THE MAINTENANCE AND REPLICATION OF THE APICOPLAST GENOME IN TOXOPLASMA GONDII by SARAH BAKER REIFF (Under the Direction of Boris Striepen) ABSTRACT The phylum Apicomplexa comprises a group of intracellular parasites of significant global health and economic concern. Most apicomplexan parasites possess a relict plastid organelle, which no longer performs photosynthesis but still retains important metabolic functions. This organelle, known as the apicoplast, also contains its own genome which is required for parasite viability. The apicoplast and its genome have been shown to be useful as therapeutic targets. However, limited information is available about the replication and maintenance of plastid DNA, not just in apicomplexan parasites but also in plants and algae. Here we use the genetic tools available in the model apicomplexan Toxoplasma gondii to examine putative apicoplast DNA replication and condensation factors, including homologs of DNA polymerase I, single-stranded DNA binding protein, DNA gyrase, and the histone-like protein HU. We confirm targeting to the apicoplast of these candidates, which are all encoded in the nucleus and must be imported. We created a genetic knockout of the Toxoplasma HU gene, which encodes a homolog of a bacterial protein that helps condense the bacterial nucleoid. We show that loss of HU in Toxoplasma results in a strong decrease in apicoplast DNA content, accompanied by biogenesis and segregation defects of the organelle. We were also interested in examining the roles of enzymes that might be more directly involved in DNA replication. To this end we constructed conditional mutants of the Toxoplasma gyrase B homolog and the DNA polymerase I homolog, which appears to be the result of a gene fusion and contains multiple different catalytic domains. We find that these proteins are essential to the parasite and required for apicoplast DNA replication. Together these data highlight the importance of the apicoplast DNA in apicomplexan cell biology and increase our understanding of plastid genome biology. INDEX WORDS: Apicoplast genome, DNA replication, HU. THE MAINTENANCE AND REPLICATION OF THE APICOPLAST GENOME IN TOXOPLASMA GONDII By SARAH BAKER REIFF B.S., University of Massachusetts-Amherst, 2006 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2012 © 2012 Sarah Baker Reiff All Rights Reserved THE MAINTENANCE AND REPLICATION OF THE APICOPLAST GENOME IN TOXOPLASMA GONDII By SARAH BAKER REIFF Major Professor: Boris Striepen Committee Members: Mark Farmer Jacek Gaertig Stephen Hajduk Robert Sabatini Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2012 DEDICATION This work is dedicated to my sister, Alison. Her journey has been an inspiration and shows that hard work and persistence always pay off. iv ACKNOWLEDGEMENTS First I would like to thank my advisor, Dr. Boris Striepen. Boris has been an exemplary advisor and I have been fortunate to be able to use his creative and innovative approaches to scientific problems as an example in my own scientific development. He has always taken the time to help me through scientific problems that arise, but has also supported and encouraged my independence in the laboratory and in my ideas for the direction of my projects. I would not be the scientist I am today without his guidance and direction. I would also like to thank my committee members, Mark Farmer, Stephen Hajduk, Jacek Gaertig, and Robert Sabatini for all the helpful conversations and encouragement during my committee meetings. Mark Farmer especially has made time for me on numerous occasions outside of committee meetings to discuss ideas about science and give important career advice. I also need to thank all the members of the Striepen Lab, both past and present. Our lab has always been a fun, positive, and collaborative work environment. It has always sustained my excitement for science and research, even through stressful times, and the lab members have always been quick to lend help and suggestions. I’ve enjoyed working with everyone here but I especially want to thank Carrie Brooks and Lilach Sheiner. Carrie, our lab manager, has always been extremely helpful with experimental advice and has done numerous favors for me over the years. And Lilach, in addition to lots of good conversations about science, is a great friend and was there to be supportive when I needed it the most. v Lastly, I want to thank my family, my sister Alison and most of all my parents Kathy and Greg Reiff. From a young age they have always supported my independence and career goals. My whole life they have both been amazing role models and I would never have been able to get this far without their love and support. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ................................................................................................. v LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES ............................................................................................................ x CHAPTER 1 INTRODUCTION .............................................................................................. 1 1.1 Introduction ............................................................................................. 1 1.2 The Structure of this Dissertation ........................................................... 4 2 REVIEW OF THE LITERATURE ...................................................................... 8 2.1 Endosymbiosis ........................................................................................ 8 2.2 The Apicoplast ...................................................................................... 13 2.3 Plastid Genomes .................................................................................. 15 2.4 Plastid DNA Replication ........................................................................ 18 3 THE HU PROTEIN IS IMPORTANT FOR APICOPLAST GENOME MAINTENANCE AND INHERITANCE IN TOXOPLASMA GONDII ............... 39 3.1 Abstract ................................................................................................ 40 3.2 Introduction ........................................................................................... 40 3.3 Materials and Methods ......................................................................... 43 3.4 Results .................................................................................................. 49 3.5 Discussion ............................................................................................ 58 4 CHARACTERIZATION OF APICOPLAST DNA REPLICATION PROTEINS IN TOXOPLASMA GONDII ................................................................................. 77 vii 4.1 Introduction ........................................................................................... 77 4.2 Materials and Methods ......................................................................... 80 4.3 Results .................................................................................................. 82 4.4 Discussion ............................................................................................ 85 5 CONCLUSIONS ............................................................................................. 96 APPENDIX A1 BUILDING THE PERFECT PARASITE: CELL DIVISION IN APICOMPLEXA ..................................................................................................................... 105 A2 A NOVEL DYNAMIN RELATED PROTEIN HAS BEEN RECRUITED FOR APICOPLAST FISSION IN TOXOPLASMA GONDII .................................. 128 viii LIST OF TABLES Page Table 3.1: Mean vacuole sizes in parental and mutant strains ....................................... 75 Table 3.2: Apicoplast DNA levels from quantitative PCR and Southern blot .................. 75 Table 3.S1: List of Primers .............................................................................................. 76 Table 3.S2: Percentage of Duplicated Centrosomes Associating with Apicoplasts in Ciprofloxacin- or Clindamycin-treated parasites ............................................................. 76 Table 4.1: Primers ........................................................................................................... 95 Table A1.1: Apicomplexan Parasites ............................................................................ 124 ix LIST OF FIGURES Page Figure 3.1: TgHU complements HupA- mutant in E. coli ................................................. 68 Figure 3.2: TgHU protein localizes to the apicoplast ....................................................... 69 Figure 3.3: Overexpression of HU-YFP results in unequal plastid distribution ................ 70 Figure 3.4: Deletion of the TgHU locus ........................................................................... 71 Figure 3.5: Distribution of vacuole sizes in parental and ΔHU parasites......................... 72 Figure 3.6: ΔHU parasites display reduced apicoplast genome copy numbers .............. 72 Figure 3.7: Apicoplast loss in ΔHU parasites .................................................................. 73 Figure 3.S1: HU loss results in reduced association with centrin during cell division ..... 74 Figure 3.S2: Defective apicoplast segregation in Ciprofloxacin- and Clindamycin-treated