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Taming the Wild Rubisco: Explorations in Functional Metagenomics

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Taming the Wild RubisCO: Explorations in Functional Metagenomics DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Brian Hurin Witte, M.S. Graduate Program in Microbiology The Ohio State University 2012 Dissertation Committee : F. Robert Tabita, Advisor Joseph Krzycki Birgit E. Alber Paul Fuerst Copyright by Brian Hurin Witte 2012 Abstract Ribulose bisphosphate carboxylase/oxygenase (E.C. 4.1.1.39) (RubisCO) is the most abundant protein on Earth and the mechanism by which the vast majority of carbon enters the planet’s biosphere. Despite decades of study, many significant questions about this enzyme remain unanswered. As anthropogenic CO2 levels continue to rise, understanding this key component of the carbon cycle is crucial to forecasting feedback circuits, as well as to engineering food and fuel crops to produce more biomass with few inputs of increasingly scarce resources. This study demonstrates three means of investigating the natural diversity of RubisCO. Chapter 1 builds on existing DNA sequence-based techniques of gene discovery and shows that RubisCO from uncultured organisms can be used to complement growth in a RubisCO-deletion strain of autotrophic bacteria. In a few short steps, the time-consuming work of bringing an autotrophic organism in to pure culture can be circumvented. Chapter 2 details a means of entirely bypassing the bias inherent in sequence-based gene discovery by using selection of RubisCO genes from a metagenomic library. Chapter 3 provides a more in-depth study of the RubisCO from the methanogenic archaeon Methanococcoides burtonii. Mc. burtonii RubisCO (MBR) is unique in being intermediate between two previously- recognized families of RubisCO, as well as having an unprecedented C-terminal loop ii structure. Deletion of all or part of the loop appears to improve the oxygen tolerance of MBR, while simultaneously disrupting ability of the protein to form a decameric holoenzyme. This is the first report of a structural feature in RubisCO that can prevent the association of RubisCO dimers into higher-order structures without eliminating the catalytic activity of the enzyme. ii Dedication I would like to dedicate this document to the friends and family who have supported me through what seemed at times a Sisyphean project. In particular, I want to thank Julia for her steadfast belief that I would someday roll that rock over the crest of the hill. Thank you. iii Acknowledgments I would like to acknowledge the help and guidance I received from members of my lab and department over the course of this research. In particular, I would like to thank Dr. Sriram Satagopan for his insight in to the mysteries of RubisCO and structural modeling. My undergraduate assistant Kim McCabe also made several valuable contributions to the study of Methanococcoides burtonii RubisCO. Drs. John Paul and David John of the University of South Florida and Dr. Boris Wawrik, currently of the University of Oklahoma, provided invaluable assistance in metagenomics research. My fellow graduate students, now Drs., David Longstaff and Jitesh Soares provided discussions on the nature of research and helped troubleshoot many problems large and small. And, of course, I would like to thank my committee for their help, insight and patience. In particular, I appreciate the opportunity to be a member of Dr. Tabita’s research group, and the insight, encouragement and interesting research projects he has provided. This work was supported in part by a National Science Foundation Pre-Doctoral Fellowship. iv Vita 1992................................................................Columbia River H.S. 1998................................................................B.S. Botany, University of Washington 2002 to present ..............................................Graduate Teaching Associate/Graduate Research Associate, Department of Microbiology, The Ohio State University 2002-2003 ......................................................University Fellowship, The Ohio State University 2003-2006 ......................................................National Science Foundation, Pre-doctoral Fellowship 2008................................................................M.S. Microbiology, The Ohio State University v Publications Blum JS, Han S, Lanoil B, Saltikov C, Witte B, Tabita FR, Langley S, Beveridge TJ, Jahnke L, Oremland RS. 2009. Ecophysiology of "Halarsenatibacter silvermanii" strain SLAS-1T, gen. nov., sp. nov., a facultative chemoautotrophic arsenate respirer from salt-saturated Searles Lake, California. Appl Environ Microbiol 75(7):1950-60. Hoeft SE, Blum JS, Stolz JF, Tabita FR, Witte B, King GM, Santini JM, Oremland RS. 2007. Alkalilimnicola ehrlichii sp. nov., a novel, arsenite-oxidizing haloalkaliphilic gammaproteobacterium capable of chemoautotrophic or heterotrophic growth with nitrate or oxygen as the electron acceptor. Int J Syst Evol Microbiol 57(Pt 3):504-12. Tabita FR, Hanson TE, Satagopan S, Witte BH, Kreel NE. 2008. Phylogenetic and evolutionary relationships of RubisCO and the RubisCO-like proteins and the functional lessons provided by diverse molecular forms. Philosophical Transactions of the Royal Society of London.Series B, Biological Sciences 363(1504):2629-40. Witte B, John D, Wawrik B, Paul JH, Dayan D, Tabita FR. 2010. Functional prokaryotic RubisCO from an oceanic metagenomic library. Appl Environ Microbiol 76(9):2997-3003. Fields of Study Major Field: Microbiology vi Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iii Acknowledgments.............................................................................................................. iv Vita ...................................................................................................................................... v Publications ........................................................................................................................ vi Fields of Study ................................................................................................................... vi Table of Contents .............................................................................................................. vii List of Tables ..................................................................................................................... ix List of Figures ..................................................................................................................... x Introduction ......................................................................................................................... 1 Chapter 1 – RubisCO from a BAC Library ...................................................................... 22 Introduction ................................................................................................................... 22 Materials and Methods .................................................................................................. 26 Results and Discussion .................................................................................................. 38 Chapter 2 – Functional RubisCO Selection ...................................................................... 46 Introduction ................................................................................................................... 46 Materials and Methods .................................................................................................. 49 Results and Discussion .................................................................................................. 70 vii Chapter 3 – Methanococcoides burtonii RubisCO ........................................................... 86 Introduction ................................................................................................................... 86 Materials and Methods .................................................................................................. 92 Results and Discussion ................................................................................................ 106 Chapter 4 – Future Directions ......................................................................................... 129 Metagenome Discovery............................................................................................... 129 Methanococcoides burtonii RubisCO ......................................................................... 139 Appendix A: Sequences Used for Phylogenetic Trees ................................................... 142 Appendix B: pBLAST Results for ORFS on BAC15 ..................................................... 145 Appendix C: Strains and Vectors .................................................................................... 146 Appendix D: Codon Usage Charts .................................................................................. 150 Appendix E: Abbreviations............................................................................................ 154 References ....................................................................................................................... 156 viii List of Tables Table 1 - Diversity of RubisCO Kinetic Constants .........................................................
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