DISTRIBUTION AND CONSERVATION OF REDUCED P METABOLISM OPERONS IN BACTERIA ____________ A Thesis Presented to the Faculty of California State University, Chico ____________ In Partial Fulfillment of the Requirements for the Degree Master’s of Science in Biological Sciences ____________ by Betsey Renfro Fall 2012 ACKNOWLEDGEMENTS The completion of this thesis concludes a decade long quest for a Master of Science degree in Biology. I would never have attained this degree without encouragement and support of numerous faculty. I would like to than Dr. Ailsie McEnteggart and Dr. Jeff Bell, past and present Chairs of the Biology Department, for allowing me the release time required from the media lab to complete the course work for this degree. Than you Dr. Kris Blee, Dr. Larry Hanne, and Dr. Patricia Edelmann, for your help with experimental design, troubleshooting, data analysis and for your encouraging words. Special thanks to Dr. Jeff Bell, who was so generous with his time and helped me with this project in more ways than I can enumerate. The biggest thanks however, must go to my advisor, co-worker and dear friend Dr. Andrea White. I have learned so much under your guidance. You were the perfect advisor for me, and I appreciate your support more than you will ever know. Finally, I would like to thank my family, especially my husband James and my children, Zach, Lauren and Michael. You guys always believed in me, and I love you so much. A huge thanks you to my parents, Craig and Susie Hawes, for all of your help with the kids and your willingness to act as a taxi service while I was busy pursuing my dream. This work was funded by the Office of Research and Sponsored Programs at California State University, Chico. iii TABLE OF CONTENTS PAGE Acknowledgements ...................................................................................... iii List of Tables ................................................................................................ vi List of Figures............................................................................................... vii Nomenclature ............................................................................................... ix Abstract ........................................................................................................ x CHAPTER I. Introduction ......................................................................................... 1 Questions........................................................................................ 23 II. Methods .............................................................................................. 26 Pseudomonas stutzeri WM88 Chromosomal Library Construction............................................................................... 26 Selection of Hpt+ and Pt+ Library Clones ....................................... 27 Restriction Analysis of Cosmid Clones............................................ 28 Database Mining for Hypophosphite, Phosphite and Phosphonate Oxidation Operons............................................... 28 III. Results ................................................................................................ 31 Reduced P Oxidation Pathways in Pseudomonas putida......................................................................................... 31 Distribution and Conservation of Hypophosphite Oxidation Pathways ................................................................... 36 Distribution of Phosphite Oxidation Pathways................................. 44 Conservation of Phosphite Oxidation Pathways.............................. 47 Distribution of C-P Lyase Operons.................................................. 52 Conservation of C-P Lyase Operons............................................... 55 iv CHAPTER PAGE IV. Discussion........................................................................................... 65 Overview ......................................................................................... 24 References ................................................................................................... 77 Appendix A. Reduced Oxidizing Bacteria Identified in this Study........................ 85 v LIST OF TABLES TABLE PAGE 1. Commercial Products Containing Phosphate that are Marketed as Fungicides and Fertilizers ................................... 9 2. Substrate Ranges Cosmid Clones in Glucose Mops minimal media +100 ug/ml Carbenicillin + 0.5mM Phosphorus source after 36 Hours of growth............................................... 32 3. Primers and expected product size for amplified htxA and ptxD products .............................................................................. 