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Scanned by Scan2net MOLECULAR DIVERSITY OF BACTERIA FROM THREE DISTINCT ECOSYSTEMS WITH IN GREAT SMOKY MOUNTAINS NATIONAL PARK By Meli ssa Brooke Collins A Thesis Submitted to th e Faculty of the Graduate School o f Western Carolina Uni versity In Parti al Fulfillment of th e Requirements for th e Degree of Master of Science Commillee: Director Dean of th e Graduate School Date: h 3? Summer 2006 Western Carolina Uni versity Cullowhee. orlh arolina MOLECULAR DIVERSITY OF BACTERIA FROM THREE DISTINCT ECOSYSTEMS WITHIN GREAT SMOKY MOUNTAINS NATIONAL PARK A thesis presented to the faculty of the Graduate School of Western Carolina University in partial ful fillment of the requirements for the degree of Master of Science By Melissa Brooke Collins Director: Dr. Sean O'Connell Assistant Professor of Biology Department of Biology July 2006 HU NTER LI BRARY WESTERN CAROLINA UNIVERSITY Acknowledgements I would like to thank my advisor Sean O 'Conncll for a ll of his hard work, help, and dedication to not onl y Ill yselfand this project, but also any student in need. Hi s passion for science is inspiring and makes working with him exciting and rewarding. would also like to thank the Bi ology department and the Office of Graduate Studies at WCU for providing equipment and funding without whi ch thi s research would not have been possible. Thank you to Kri stina Reid , Heath er Sink, Darby Harris, Barclay Taylor, and especiall y Philip Drummond for all of the time and energy that they donated to helping inside the lab. Finall y, 1 would like to thank my family and Peter Gut for a ll o f th eir love, support, and help with Aubrie during this time. 11 Table of Contents Pagc List of Tables ... .. ........ ............... ..................................................................................... .. ......... iv List of Figures .... ... ...... ............................ ........................................ .................. ............... ... .. .. .. v Abstract ............................................ .... .. .................................................................................. vi Introduction ........ .. ............................. ............... ... ................... ................................................... I Methods ................................................................................................................................... 18 Soil Sample Coll ccti on ................................................................. ... .............. .. ... ........ 18 Methods Development .. ......... .. .... .. .... .. ................................ ................ ...................... 18 DNA Extraction ........................... ... .. ........................... ... ....... ................ .. ................. .. 19 PCR Amplification ............. ........................ ................................................................ 19 Denaturing Gradicnt Gel Electrophorcsis (DGG E) ..................................... .. ........... 20 Methods used in Soil Study ............ ............................................. .. ... .... .. .................. .. ........ 20 DNA Extraction ........................ .. ........................... ... ........ ........ ..... .... .... .. .. .......... .. .. ... 20 PCR Amplification (1500bp) .. .. ..... .......................................................................... 20 PCR Clean-Up ..................... .. ................................ .. .......... ................... ...................... 2 1 Molecular Cloning ... .. ............. ........... .. ..................... .... ........................ .. .. .................. 2 1 Whole el l PCR ............................... .. ................ .. ... ...... ........................... ........... .. ... .. 22 Restriction Fragment Length Pol ymorphism (RFLP) ........................ .. ...... .............. 23 Sequencing ................ .......................... .............. ......................................... ................. 24 Results ...................... .. ....................... ....................................... ... ............ .. .. .. .... ... ... ......... ..... .. 26 Methods Development ... ..... .. ........................................................... ................. ......... 26 Methods uscd in Soil Study ...... .......................... ........... ...... ...... ..................... .. ... ...... 26 Molecular Cloning Results .. .. ... ... ............................................................ .. .... .... .. .... ... 26 RFLP Results ................... ..................................... .. ... ............................ ..... ... .. .. ......... 29 Sequencing and RDP II Results ........ .. ........ .......... .. ............................................ ..... .. 30 Di scussion .......... ............ .. .................. .. ...................... ....................... .. ............... .. .. .... ......... .... 51 Conclusions and Possible Future Work ................................. .......... .. .................................... 63 Li terature Cited ..................................................................... ... ..... ......... .. ... ............................ 66 Appendix ................. ................ ...................... .. ................... .................................. ................... 75 List of Tables Page I. A TBI Plot Characteri sti cs ... .............................. .. .. .................................. .... ..... ... .......... .. ... 14 2. Number of Clones per Replicate ................. .... .... ........................ .. ..... ..... .. ... .... ................. 29 3. Phylum Level Diversity fTom All Sites .......................... .. .................. .. ................ .. ........... 33 4. Genera Represented in Molecul ar Clone Libraries .. ................... ..... .... ... ......... .. ............... .42 5. Sequence Id entities Compared Within Divisions ..... ..... ....... .. .... ....................... ..... ... .. .... .45 6. Percentages ofNon-Acidobacteria Divisions within Sites ....................... ... .... ..... .... ... .... 58 IV List of Figures Page I. Uni versal Phylogenetic Tree .................... .. ................................................... ... .... .. .... ......... 2 2. Phylogenetic Tree of Bacteria .. ... .... .. .. ............... ... ............................... .. ... .. ... .. ... ................ 6 3. Phylum Level Di versity for Bacteria Cultivated from Soil ............. .... .. ...... ... ... ..... ..... .... 13 4. Map of Great Smoky Mountains National Park ...... .. ............ .. ............................. ........ .. .. 15 5. DNA Extraction Kits Comparison Gel ............................................................................. 27 6. PCR Amplification Gcl Comparin g DNA Ex tracti on Kits .............................................. 28 7. Example of Restriction Fragment Length Polymorphism Gel ...... .. ............ ..................... 30 8. RDP \I "Classifier" Results for All Three Si tes ............................ .. .. ................................ 32 9. RDP [! "Classifi er" Results for Albright Grove .. ...... ... ................. ...... .. ........................... 34 10. RDP II " lassifi er" Results for Cataloochee ............ .................................. ..................... 35 II . RDP II "Classifier" Results for Purchase Knob ...................... ..... .. ................................. 36 12. Phylum Diversity Patterns for Albright Grove Rep li cates ............ .... .............................. 38 13. Phylum Di versity Patterns [or Cataloochee Repli cates ................................................... 38 14. Phylum Diversity Pattems for Purchase Knob Repl icates .................... ........ ...... .. .. ....... .40 15. Simple Comparative Tree for Acidobacteria Clones ...................................................... .46 16. Simple Comparati ve Tree for Firmiclltes Clones .. .. ...... ...................... ........................... .47 17. Simple Comparati ve Tree for Planclomycetes Clones .................................. ................. .48 18. Simple Comparative Tree for Verrucomicrobia Clones ................................................. .48 19. Simple Comparative Tree for Alphaproteobacleria Clones .... ........ .. .. ........................... .49 20. Simple Comparative Tree for Dellaproteobacleria Clones ............................................ 50 2 1. Simple Comparative Tree for Gammaproteobacteria Clones .... .................................... 50 v Abstract MOLECULAR DIVERSITY OF BACTERlA FROM THREE DISTINCT ECOSYSTEMS WJTI-IIN GREAT SMOKY MOUNTAlNS NATIONAL PARK Melissa B. Collins M.S. Western Carolina University (August 2006) Director: Dr. Sean O'Connell The number of microbial species in nature may be in the millions, but most have never been observed or detected (Hong et a!. 2006). For over 100 years, studies have focused primari ly on culturing species from environm ental sampl es in order to examine diversity of th e community. With advancements in molecular techniques, a shift has occurred in both the approaches used to create community profiles and to ex pl ain what these profiles look like. This knowledge of microbial diversity is crucial for our understanding of the structure, function, and evoluti on of biological communities. The bi odiversity of several thousand organi sms has been catalogued throughout Great Smoky Mountains
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