Incomplete Denitrification in Thermus Species
Total Page：16
File Type：pdf, Size：1020Kb
UNLV Theses, Dissertations, Professional Papers, and Capstones August 2016 Incomplete Denitrification in Thermus Species Chrisabelle Mefferd University of Nevada, Las Vegas Follow this and additional works at: https://digitalscholarship.unlv.edu/thesesdissertations Part of the Biology Commons, Environmental Sciences Commons, Microbiology Commons, and the Terrestrial and Aquatic Ecology Commons Repository Citation Mefferd, Chrisabelle, "Incomplete Denitrification in Thermus Species" (2016). UNLV Theses, Dissertations, Professional Papers, and Capstones. 2793. http://dx.doi.org/10.34917/9302950 This Thesis is protected by copyright and/or related rights. It has been brought to you by Digital Scholarship@UNLV with permission from the rights-holder(s). You are free to use this Thesis in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Thesis has been accepted for inclusion in UNLV Theses, Dissertations, Professional Papers, and Capstones by an authorized administrator of Digital Scholarship@UNLV. For more information, please contact [email protected]. INCOMPLETE DENITRIFICATION IN THERMUS SPECIES By Chrisabelle R. Cempron Bachelor of Sciences - Biology Montclair State University 2013 A thesis submitted in partial fulfillment of the requirements for the Master of Science - Biological Science School of Life Sciences College of Sciences The Graduate College University of Nevada, Las Vegas August 2016 Thesis Approval The Graduate College The University of Nevada, Las Vegas May 25, 2016 This thesis prepared by Chrisabelle R. Cempron entitled Incomplete Denitrification in Thermus Species is approved in partial fulfillment of the requirements for the degree of Master of Science – Biological Science School of Life Sciences Brian Hedlund, Ph.D. Kathryn Hausbeck Korgan, Ph.D. Examination Committee Chair Graduate College Interim Dean Eduardo Robleto, Ph.D. Examination Committee Member Laurel Raftery, Ph.D. Examination Committee Member Elisabeth Hausrath, Ph.D. Graduate College Faculty Representative ii ABSTRACT INCOMPLETE DENITRIFICATION IN THERMUS SPECIES by Chrisabelle Cempron Brian Hedlund, PhD, Committee Chair Professor of Biology University of Nevada, Las Vegas Members the bacterial genus Thermus have been shown to be incomplete denitrifiers, terminating with nitrite or nitrous oxide (N2O). However, the ability to carry out denitrification and the evolution of nitrogen oxide reductase genes in Thermus remains poorly understood. This study tests the hypothesis that incomplete denitrification is common in Thermus and seeks to uncover patterns in the evolution of denitrification pathways in Thermus. Denitrification capacity was determined in a collection of 25 strains representing ten species of Thermus and phylogenetic analysis was performed to determine whether denitrification genes evolved horizontally in Thermus. No strains in this study reduced nitrate to dinitrogen (N2). Terminal products were nitrite, nitric oxide (NO), or nitrous oxide (N2O), with most strains ending with N2O as a final product. In most cases, denitrification phenotypes were consistent with the presence of denitrification genes and strains of the same species typically had the same denitrification phenotypes. Phylogenetic analysis and the pattern of extant nitrogen oxide reductases showed evidence for horizontal gene transfer (HGT) and gene loss/gain within Thermus. These results show that incomplete denitrification is prominent in the genus Thermus, which suggests Thermus may play a role in consortial denitrification at high temperatures. iii ACKNOWLEDGMENTS I would first like to thank my thesis advisor Brian Hedlund of the School of Life Sciences at the University of Nevada, Las Vegas. The door to Brian’s office was always open whenever I ran into trouble, or had a question about my research or writing. He allowed this project to be my original work, but gave me direction whenever he thought I needed it. I would also like to thank my very supportive Thesis Committee who were involved in the validation of my work for this research project: Elizabeth Hausrath, Laurel Raftery, and Eduardo Robleto. Without their passionate participation and time, this work could not have been successfully completed. The expertise of the following individuals were so very necessary for fruitful conclusion of this project, for this I thank Enmin Zhou, Wen-Jun Li, Shreya Srivastava, Quiyuan Huang, Mira Han and her Lab, Ali Pour Yazdanpanah, and Klaus Schliep. I thank all the members of the Hedlund Lab for their valuable assistance and input, specifically Adrian Bernardo, Jacob Villarama, Michele