35 4. Function of Pseudomonas stutzeri WM88 Phn and Htx Orthologues of the C-P Lyase Operons................................... 56 5. Reduced P Oxidation Genes in bacteria with htx Encoded C-P Lyase Operons vi LIST OF FIGURES FIGURE PAGE 1. Diagram Depicting the Traditional P Cycle............................................. 3 2. Chemical Structure and Oxidation State of Common Organic and Inorganic Phosphorus Compounds .................................................. 6 3. Pathways for the Microbial Metabolism of Phosphonates...................... 11 4. Model for the Degradation of Methylphosphonate Via the C-P Lyase Pathway ................................................................................. 12 5. Biochemical Pathway for the Oxidation of Hypophosphite and Phosphite ......................................................................................... 17 6. Arrangement of Genes Involved In Catalysis of Hypophosphite, Phosphite and Phosphonates in Diverse Bacterial Species ............ 19 7. Pseudomonas Putida AW2 Phosphite Oxidation Pathway .................... 33 8. Growth of wild type P. putida AW2, E. coli Epi T100 +pBR20 and wild type E ........................................................................................ 34 9. Phylogenetic Tree of the HtxA Hypophosphite-2-Oxoglutarate Dioxygenase Catalytic Protein.......................................................... 38 10. Gene Organization and Protein Similarity of Phosphite Oxidation Pathways Indicate Recent Horizontal Gene Transfer. ...................... 39 11. Comparison of the htx and phn Encoded C-P Lyase Pathways in Pseudomonas stutzeri WM88 with the htx Encoded C-P Lyase Pathway in Pseudomonas aeruginosa PADK-CF510 ....................... 41 12. Structure of Two Potential Hypophosphite Oxidation Pathways in Bradyrhizobium BTAi1 and Their Similarity to Hypophophite Oxidation Clusters in Xanthobacter flavis and Alcaligenes faecalis ............................................................................................. 43 13. Bacteria with Phosphite Oxidation Enzymes are Found in Chemically and Physically Diverse Environments ............................ 45 vii FIGURE PAGE 14. Schematic of Common Arrangements of the ptx Operon and Surrounding Genes in Bacteria.................................................. 48 15. Phylogenetic Tree of the NAD-Dependent Phosphite Dehydrogenase Enzyme PtxD ......................................................... 51 16. Maximum Likelihood Tree of Catalytic C-P Lyase Protein PhnJ and HtxH with 100 Bootstrap Replicates .......................................... 58 17. The distribution of the organisms into two separate clades indicates separate evolution of the Phn and Htx proteins................. 59 18. Maximum Likelihood Tree of Catalytic Proteins PhnM and HtxL with 100 Bootstrap Replicates .......................................................... 60 viii NOMENCLATURE P Phosphorus or any compound containing phosphorus Pi Inorganic phosphate. Inorganic reduced phosphorus compound (+5 oxidation state) Hpt Hypophosphite. Inorganic reduced phosphorus compound (+1 oxidation state) Pt Phosphite Inorganic reduced phosphorus compound (+3 oxidation state) AePn Aminoethylphosphonate. Organic reduced phosphorus compound (+3 oxidation state) PH3 Phosphine gas. (-3 oxidation state) htxA Gene encoding 2-oxoglutarate-dependent hypophosphite dioxygenase, which is the enzyme that catalyzes the oxidation of hypophosphite to phosphite. ptxD Gene encoding a NAD-dependent phosphite dehydrogenase, which is the enzyme that catalyzes the oxidation of phosphite to phosphate. HGT Horizontal gene transfer.PET—photosynthetic electron transport ix ABSTRACT DISTRIBUTION AND CONSERVATION OF REDUCED P METABOLISM OPERONS IN BACTERIA by Betsey Renfro Master of Science in Biological Sciences California State University, Chico Fall 2012 P has long been considered a biologically inert yet essential element to all living organisms. However, our understanding of how P compounds are converted and made available for growth in the environment is greatly lacking. This deficit in our knowledge of P metabolism in an environmental context is highlighted by recent studies demonstrating that common soil bacteria are capable of oxidizing and reducing P compounds, thus altering P bioavailability in the environment. Understanding the interactions between these reduced P compounds and microbial populations is crucial to our understanding of P nutrient availability and management in the environment. These deficits in our knowledge led to our desire to identify novel reduce P oxidation pathways. Towards this end, DNA sequencing and analysis of the phosphite oxidation pathway in Pseudomonas putida AW2 were completed. The similarity of this x pathway to previously characterized ptx operons