditions ............................................................................ 26 2.2. Measurement of bacterial growth kinetics ..................................................................................... 28 2.3. PCR methods and cloning techniques............................................................................................. 28 2.3.1. Agarose gel electrophoresis ................................................................................................ 29 2.3.2. Plasmid extraction, restriction enzyme digestion, and DNA ligation ................................. 30 2.3.3. Construction of the ancestral ΔmreB strain ........................................................................ 30 2.3.4. Tri-parental conjugation ..................................................................................................... 31 2.3.5. Cycloserine enrichment ...................................................................................................... 31 2.3.6. Gene complementation ...................................................................................................... 32 2.3.7. Construction of GFP-labeled P. fluorescens SBW25 ........................................................... 32 iii 2.4. Experimental evolution ................................................................................................................... 32 2.5. Competitive fitness assay using flow cytometry ............................................................................. 33 2.6. Mutation Detection......................................................................................................................... 33 2.7. Microscopy and Image analysis ...................................................................................................... 34 2.7.1. Cell Shape Quantification using CMEIAS ............................................................................. 36 2.7.2. Cell shape (compactness vs volume) charts ....................................................................... 37 2.8. PG isolation and UPLC analysis ......................................................................................................