UC Berkeley UC Berkeley Electronic Theses and Dissertations Title Cellular organization in Agrobacterium tumefaciens Permalink https://escholarship.org/uc/item/1sk3q6hn Author Cameron, Todd A. Publication Date 2013 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California Cellular organization in Agrobacterium tumefaciens By Todd A. Cameron A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy In Microbiology in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Patricia C. Zambryski, Chair Professor Arash Komeili Professor David G. Drubin Fall 2013 Abstract Cellular organization in Agrobacterium tumefaciens by Todd A. Cameron Doctor of Philosophy in Microbiology University of California, Berkeley Professor Patricia C. Zambryski, Chair Bacterial cell structures are macromolecular features found among all types bacteria. They often serve essential or specialized functions, and are important contributors to the widespread diversity and success of bacteria. I have focused my research on the formation and localization of two of these structures in the plant pathogen Agrobacterium tumefaciens: the type IV secretion system (T4SS) and the bacterial cell envelope. A. tumefaciens is an α-proteobacterium uniquely capable of genetically transforming plant host cells during its infection process. DNA and protein substrates are transferred into plant cells via the T4SS, which forms multiple complexes around the bacterial circumference. To understand the mechanisms driving T4SS positioning, I assessed their spatial distribution by quantitative analysis and modeling. My findings indicate that these secretion complexes localize in a non-random periodic pattern along the cell perimeter. These results, along with additional plant infection studies, suggest this spatial organization serves to promote efficient delivery of substrates into host cells A potential mechanism behind this periodic arrangement is the unipolar cell growth recently described for A. tumefaciens. To better understand this unusual growth mechanism, I carefully scrutinized growing cells, nascent peptidoglycan, and peptidoglycan synthesis components by electron and fluorescence microscopy. My results indicate that the unipolar growth process is surprisingly dynamic, multi- dimensional, and involves both novel and familiar components. These findings thus represent a significant departure from the canonical growth mechanism of E. coli and other well-studied bacilli. 1 Table of Contents List of Figures and Tables ............................................................................................. ii! Acknowledgements ...................................................................................................... iii! CHAPTER 1 Introduction ............................................................................................... 1! 1.1 Type IV Secretion ................................................................................................... 2! 1.2 Peptidoglycan ......................................................................................................... 6! 1.3 Dissertation Overview ............................................................................................. 9! CHAPTER 2 The Type IV secretion system of Agrobacterium tumefaciens is arranged as multiple foci around the bacteria cell, and mediates lateral attachment to host plant cells ..................................................................................... 10! 2.1 Summary .............................................................................................................. 11! CHAPTER 3 Quantitative image analysis and modeling indicate the Agrobacterium tumefaciens type IV secretion system is organized in a periodic pattern of foci. .............................................................................................................. 13! 3.1 Introduction ........................................................................................................... 14! 3.2 Results .................................................................................................................. 16! 3.3 Discussion ............................................................................................................ 19! 3.4 Materials and Methods ......................................................................................... 21! CHAPTER 4 Dynamic FtsA and FtsZ localization and outer membrane alterations during polar growth and cell division in Agrobacterium tumefaciens .................... 24! 4.1 Summary .............................................................................................................. 25! CHAPTER 5 Characterization of the peptidoglycan synthesis machinery in Agrobacterium tumefaciens during unipolar growth and cell division .................. 30! 5.1 Introduction ........................................................................................................... 31! 5.2 Results .................................................................................................................. 32! 5.3 Discussion ............................................................................................................ 41! 5.4 Materials and Methods ......................................................................................... 43! CHAPTER 6 Concluding Remarks .............................................................................. 51! 6.1 Summary of findings ............................................................................................. 52! 6.2 Conclusions and future directions ........................................................................ 53! References .................................................................................................................... 57! i List of Figures and Tables Figure 1-1 A model of the architecture of the T4SS ........................................................ 4! Figure 3-1 GFP-VirB8 localizes as multiple foci along the cell periphery ...................... 15! Figure 3-2 Fourier analysis reveals periodicity of GFP-VirB8 foci ................................. 16! Figure 3-3 Nearest neighbor distributions of GFP-VirB8 foci ........................................ 17! Figure 3-4 Maximum likelihood modeling and Monte Carlo simulations support periodic placement ............................................................................................................... 18! Figure 3-5 T4SS-mediated attachment of A. tumefaciens ............................................ 20! Figure 4-1 FtsA-GFP localization .................................................................................. 27! Figure 4-2 FtsZ localization and model ......................................................................... 28! Figure 5-1 FtsZ and FtsA cell cycle localization ............................................................ 33! Figure 5-2 BocillinFL and citrine-PBP1a exhibit growth pole and midcell fluorescence 34! Figure 5-3 PBP3a and PBP3b do not exhibit strong polar localization ......................... 35! Figure 5-4 Unrooted phylogenetic tree of LDTs from representative Proteobacteria species. ................................................................................................................... 36! Figure 5-5 LDT Atu0845 localizes intensely to the growth pole .................................... 37! Figure 5-6 Localization of additional LDTs .................................................................... 38! Figure 5-7 LDTs are active over an increasing area of the new cell compartment ....... 40! Figure 5-8 New cell compartments increase in width as they grow in length ................ 41! Figure 5-9 Model of A. tumefaciens cell growth dynamics ............................................ 42! Table 5-1 Peptidoglycan synthesis and cell division genes in A. tumefaciens ............ 46! Table 5-2 Strains used in phylogenetic studies ........................................................... 49! Table 5-3 Strains and plasmids used in this study ...................................................... 50! Figure 6-1 Effect of ∆minCDE on FtsZ localization ....................................................... 54! Figure 6-2 A. tumefaciens growth characteristics ......................................................... 55! ii Acknowledgements I thank my graduate advisor Dr. Pat Zambryski for her guidance and mentoring throughout my graduate career. I have learned much about the scientific process, discourse, and writing through our work together. I also thank Dr. Arash Komeili for his assistance and many insights, during both committee meetings and informal discussions. I am further grateful for the guidance of the additional faculty that helped shape my dissertation research through the qualifying exam and subsequent committee meetings: Dr. David Drubin, Dr. David Zusman, and Dr. Kathleen Ryan. I wish to thank the current and past members of the Zambryski lab. Dr. John Zupan has especially been a great mentor and collaborator on many projects. I also appreciate and enjoyed the collaborations with Dr. Julieta Aguilar, James Anderson- Furgeson, and Justin Zik. I am grateful for the advice and comments from Dr. Tessa Burch-Smith, Dr. Solomon Stonebloom, Jacob Brunkard, and Anne Runkel. I also thank the many researchers outside of the Zambryski lab who