RIBOSWITCH-BASED SENSORS OF SMALL MOLECULES IN BACTERIA THE ENGINEERING OF RIBOSWITCH-BASED SENSORS OF SMALL MOLECULES IN BACTERIA AND THEIR APPLICATION IN THE STUDY OF VITAMIN B12 BIOLOGY By CASEY C. FOWLER B.Sc., Queen’s University, Kingston Ontario, 2004 A Thesis Submitted to the School of Graduate Studies in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy McMaster University © by Casey C. Fowler, August 2011 DOCTOR OF PHILOSOPHY (2011) McMaster University (Biochemistry & Biomedical Sciences) Hamilton, Ontario TITLE: The Engineering of Riboswitch-Based Sensors of Small Molecules in Bacteria and Their Application in the Study of Vitamin B12 Biology AUTHOR: Casey C. Fowler, B.Sc. (Queen’s University) SUPERVISOR: Dr. Yingfu Li NUMBER OF PAGES: xiii, 191 ii Abstract Small molecule metabolites have important and diverse roles in every major cellular function. To study the activities of metabolites and the biological processes in which they are involved, it is important to be able to detect their levels within cells. Technologies that measure the concentrations of small molecules within the context of living, growing cells are highly advantageous but are challenging to produce. In this thesis, a novel class of intracellular small molecule sensors is produced, characterized and applied to address novel and relevant research questions. These sensors detect a specific target molecule within bacterial cells using RNA regulatory elements known as riboswitches and one of many possible reporter proteins. In addition to a project that yielded new methodology to create custom riboswitches, two projects that assess the capabilities of sensors that detect an active form of vitamin B12 are described. These projects present an abundance of data that provide novel insights into the transport and metabolism of vitamin B12 in E. coli cells. Overall, the results presented indicate that riboswitch-based sensors represent valuable and unique tools for the study of microbial biology. The thesis is concluded with a discussion that describes design strategies and several exciting potential applications for future riboswitch sensors. iii Acknowledgements First and foremost I would like to thank my supervisors Dr. Yingfu Li and Dr. Eric Brown. Their contributions to the research presented in this thesis and to my development as a scientist have been monumental. My interactions with both of these fantastic mentors will continue to benefit me for the remainder of my career. I would also like to recognize the contributions of my other supervisory committee member, Dr. David Andrews. His vigorous and carefully considered questions and suggestions kept me on my toes during committee meetings and were very helpful in addressing problems that arose over the course of my research. I would also like to acknowledge Dr. Murray Junop for the many long discussions we’ve had regarding the work described in Chapter 5 of this thesis. With his guidance I have learned a great deal about structural biology. The Li lab has provided a pleasant and productive research environment throughout my time at McMaster. I would like to recognize Simon Mcmanus, Naveen Kumar, Wendy Mok and Kacper Kuryllo for the useful discussions, advice and assistance they have provided in the lab. On a more personal level, I would like to thank Naomi as well as my family and friends for making my life outside of the lab so enjoyable throughout my time in graduate school. iv To my parents, who have set and kept me on a happy path in life. v Table of Contents Abstract.............................................................................................................................iii Acknowledgements...........................................................................................................iv List of Tables.....................................................................................................................ix List of Figures.....................................................................................................................x List of Abbreviations.......................................................................................................xii Chapter 1. A General Introduction.................................................................................1 1.1 Overview of the problem to be addressed…………..............................................1 1.2 The isolation, properties and applications of aptamers .........................................9 1.3 Riboswitches: metabolite-sensing RNA regulatory elements..............................18 1.4 The engineering and applications of bacterial riboswitches................................35 1.5 Vitamin B12 and its synthesis, uptake and utilization in E. coli cells.................44 1.6 Specific research objectives and thesis organization...........................................52 Chapter 2. A FACS-Based Approach to Engineering Artificial Riboswitches.........57 2.1 Author’s preface……………………………....................................................57 2.2 Abstract...............................................................................................................59 2.3 Introduction........................................................................................................59 2.4 Materials and Methods......................................................................................63 2.4.1 Growth media, strains, plasmids and molecular cloning.............................63 2.4.2 Fluorescence activated cell sorting and colony isolation.............................64 2.4.3 Fluorescence assays.....................................................................................65 vi 2.5 Results.................................................................................................................66 2.5.1 Selection scheme and library design............................................................66 2.5.2 FACS to isolate candidate riboswitch sequences.........................................67 2.5.3 Assaying candidate riboswitches for theophylline response.......................70 2.5.4 Mechanistic characterization of RS11A......................................................72 2.6 Discussion...........................................................................................................75 2.7 References...........................................................................................................78 Chapter 3. Using a Riboswitch Sensor to Examine Coenzyme B12 Metabolism and Transport in E. coli .........................................................................................................80 3.1 Author’s preface……………………………....................................................80 3.2 Abstract...............................................................................................................81 3.3 Introduction........................................................................................................82 3.4 Materials and Methods......................................................................................85 3.4.1 Plasmids and cell strains..............................................................................85 3.4.2 Molecular cloning........................................................................................86 3.4.3 Growth conditions and reporter assays........................................................86 3.4.4 Ethanolamine utilization assays...................................................................88 3.5 Results.................................................................................................................89 3.5.1 Construction of riboswitch-based AdoCbl sensors......................................89 3.5.2 Riboswitch-based sensors can be used to monitor AdoCbl metabolism.....93 3.5.3 Riboswitch-based sensors can detect the availability of B12 lower axial ligands.........................................................................................................................98 3.5.4 Riboswitch-based sensors can be used to monitor AdoCbl transport........100 3.6 Discussion.........................................................................................................103 vii 3.7 References.........................................................................................................107 Chapter 4. Characterization of Intermolecular Interactions Involving the Periplasmic Binding Protein BtuF Using Riboswitch-Based Sensors.......................113 4.1 Author’s preface………………………..........................................................113 4.2 Abstract.............................................................................................................114 4.3 Introduction......................................................................................................115 4.4 Materials and Methods....................................................................................119 4.4.1 Strains, plasmids and molecular cloning...................................................119 4.4.2 Low-throughput reporter assays................................................................120 4.4.3 Reporter assays for saturation mutagenesis libraries.................................122 4.4.4 Ethanolamine utilization assays.................................................................123 4.5 Results...............................................................................................................123