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University of New Hampshire University of New Hampshire Scholars' Repository Master's Theses and Capstones Student Scholarship Winter 2007 The identification, characterization, and expression of truncated hemoglobin genes in the nitrogen-fixing actinorhizal symbiont Frankia James Michael Niemann University of New Hampshire, Durham Follow this and additional works at: https://scholars.unh.edu/thesis Recommended Citation Niemann, James Michael, "The identification, characterization, and expression of truncated hemoglobin genes in the nitrogen-fixing actinorhizal symbiont rF ankia" (2007). Master's Theses and Capstones. 340. https://scholars.unh.edu/thesis/340 This Thesis is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Master's Theses and Capstones by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. THE IDENTIFICATION, CHARACTERIZATION, AND EXPRESSION OF TRUNCATED HEMOGLOBIN GENES IN THE NITROGEN-FIXING ACTINORHIZAL SYMBIONT FRANKIA BY JAMES MICHAEL NIEMANN Bachelor of Science, University of New Hampshire, 2001 THESIS Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of Master of Science in Microbiology December, 2007 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 1449598 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ® UMI UMI Microform 1449598 Copyright 2008 by ProQuest LLC. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 E. Eisenhower Parkway PO Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This thesis has been examined and approved. / / A. Thesis Director, Dr. Louis Tisa, Associate Professor of Microbiology Dr. Robert Zsigfay, sor Emeritus of Microbiology x ' iT T Dr, Ste n Torosian, Afflliliate Assistant Professor of Microbiology Date Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEDICATION This thesis and all of the work accomplished during this research could not have been possible without the continued love and support of my parents and my wife. I am forever indebted to their never-ending belief in me. iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS I would like to thank Bob Mooney for all of his technical support during this research. I could not have asked for better thesis committee members than Dr. Robert Zsigray and Dr. Steven Torosian. All of their input was greatly valued. Thanks to Dr. John Tjepkema whose feedback as our collaborator on some of this research was of great help. I would also like to acknowledge all of the members of the Tisa Lab, especially Erik Janicki and Dr. Brandye Michaels for all of there constructive input during my graduate research. Last, but certainly not least, I want to thank my graduate advisor Dr. Louis Tisa for his knowledge and guidance that helped shape me to think and act as a scientist, but most of all for his patience. iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS DEDICATION.........................................................................................................iii ACKNOWLEDGEMENTS...................................................................................... iv TABLE OF CONTENTS..........................................................................................v LIST OF TABLES................................................................................................. viii LIST OF FIGURES................................................................................................ix ABSTRACT............................................................................................... x CHAPTER PAGE I. INTRODUCTION................................................................................................ 1 General Overview of Frankia......................................................................1 General Overview of Truncated Hemoglobins...........................................3 Hemoglobin in Frankia.............................................................................. 10 Research Goals ......................................................................................... 11 II. MATERIALS AND METHODS......................................................................... 13 Bacterial Strains and Growth Conditions .................................................. 13 Gene Sequences Used............................................................................. 13 Polymerase Chain Reaction (PCR) ...........................................................15 DNA Sequencing and Phylogenetic Analyses.......................................... 17 Sequence Analyses: Gene Neighborhoods and Promoter Analyses 18 RNA Extraction Methods........................................................................... 18 Determination of RNA Quality and Quantity ............................................. 21 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Environmental Test Conditions.............................. 21 One-Step Reverse Transcriptase PCR (RT-PCR) ....................................22 Preparation of cDNA for Real-Time RT-PCR............................................24 Real-Time RT-PCR ................................................................................... 26 III. RESULTS...................................................................................................... ,29 Molecular Identification of Truncated Hemoglobin in Frankia.................. 29 Identification of TrHb Amplicons.................................................... 29 Sequencing TrHbO Amplicons from Frankia................................. 32 Phylogentic Analyses of TrHb Sequences.....................................32 Analysis of TrHb Gene Neighbors and Potential Promoter Regions.......................................................................................... 37 The Effect of Environmental Stimuli on TrHb Expression.........................44 Optimizing RNA Extraction from Frankia....................................... 45 RT-PCR and Relative Expression of Frankia TrHbs...................... 48 Real-Time PCR and Expression of Frankia TrHbs........................ 55 IV. DISCUSSION..................................................................................................60 Molecular Identification of Truncated Hemoglobin in Frankia.................. 62 Sequencing the TrHb Amplicons................................................... 62 The Phylogeny of TrHbN and TrHbO in Frankia............................63 The Gene Neighborhoods of TrHbN and TrHbO in Frankia.......... 64 Characterizing the Promoter Regions of TrHbN and TrHbO in Frankia................................................. 67 The Effect of Environmental Stimuli on TrHb Expression.........................68 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The Triton X-100 Boiling RNA Extraction Method..........................68 Nitrogen Limitation Does Not Affect TrHb Expression in Frankia.................................................. 69 Oxidative Stress Does Not Affect TrHb Expression in Frankia.......................................................................................70 TrHbO Expression in Frankia Increases Under Low O2 Environments................................................................................ 71 TrHbN Expression in Frankia Increases Under Nitrosative Stress..............................................................................................73 LIST OF REFERENCES...................................................................................... 77 v ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES TABLE PAGE 1. Bacterial Strains and Growth Conditions ..........................................................14 2. Primers Used for PCR, RT-PCR, and Real-Time PCR.................................... 16 3. Percent Identity of TrHb DNA Sequences....................................................... 34 4. RNA Extraction Quality and Yield .................................................................... 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES FIGURE PAGE 1. Stereo Ribbon Structure of Truncated Hemoglobins......................................... 5 2. Cluster Analyses for Primer Design.................................................... 30 3. PCR of Frankia TrHb Amplicons......................................................................31

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