Louisiana State University LSU Digital Commons LSU Master's Theses Graduate School 2012 The Distribution and Diversity of Functional Gene Pathways Controlling Sulfur Speciation in Lower Kane Cave, Wyoming Audrey Tarlton Paterson Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_theses Part of the Earth Sciences Commons Recommended Citation Paterson, Audrey Tarlton, "The Distribution and Diversity of Functional Gene Pathways Controlling Sulfur Speciation in Lower Kane Cave, Wyoming" (2012). LSU Master's Theses. 2877. https://digitalcommons.lsu.edu/gradschool_theses/2877 This Thesis is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. THE DISTRIBUTION AND DIVERSITY OF FUNCTIONAL GENE PATHWAYS CONTROLLING SULFUR SPECIATION IN LOWER KANE CAVE, WYOMING A Thesis Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Master of Science in The Department of Geology and Geophysics by Audrey Tarlton Paterson B.S., Louisiana State University, 2009 May 2012 ACKNOWLEDGEMENTS I would like to acknowledge Dr. Annette Summers Engel who was the major advisor for the project. Thank you for bringing me into your lab group as an undergrad, introducing me to an amazing group of people, accepting me as a graduate student, and encouraging me to ‘stick my neck out.’ I cannot imagine where I would be without your guidance. I sincerely appreciate the time, effort, and funding you provided that made this project an enjoyable and memorable experience for me. I also especially thank Dr. Amitava Roy, Dr. Huiming Bao, Dr. Sam Bentley, and Dr. Rachel Beech for patience and guidance while serving on my thesis committee. I am very proud to have such inspiring, interdisciplinary committee members. Technical support at CAMD was provided by Greg Merchan, Henning Lichtenberg, and Lisa Bovenkamp. Partners in the field and lab included Kathleen Brannen, Terri Brown, Shane Cone, Chang Liu, Ben Maas, Axita Neema, Sarah Keenan, and Brendan Headd. Thank you all so much for your brilliance, energy and enthusiasm. To my family, Malcolm and Dorothy Paterson, Andrew Paterson, Helen and Alec Paterson, Damon and Dot Slator, Terry Huffington and the Dittman family, William Grierson, the Wilds, the Rickmans, the Bakers, the Youngs, the Skinners, the O’Connors, the Herkenhoffs, thank you for emotional and technical support, love and encouragement! This research was partially supported by two grants from the National Science Foundation (DEB-0640835 and EAR-0844364) and a grant from the Louisiana Board of Regents Pilot fund for New Research Program [NSF(2010)-PFUND-174]. Support for the project also came from receiving the James G. Mitchell award for best student presentation at the 2011 National Speleological Society convention, Glenwood Springs, Colorado. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................................................................................................... ii LIST OF TABLES ......................................................................................................................... v LIST OF FIGURES ...................................................................................................................... vi ABSTRACT ................................................................................................................................ vii CHAPTER 1: INTRODUCTION ................................................................................................ 1 Research Objectives and Hypotheses .............................................................................. 1 Scientific Importance ....................................................................................................... 5 Thesis Organization ......................................................................................................... 6 CHAPTER 2: REVIEW OF LITERATURE ............................................................................... 7 Introduction ...................................................................................................................... 7 Lower Kane Cave ............................................................................................................. 7 Microbial Roles in the Sulfur Cycle ................................................................................. 9 The Sulfur Oxidation Multienzyme (Sox) System ........................................................... 10 Adenosine-5’-phosphosulfate (APS) Reductase .............................................................. 13 Biological Sulfur Speciation in Biological and Geological Materials ............................. 14 The Sulfur Cycle in Karst Systems .................................................................................. 16 CHAPTER 3: MATERIALS AND METHODS .......................................................................... 18 Sample Collection and Aqueous Geochemistry .............................................................. 18 XANES Spectroscopy, Spectral Fitting, and Quantitative Analyses .............................. 21 Microbial Mat DNA Extraction ....................................................................................... 22 Functional Gene PCR Amplification and Cloning .......................................................... 24 Functional Gene Sequence Analysis ................................................................................ 28 Pyrosequencing of 16S rRNA Genes ............................................................................... 28 Statistical Analyses .......................................................................................................... 30 CHAPTER 4: RESULTS ............................................................................................................. 32 Upper Spring Microbial Mat Morphology and Aqueous Geochemistry ......................... 32 Sulfur Speciation in the Microbial Mats ......................................................................... 34 Functional Gene Diversity and Distribution .................................................................... 36 soxB ................................................................................................................................. 36 aprA ................................................................................................................................. 38 Diversity of 16S rRNA Gene Sequences ......................................................................... 46 Statistical Comparisons .................................................................................................... 50 CHAPTER 5: DISCUSSION ....................................................................................................... 57 Links between Geochemistry and Biodiversity ............................................................... 57 Importance of Missing Functional Representation for Epsilonproteobacteria ................ 59 iii Microbial Metabolisms in Industrial Settings .................................................................. 60 Conclusions and Future Directions .................................................................................. 60 REFERENCES ............................................................................................................................ 62 APPENDIX A: LOWER KANE CAVE SPRING GEOCHEMISTRY ...................................... 74 APPENDIX B: DIVERSITY OF 16S RRNA GENE SEQUENCES .......................................... 77 VITA .......................................................................................................................................... 109 iv LIST OF TABLES 3.1 Functional gene-targeting primers used in PCR assays in this study ............................. 25 4.1 Geochemical results from Lower Kane Cave, listed in meters from the back of the cave to the front of the cave ........................................................................................................ 33 4.2 Percentages of chemical sulfur species contribution based on the best fittings of the XANES analyses of microbial mat samples ................................................................... 34 4.3 Lower Kane Cave soxB sequence diversity, as number of sequences with maximum shared identity to GenBank sequences with taxonomic affiliations ............................... 39 4.4 Lower Kane Cave soxB sequence diversity, as number of sequences with maximum shared identity to GenBank sequences with and without taxonomic affiliations ........... 48 4.5 Distribution of 16S rRNA pyrosequences among classes of Proteobacteria .................. 50 v LIST OF FIGURES 2.1 Location of Lower Kane Cave in Wyoming, USA, and schematic diagram of the cave with springs and mat types ............................................................................................... 9 3.1 Images from the Upper Spring area of Lower Kane Cave ............................................... 19 3.2 Sulfur K-edge XANES spectra (normalized and stacked) for standard reference compounds used for linear combination fittings of microbial mat