Experimental and Bioinformatic Analyses of Amino Acid Metabolism
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EXPERIMENTAL AND BIOINFORMATIC ANALYSES OF AMINO ACID METABOLISM IN THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS FURIOSUS by ANGELA KATHLEEN SNOW (Under the Direction of Michael W. W. Adams) ABSTRACT Pyrococcus furiosus is a hyperthermophilic archaeon that grows well on complex media containing peptides and various carbohydrates. Its growth on defined media is less well characterized. In this study, the growth of P. furiosus was investigated on various defined media. P. furiosus grows well on a defined medium containing all 20 amino acids. It also shows consistent growth on a defined medium containing arginine, cysteine, glycine, lysine, proline, and serine as the only amino acids. P. furiosus grows inconsistently in defined media with cysteine as the only amino acid. The experimental results are compared with bioinformatics data to better understand the amino acid biosynthesis capabilities of this fascinating organism. INDEX WORDS: archaea, hyperthermophile, Thermococcales, Pyrococcus furiosus, amino acid biosynthesis, amino acid anabolism, metabolism, defined medium EXPERIMENTAL AND BIOINFORMATIC ANALYSES OF AMINO ACID METABOLISM IN THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS FURIOSUS by ANGELA KATHLEEN SNOW B.S., California Institute of Technology, 2002 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2008 © 2008 Angela Kathleen Snow All Rights Reserved EXPERIMENTAL AND BIOINFORMATIC ANALYSES OF AMINO ACID METABOLISM IN THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS FURIOSUS by ANGELA KATHLEEN SNOW Major Professor: Michael Adams Committee: Anne Summers William Whitman Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2008 DEDICATION I dedicate this work to my undergraduate mentor, Dr. Dianne Newman. She provided me with my first opportunity to work in a microbiology lab. Her patience and encouragement made the learning process an absolute joy. More importantly, she introduced me to the wonderous diversity of prokaryotes. She instilled in me the desire to learn about every novel environment in which microorganisms thrive and every peculiar adaptation that allows them to flourish. My experience working in her lab is what inspired me to join graduate school and pursue a career in microbiology. I also dedicate this work to our mutual friend, Shewanella sp. strain ANA-3, who taught me that when life gives you arsenic, sometimes you just have to adapt to it the best you can. Who knows? Maybe that element in your life that was once so toxic will end up being the only thing that sustains you. iv ACKNOWLEDGEMENTS I would like to express my gratitude to my advisor Dr. Adams for accepting me as a student and for continuing to support me even when I was spending more on supplies than I was yielding in results. Thank you for giving me the opportunity to work in your lab. I would also like to thank my committee members Dr. Summers and Dr. Whitman for their advice on my research and for their assistance in preparing my dissertation. I would like to thank my colleagues in the Adams lab. I would especially like to Sonya Clarkson, who taught me how to grow P. furiosus when I first joined the lab, and Chris Hopkins, who worked with me on the Genetics and Hydrogenase projects, and who was always able to convince me that my experiments were working, no matter what the results showed. I would also like to thank some former members of the Adams lab; in particular, Dr. Francis Jenney, who always seemed to find the time to help me out with my project no matter how busy he was with his own work, and Jim Griffin, who worked with me on the P. furiosus Genetics project while he was an undergraduate. Finally, I would like to thank my colleagues in the Microbiology Department, who were responsible for creating an atmosphere of collaboration that is one the department’s greatest strengths. Whenever I was struggling, whether it was with my research or with one of my classes, I could always count on my fellow graduate students to lend a hand. Thank you very much to everyone who helped me to prepare for my Oral Exam and my Dissertation Defense, to everyone else who helped me during my time at UGA. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.............................................................................................................v LIST OF TABLES....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix CHAPTER 1 INTRODUCTION........................................................................................................1 Pyrococcus furiosus Overview..................................................................................1 Metabolism of Pyrococcus furiosus..........................................................................5 Amino Acid Metabolism by Pyrococcus furiosus ....................................................9 Project Goals ...........................................................................................................11 2 BIOINFORMATICS ANALYSIS OF AMINO ACID BIOSYNTHETIC PATHWAYS IN PYROCOCCUS FURIOSUS....................................................14 Genomic Analysis and Predicted Amino Acid Biosynthetic Pathways..................14 Alanine ....................................................................................................................15 Lysine ......................................................................................................................17 Arginine...................................................................................................................20 Leucine ....................................................................................................................21 Valine ......................................................................................................................23 Isoleucine.................................................................................................................24 Phenylalanine, Tryptophan, and Tyrosine...............................................................25 vi Bioinformatics Summary ........................................................................................27 3 MATERIALS AND METHODS ..............................................................................32 Strains......................................................................................................................32 Media Preparation ...................................................................................................32 Growth Conditions ..................................................................................................35 Storage Conditions ..................................................................................................35 Bioinformatics Analysis..........................................................................................36 4 CHARACTERIZATION OF PYROCOCCUS FURIOSUS GROWTH IN DEFINED MEDIA ................................................................................................37 Introduction .............................................................................................................37 Defined Medium with 20 Amino Acids..................................................................38 Defined Medium with 9 Amino Acids....................................................................41 Defined Medium with 6 Amino Acids....................................................................45 Defined Medium with 1 Amino Acid......................................................................47 Comparison of Media Types ...................................................................................52 Storage Conditions ..................................................................................................54 5 DISSCUSSION...........................................................................................................58 REFERENCES ..............................................................................................................................62 APPENDICES ...............................................................................................................................76 A ABBBREVIATIONS .................................................................................................76 B P. FURIOSUS ORFS...................................................................................................79 vii LIST OF TABLES Page Table 1.1: Experimentally Determined Amino Acid Auxotrophies in P. furiosus........................12 Table 2.1: Predicted Amino Acid Biosynthetic Pathways.............................................................16 Table 2.2: P. furiosus ORFs Potentially Involved in Amino Acid Biosynthesis...........................28 Table 5.1: Comparison of Results with Previous Experiments and Bioinformatics Analyses......59 Table B.1: P. furiosus ORFs Relevant to This Study ....................................................................79 viii LIST OF FIGURES Page Figure 2.1: Lysine Biosynthesis Genes in T. thermophilus, P. horikoshii, and P. furiosus ..........19 Figure 4.1: Inoculation Procedure for Endpoint Growth Experiments..........................................39