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EUBACTERIAL ENDOSYMBIONTS of the FUNGUS PISOLITHUS TINCTORIUS a University Thesis Presented to the Faculty of California State

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EUBACTERIAL ENDOSYMBIONTS of the FUNGUS PISOLITHUS TINCTORIUS a University Thesis Presented to the Faculty of California State EUBACTERIAL ENDOSYMBIONTS OF THE FUNGUS PISOLITHUS TINCTORIUS A University Thesis Presented to the Faculty of California State University East Bay In Partial Fulfillment of the Requirements for the Degree Master of Science in Biological Science By Kaushalya Nadi Tillakarathna December 2016 Kaushalya Nadi Tillakarathna © 2016 ii Abstract The fungus Pisolithus tinctorius is a common inhabitant of Yellowstone National Park geothermal soils and appear to have formed symbiotic relationships with a diverse group of bacterial species to satisfy their nutritional and energy needs. In this study, I was able to isolate and identify 34 different eubacteria from three phyla from within the fruiting body of P. tinctorius from Yellowstone National Park. Five are members of the phylum Actinobacteria, four are members of the phylum Proteobacteria and twenty-five are members of the phylum Firmicutes. Fluorescent in situ hybridization (FISH) using probes for both eubacteria and archaea, I confirmed the presence and provided a visual idea of the localization of eubacterial and archaea endosymbionts present within the tissues of fruiting body P. tinctorius. At this point, further metabolic studies are needed to confirm the role of each endosymbiotic bacterium found within the fungus P. tinctorius. The phylogenetic analysis of 16S rRNA gene sequence indicated that Piso NA-SE-3 isolate clustered with Paenibacillus species with a bootstrap value of 82 % and exhibited 16S rRNA gene sequence similarity of 96% with Paenibacillus cineris. The isolate Piso NP3- KT 25 was closer to Corynebacterium species and clustered with a poor bootstrap value of 74 %. The isolate showed 16S rRNA gene sequence similarity of 88% with Corynebacterium mucifaciens. iii EUBACTERIAL ENDOSYMBIONTS OF THE FUNGUS PISOLITHUS TINCTORIUS By Kaushalya Nadi Tillakarathna Approved: Date: r ' a!fAA, YV1 t- fo.:;;t;;J Dr. Ann McPartland r 1 IV Acknowledgements I would first like to thank my professor and mentor, Dr. Carol Lauzon at California State University East Bay, for all the advice, support and encouragement offered to me during my thesis project. Thank you for been patient with me. I would have been lost without your help. Also, thank you for letting me participate in an opportunity of a lifetime. I would also like to thank Dr. Ken Cullings at Astrobiology department, NASA Ames research center, Mountain View, California for all his time and expertise. Thank you to all my thesis committee members, Dr. Maria Gallegos and Dr. Ann McPartland for their expert analysis of my thesis research. Finally, I would like to express my gratitude towards my parents, my aunt and my husband for their unconditional love, support, and encouragements. I would not have gotten to the point I am at today without anyone of you. This work was generously funded by the Astrobiology department, NASA Ames research center, Mountain View, California. v Table of Contents Page Abstract ............................................................................................................................. iii Acknowledgement ..............................................................................................................v List of Figures ................................................................................................................. viii List of Tables .................................................................................................................... xi Chapter 1: Introduction The Primitive Earth ..................................................................................................1 Hydrothermal Vents .................................................................................................2 Symbiotic Interactions .............................................................................................3 Kingdom Fungi ........................................................................................................7 Genus Pisolithus ...................................................................................................12 Pisolithus tinctorius ...............................................................................................12 Yellowstone National Park ....................................................................................13 Objectives ..............................................................................................................15 References ..............................................................................................................15 Chapter 2: Bacterial Culturing Introduction ............................................................................................................25 Bacterial Culture Media .........................................................................................26 The 16S rRNA Gene ..............................................................................................26 Materials and Methods ...........................................................................................27 Results ....................................................................................................................34 Discussion ..............................................................................................................44 References ..............................................................................................................51 Chapter 3: Phylogenetics Introduction ............................................................................................................67 Basic Concepts .......................................................................................................68 Phylogenetic Tree Construction Methods ..............................................................69 Materials and Methods ...........................................................................................71 Results ....................................................................................................................72 Discussion ..............................................................................................................77 References ..............................................................................................................78 vi Chapter 4: Fluorescent in situ Hybridization Introduction ............................................................................................................81 Fluorescent in situ Hybridization (FISH) ............................................................. 81 FISH in Microbiology ............................................................................................82 Confocal Laser Scanning Microscopy ...................................................................84 Autoflorescence .....................................................................................................84 Materials and Methods ...........................................................................................85 Results ....................................................................................................................88 Discussion ............................................................................................................104 References ............................................................................................................108 Chapter 5: Thesis Summary ...........................................................................................115 Complete References .....................................................................................................122 Appendix A .....................................................................................................................154 Appendix B .....................................................................................................................161 Appendix C .....................................................................................................................194 Appendix D .................................................................................................................... 201 vii List of Figures Figure Heading Page number number 1 The four main divisions within the Kingdom fungi. 8 2 (A) The fungal body structure (B) The fungal life cycle. 10 3 (A) Landscape of Norris Geyser Basin in Yellowstone National 27 - 28 Park (B) and (C) Pisolithus tinctorius in Norris Geyser Basin (D) and (E) Interior of the P. tinctorius fruiting body. 4 Replica plating. 43 5 Tree of life. 68 6 Phylogenetic tree based on 16S rRNA sequences showing the 74 relationships between isolate PisoNA-SE-3 and related taxa. 7 Phylogenetic tree based on 16S rRNA sequences showing the 76 relationships between isolate Piso-NP3-KT-25 and related taxa. 8 P. tinctorius 10 sample, no probes (to detect autofluorescence) 90 (A) Laser 405nm (excitation), Emission spectra :420 - 600 nm (B) Laser 488 nm (excitation), Emission spectra :498 -676 nm (C) Laser 638 nm (excitation), Emission spectra: 650 -754 nm. With probe (D) NONEUB338 (negative control) with ALEXA 405. Laser 405 nm (excitation) Emission spectra :420 - 473 nm. 9 P. tinctorius sample 10, EUB338 with ALEXA 488. Laser 488 91 nm (excitation), Emission spectra :537 -557 nm. (A) Replicate 1 (B) Replicate 2 and (C) Replicate 3. 10 P. tinctorius sample 10, SRB385 with ALEXA 488. Laser 488 92 nm (excitation), Emission spectra: 537 -557 nm. (A) Replicate 1 (B) Replicate 2 and (C) Replicate 3. 11 P. tinctorius
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