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Rhodobacter Sphaeroides and Escherichia Coli

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Rhodobacter Sphaeroides and Escherichia Coli Assembly of modified and non-native proteins in Rhodobacter sphaeroides and Escherichia coli Katie Jane Grayson A thesis submitted for the degree of Doctor of Philosophy at the University of Sheffield Department of Molecular Biology and Biotechnology September 2015 Summary Photosynthesis is a highly efficient and productive process that converts solar energy into chemical energy. Much of the visible and near-infrared radiation falling on the surface of the Earth is not absorbed by photosynthesising organisms, which occupy particular spectral niches depending on the absorption of the particular pigments they synthesise. For synthetic biology applications it would be worthwhile to design and construct bacteria that could utilise a greater range of wavelengths than naturally-evolved photosynthetic bacteria. Although the incorporation of synthetic chromophores to complement native light-harvesting proteins is promising, the approach generally involves in vitro reassembly. In order to create tailor-made light harvesting antennas in vivo, we must make use of the toolbox of proteins and pigments available in nature, or create synthetic elements that are able to be created by the host organism. To investigate the possibility of creating artificial light-harvesting antennas in vivo, the yellow fluorescent protein, YFP, was incorporated as a chromophore into the photosynthetic apparatus of the purple photosynthetic bacterium Rhodobacter (Rba.) sphaeroides. It is shown that energy absorbed by YFP can transfer to the native reaction centre and LH1 proteins, sufficient to enhance the photosynthetic growth rate in a Rba. sphaeroides carotenoidless mutant. The light-driven proton pump, proteorhodopsin (PR), also has potential to augment the proton motive force in Rba. sphaeroides and drive downstream metabolism. Rba. sphaeroides was engineered to express PR and its chromophore retinal. The gene for a transmembrane synthetic peptide maquette was designed and expressed in Rba. sphaeroides. This work forms the basis of the bottom-up redesign of photosystem components with the aim of augmenting photosynthesis in Rba. sphaeroides and in the long term to create new photosynthetic complexes and membrane assemblies. In addition, the plasticity of the E. coli Tat pathway for the export of maquettes was investigated, as the quality control mechanism of the Tat transporter makes it a desirable system for efficient large-scale protein production to facilitate further characterisation of maquette proteins. i Acknowledgements First I would like to thank my supervisor Neil Hunter for his continued support and advice through the years that I have worked in the lab. I would also like to thank Graham Leggett for the opportunity to work as part of the Low-Dimensional Chemistry project. I have had the pleasure of working with and visiting several collaborators with whom I have gained many new skills and experiences. The maquette work was done in collaboration with Les Dutton, Goutham Kodali and Bohdana Discher at the University of Pennsylvania. The Tat work was done with Colin Robinson, Alex Jones and Cristina Matos at University of Kent. Thank you to everyone at Washington University in St Louis who contributed to the RC-YFP work, including Christine Kirmaer, Dewey Holten, Preston Dilbeck and Jon Yuen. I would particularly like to thank Kaitlyn Faries for the ultrafast transient absorbance data on the RC- YFP complexes. Thank you for making me feel so welcome and at home when I made my visits. Thank you to PARC who funded my visit to St Louis. I would like to thank all the people in the Hunter lab, past and present, who have helped me with their expertise and wise advice over the course of my PhD. In particular I would like to thank Dr Pu Qian for producing the EM images and model of the LH1-RC-YFP complex, Xia Huang for the YFP lifetimes data, Dr John Olsen for help with the 77 K membrane data, Dr Dan Canniffe for help with gene design and synthesis, Dr Sarah Hollingshead for help with the Synechocystis work, Shuang Chi for carotenoid expertise, and Dr. Amanda Brindley and Dr Pu Qian for all their help with protein purification. Thank you to Elizabeth Martin (with magic cloning skills), Dr Dave Mothersole and Dr Michaёl Cartron for teaching me all they know. Thank you to Dr Andy Hitchcock for helping me out with a new short loop version of the transmembrane maquette as I reached the last stages of thesis writing. Thank you to all my friends in Sheffield and back at home. Thank you to Wednesday Club: Abi, Sophie, Suze, Simon, Lucy and Sarah for getting me through my PhD with weekly beers in the University Arms. Thanks for all the lunches in the park, duck watching and for always being there to answer my questions regarding thesis writing and science knowhow. Thank you also to the Janets and the Goons for cheering me on all the way. Thank you to Will Wood for keeping life in E11 lively. Finally, I would like to say a special thank you to my family for their love, belief and support. My parents, who can now pronounce “Rhodobacter sphaeroides” perfectly, have always taken a strong interest in my research and bravely sat through many practice presentations and proof read sections of my thesis – thank you! Thank you to my sister, Hannah, for ii providing me with time-out from my thesis by taking me to wacky art exhibits. To Alex, who also diligently sat through so many practice presentations, thank you for all your love and patience over the last 4 years – here’s to the future! iii Table of Contents Chapter 1: Introduction 1.1 Photosynthesis ............................................................................................................ 1 1.2 Photosynthetic organisms ............................................................................................ 1 1.2.1 Classification of photosynthetic organisms ................................................................... 1 1.2.1.1 Purple photosynthetic bacteria .............................................................................. 2 1.2.1.2 Green bacteria ........................................................................................................ 2 1.2.1.3 Heliobacteria ........................................................................................................... 3 1.2.1.4 Cyanobacteria ......................................................................................................... 3 1.2.1.5 Acidobacteria .......................................................................................................... 3 1.2.1.6 Algae and plants ...................................................................................................... 3 1.2.1.7 Rhodopsin-based photosynthesis ........................................................................... 3 1.2.2 Rhodobacter sphaeroides .............................................................................................. 4 1.3 Pigments in photosynthesis ......................................................................................... 4 1.3.1 The role of pigments in photosynthesis......................................................................... 4 1.3.2 Bacteriochlorophyll a biosynthesis ................................................................................ 5 1.3.3 Carotenoid biosynthesis ................................................................................................ 7 1.4 Light harvesting in Rhodobacter sphaeroides .............................................................. 10 1.4.1 The photosynthetic unit of Rhodobacter sphaeroides ................................................ 10 1.4.2 The peripheral light-harvesting complex LH2 .............................................................. 10 1.4.3 The major light-harvesting complex LH1 ..................................................................... 12 1.4.4 The reaction centre ...................................................................................................... 12 1.4.5 The core complex ......................................................................................................... 14 1.5 Transduction of excitation energy and the formation of ATP in photosynthesis ........... 15 1.5.1 Excitation energy transfer within LH1 and LH2 ........................................................... 16 1.5.2 Excitation energy transfer to the reaction centre ....................................................... 16 iv 1.5.3 Transduction of excitation energy transfer to electron flow in the reaction centre ... 17 1.5.4 Completion of electron flow at the cytochrome bc1 complex ..................................... 17 1.5.5 Formation of ATP by ATP synthase .............................................................................. 18 1.6 The genetics of Rhodobacter sphaeroides ................................................................... 18 1.6.1 The photosynthesis gene cluster of Rhodobacter sphaeroides ................................... 18 1.6.2 Control of gene expression .......................................................................................... 20 1.7 The ultrastructure of the Rhodobacter sphaeroides photosynthetic membrane ........... 20 1.7.1 The intracytoplasmic membrane ................................................................................. 20 1.7.2 Maturation of intracytoplasmic membranes and precursor membranes. .................. 21 1.7.3 The arrangement of the photosynthetic membrane ..................................................
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