CHARACTERIZATION OF IRON-RESPONSIVE TRANSCRIPTION REGULATORS IN PYROCOCCUS FURIOSUS by YIXUAN ZHU Under the Direction of ROBERT A. SCOTT and MICHAEL W.W. ADAMS ABSTRACT Iron is an essential element for the growth of Pyrococcus furiosus since limited medium iron causes impaired growth. In previous iron-limitation microarray experiments, two putative iron transporters Ftr1 and FeoAB were significantly up-regulated in response to limited iron in the growth medium. This change in the iron acquisition system raises the question: what is responsible for the iron-dependent regulation? Two putative iron-responsive transcription factors were found in P. furiosus, Fur and DtxR. Studies on Fur-DNA binding activity and the characterization of its deletion mutant strain revealed no demonstrable role for the Fur homolog in the regulatory response of the cells to iron. Analysis of the DtxR genomic sequence suggested an incorrect translation start site prediction by NCBI and TIGR; another start site resulted in a 12 amino acid extension on the N-terminus. Recombinant DtxR proteins were expressed (with "full- length" and without the N-terminal extension) and their DNA-binding affinity was determined using electrophoretic mobility shift assay. The full-length DtxR was verified to bind specifically to ftr1 and feoAB promoters, indicating the function of DtxR as a potential transcriptional factor. The truncated DtxR failed to recognize any of the promoters. The dtxR deletion mutant (DTXR) did not show any growth phenotype compared to the control strain COM1C2 under iron-rich and iron-limited conditions. Microarray analysis of the iron-dependent regulation in DTXR and COM1C2 provided evidence that DtxR controls the expression of Ftr1 and FeoAB in P. furiosus since both iron transporters were no longer iron-regulated in the deletion strain. Results from the quantitative PCR experiment confirmed that DtxR is an iron-dependent repressor for the putative iron transporters in P. furiosus. Meanwhile, the ftr1 deletion mutant (FTR1) was constructed to investigate its role in iron uptake. However, no phenotype was observed for its growth in iron- limited and metal-limited medium; the similar intracellular iron content detected by ICP-MS in FTR1 and parent strain indicates that Ftr1 might not be an essential iron transporter despite its highly iron-responsive expression in P. furiosus. INDEX WORDS: Archaea, Pyrococcus furiosus, transcription factor, transcriptional regulation, iron acquisition, Fur, DtxR, DNA microarray, EMSA, deletion mutant, ICP-MS. CHARACTERIZATION OF IRON-RESPONSIVE TRANSCRIPTION REGULATORS IN PYROCOCCUS FURIOSUS by YIXUAN ZHU B.S., Tsinghua University, 2005 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 2011 ©2011 YIXUAN ZHU All Rights Reserved CHARACTERIZATION OF IRON-RESPONSIVE TRANSCRIPTION REGULATORS IN PYROCOCCUS FURIOSUS by YIXUAN ZHU Major Professor: Robert A. Scott Michael W.W. Adams Committee: I. Jonathan Amster John Rose Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia December, 2011 DEDICATION To my parents who gave life to me, love me and support me for all time. To my dear grandfather who has been an inspiration to me, passed away during my pursuit of Ph.D., R.I.P. iv ACKNOWLEDGEMENTS I would like to thank my lab mate Gina Lipscomb, who shared four years time with me in three different labs, for always being patient and helpful and offering invaluable advice on my research; Gerrit Schut and Angeli Menon, for providing important experimental data and sharing useful research experiences; Karen Stirrett, for constructing the strains used in this research; Alex Cvetkovic and Sunil Kumar for running ICP-MS for my samples and providing insightful suggestions; Chris Hopkins and Junsong Sun for teaching me how to use the ÄKTA purifier and giving me advice on protein purification; Farris Poole who offered me firsthand training when I joined the lab and technical support along with my research; and Darin Cowart for providing the database to search the potential DtxR binding site. I am also thankful to Dr. Eidsness from whom I got much support and encouragement through my first year in graduate school; Dr. Westpheling and Dr. Adams who offered me workspace in their labs and wonderful suggestions on my research; and my advisor Dr. Scott, not only for the guidance to my works, but also for the patience and support that helped me overcome crisis situations and finish my dissertation. And last but not least, I express my gratitude to all those that I have been honored to work with, including previous and current lab members, faculty and departmental staffs from the departments of Chemistry and Biochemistry. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .............................................................................................................v CHAPTER 1 OVERVIEW OF TRANSCRIPTIONAL REGULATION IN ARCHAEA .....................1 1.1 Introduction to archaea ...............................................................................................1 1.2 Archaeal basal transcription........................................................................................2 1.3 Archaeal transcriptional regulation and regulatory transcription factors ...................5 1.4 The model archaeon Pyrococcus furiosus and its iron homeostasis .........................10 2 MATERIALS AND METHODS ....................................................................................15 2.1 Protein expression and purification ..........................................................................15 2.2 Gel filtration to determine protein quaternary structure ...........................................16 2.3 Electrophoretic mobility shift assay..........................................................................17 2.4 Strains and growth conditions ...................................................................................18 2.5 Mutant strain construction ........................................................................................19 2.6 Growth characterization of cultures ..........................................................................21 2.7 Quantitative PCR ......................................................................................................22 2.8 Microarray transcriptomics and data analysis ...........................................................22 2.9 Determination of intracellular iron content...............................................................24 3 CHARACTERIZATION OF THE PUTATIVE IRON-RESPONSIVE TRANSCRIPTION FACTOR FUR ...............................................................................25 3.1 Introduction to the Fur family protein ......................................................................25 3.2 Fur homolog in Pyrococcus furiosus ........................................................................29 vii 3.3 Validation of the DNA-binding affinity of PfFur .....................................................30 3.4 Characterization of the fur deletion mutant ..............................................................32 4 CHARACTERIZATION OF THE PUTATIVE IRON-RESPONSIVE TRANSCRIPTION FACTOR DTXR ............................................................................58 4.1 Introduction to the DtxR family protein ...................................................................58 4.2 DtxR homolog in Pyrococcus furiosus .....................................................................60 4.3 Reanalysis of the DtxR homolog in Pyrococcus furiosus ........................................63 4.4 Validation of the DNA-binding affinity of PfDtxR ..................................................64 4.5 Characterization of the dtxR deletion mutant............................................................70 5 CHARACTERIZATION OF THE PUTATIVE IRON PERMEASE FTR1 DELETION MUTANT .....................................................................................................................120 5.1 Introduction .............................................................................................................120 5.2 Characterization of the ftr1 deletion mutant ...........................................................121 6 CONCLUSIONS...........................................................................................................134 6.1 Effect of iron on the growth of Pyrococcus furiosus ..............................................134 6.2 PfDtxR-DNA binding affinity ................................................................................135 6.3 Physiology of DTXR mutant and the mode of regulation by DtxR .....................136 REFERENCES ................................................................................................................138 APPENDIX ......................................................................................................................162 vii CHAPTER 1 INTRODUCTION 1.1 Introduction to archaea The Archaea are microorganisms evolved as one of the three primary lineages several billion years ago. The first scientific literature came out describing the discovery of archaea dated about 200 years ago, and the Archaea were not formally proposed as the third domain of life till the 1970s [1, 2]. Molecular, genomic and phylogenetic evidences have been accumulated ever since then to strengthen the definition