Malwane, Amanda Williams, Senthil Murugapiran, and Cale Seymour. This project was funded by NSF grants OISE 0968421 and the Nevada NASA Space Grant RISE and HOP Scholarship. I must thank my Parents, Manuel and Arsenia Cempron, for providing me with life and unfailing support, even 3,000 miles away. Finally, I must express my very profound gratitude to my husband Kyle Mefferd for his devoted encouragement throughout my years of study and through the process of researching and writing this thesis. This accomplishment would not have been possible without you, love. Thank you. Totus Tuus iv TABLE OF CONTENTS ABSTRACT ....................................................................................................................... iii ACKNOWLEDGMENTS ................................................................................................. iv LIST OF TABLES ............................................................................................................. vi LIST OF FIGURES .......................................................................................................... vii CHAPTER 1 INTRODUCTION .........................................................................................1 CHAPTER 2 MATERIALS AND METHODS ..................................................................9 CHAPTER 3 MANUSCRIPT FOR FEMS MICROBIOLOGY ECOLOGY ......................16 CHAPTER 4 MANUSCRIPT FOR GENOME ANNOUNCEMENTS ..............................37 CHAPTER 5 CONCLUSION............................................................................................41 APPENDIX ........................................................................................................................44 REFERENCES ..................................................................................................................58 CURRICULUM VITAE ....................................................................................................69 v LIST OF TABLES Table 1. Primer sequences and positions ...........................................................................13 Table 2. Measured reductase activity and gene amplification ...........................................23 Table S1. Thermus master list and isolation source ...........................................................48 Table S2. Primers for PCR amplification and annealing temperatures .............................52 vi LIST OF FIGURES Figure 1. The nitrogen cycle ................................................................................................2 Figure 2. Complete denitrification pathway and corresponding reductase genes ...............2 Figure 3. Near stoichiometric conversion of nitrate to N2O during Thermus growth .......24 Figure 4. Map showing nar operon and neighboring genes ...............................................27 Figure 5. Maximum-likelihood phylogenetic tree based on Thermus 16S rRNA .............28 Figure 6A. Maximum-likelihood phylogenetic tree of Thermus narG..............................29 Figure 6B. Maximum-likelihood phylogenetic tree of Thermus nirK ...............................30 Figure 6C. Maximum-likelihood phylogenetic tree of Thermus nirS ................................31 Figure 6D. Maximum-likelihood phylogenetic tree of Thermus norB ..............................32 Figure S1. Near-stoichiometric conversion of nitrate to N2O during growth ...................45 Figure S2. Amino acid sequences of NirS and important residues ....................................46 Figure S3. Amino acid sequences of NorB and important residues ..................................47 vii CHAPTER 1 INTRODUCTION 1.1 The nitrogen cycle Microbial ecology concerns the relationship of microbes with one another and their environment. This includes the study of microbes and their roles in the cycling of nitrogen. The nitrogen cycle is the process by which nitrogen is converted between its different physical and chemical forms (Fig. 1) (Madigan et al. 2012). These transformations are often facilitated by microbial processes in an effort to accumulate nitrogen for growth or harvest energy. Assimilatory processes of the nitrogen cycle include dinitrogen (N2) fixation, in which nitrogen is combined with hydrogen other elements to form ammonia to incorporate into proteins. Ammonification describes the pathway by which ammonia is released from the decomposition of organic nitrogen compounds. Dissimilatory processes of the nitrogen cycle include dissimilatory nitrate reduction to ammonia (DNRA), nitrification, anaerobic ammonia oxidation (anammox), and denitrification. DNRA is the direct reduction of nitrate to ammonia and can occur in anoxic environments