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Development of Genetic Tools for Thermotoga Spp

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Development of Genetic Tools for Thermotoga Spp DEVELOPMENT OF GENETIC TOOLS FOR THERMOTOGA SPP. Dongmei Han A Dissertation Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY December 2013 Committee: Dr. Zhaohui Xu, Advisor Dr. Lisa C. Chavers Graduate Faculty Representative Dr. George S. Bullerjahn Dr. Raymond A. Larsen Dr. Scott O. Rogers © 2013 Dongmei Han All Rights Reserved iii ABSTRACT Zhaohui Xu, Advisor Thermotoga spp. may serve as model systems for understanding life sustainability under hyperthermophilic conditions. They are also attractive candidates for producing biohydrogen in industry. However, a lack of genetic tools has hampered the investigation and application of these organisms. We improved the cultivation method of Thermotoga spp. for preparing and handling Thermotoga solid cultures under aerobic conditions. An embedded method achieved a plating efficiency of ~ 50%, and a soft SVO medium was introduced to bridge isolating single Thermotoga colonies from solid medium to liquid medium. The morphological change of T. neapolitana during the growth process was observed through scanning electron microscopy and transmission electron microscopy. At the early exponential phase, around OD600 0.1 – 0.2, the area of adhered region between toga and cell membrane was the largest, and it was suspected to be the optimal time for DNA uptake in transformation. The capacity of natural transformation was found in T. sp. RQ7, but not in T. maritima. A Thermotoga-E. coli shuttle vector pDH10 was constructed using pRQ7, a cryptic mini-plasmid isolated from T. sp. RQ7. Plasmid pDH10 was introduced to T. sp. RQ7 by liposome-mediated transformation, electroporation, and natural transformation, and to T. maritima through liposome- mediated transformation and electroporation. Transformants were isolated, and the transformed kanamycin resistance gene (kan) was detected from the plasmid DNA extract of the recombinant strains by PCR followed by restriction digestions. The transformed DNA was stably maintained in both Thermotoga and E. coli even without iv the selection pressure. A uracil auxotrophic strain RQ7-15, with a 115 bp deletion near the 3' end of pyrE gene on T. sp. RQ7 chromosome, was isolated and used as the recipient cell for using pyrE as the selection marker. The pyrE gene from Caldicellulosiruptor saccharolyticus was expressed in RQ7-15, driven by either promoter PslpA from Thermus thermophilus or promoter PRQ7.pyr, which is the promoter of the pyrimidine synthesis operon of T. sp. RQ7. This work advanced the development of genetic tools for the study of Thermotoga. v Dedicated to my parents, Zhixiang Han and Enqiu Gao. vi ACKNOWLEDGMENTS I owe my deepest gratitude to my advisor, Dr. Zhaohui Xu. Without your support, guidance, encouragement, and patience, this work could not be accomplished successfully. I was honored to be your Ph.D. student and sincerely appreciate everything you have done for me. I would also like to thank my committee members, Dr. Scott Rogers, Dr. George Bullerjahn, Dr. Ray Larsen, and Dr. Lisa Chavers, for their advice and help on my project. I would like to thank Dr. Carol Heckman and Dr. Marilyn Cayer for your guidance and assistance with Electron Microscopes. I would like to thank Dr. Paul Morris for lending me the equipment in his lab generously. I would also like to thank the members of the Xu lab: Dr. Uksha Snini, Patel Jigar, Jingjing Cao, Rutika Puranik, Hui Xu, and Stephen Morris. Many thanks to your help, suggestion, and support during the past five years. It was a great time to work together with all of you. I am grateful to Linda Treeger, Susan Schooner, Chris Hess, Sheila Kratzer, Steve Queen, and Dee Dee Wentland for all your help and patience. Finally, I would like to thank my family for all your love and support. vii TABLE OF CONTENTS GENERAL INTRODUCTION ...................................................................................... 1 The evolutionary significance of Thermotoga ........................................................ 2 The potentials of Thermotoga in producing biomass-based fuels .......................... 4 The development of genetic tools for the study of hyperthermophiles .................. 7 Chapter I. Improvement of the cultivation method of Thermotoga spp. ..................... 11 Introduction .......................................................................................................... 11 Materials and Methods ......................................................................................... 13 Strains and growth condition ......................................................................... 13 Sensitivity of Thermotoga to oxygen ............................................................ 16 Results and Discussion ......................................................................................... 16 Sensitivity of Thermotoga to oxygen ............................................................ 16 Improved methods for handling Thermotoga cultures in an aerobic environment ................................................................................................... 18 Isolation of single colonies from plates ......................................................... 19 Conclusion ............................................................................................................ 21 Chapter II. The morphological change of Thermotoga during the growth process ..... 24 Introduction .......................................................................................................... 24 Materials and Methods ......................................................................................... 25 Growth curve of Thermotoga ........................................................................ 25 Scanning electron microscopy (SEM) ........................................................... 26 viii Transmission Electron Microscopy (TEM) ................................................... 26 Results and Discussion ......................................................................................... 27 Growth curve of T. neapolitana. ................................................................... 27 The morphological change of Thermotoga cells revealed by Scanning Electron Microscopy ..................................................................................... 28 Transmission Electron Microscopy revealed the inside structure change of Thermotoga cells ........................................................................................... 30 Conclusion ............................................................................................................ 32 Chapter III. Construction and transformation of a Thermotoga-E. coli shuttle vector pDH10 .......................................................................................................................... 34 Introduction .......................................................................................................... 34 Materials and Methods ......................................................................................... 38 Strains and growth condition ......................................................................... 38 Nucleic acid extraction .................................................................................. 39 Sensitivity of Thermotoga to different antibiotics ........................................ 41 Construction of vectors ................................................................................. 42 Transformation and selection methods of Thermotoga ................................. 44 Selection and verification of Thermotoga transformants .............................. 46 Stability assays of the transformed DNA in Thermotoga and E. coli ........... 47 Results and Discussion ......................................................................................... 48 Sensitivity of Thermotoga strains to different antibiotics ............................. 48 Thermotoga is naturally transformable ......................................................... 52 ix Transformation of Thermotoga ..................................................................... 54 Verification of Thermotoga transformants .................................................... 55 Transformed pDH10 was stably maintained in Thermotoga ........................ 57 Incorporation of pRQ7 increased the stability of pUC19 derivatives in E. coli ....................................................................................................................... 58 Conclusion ............................................................................................................ 60 Chapter IV. Isolation of uracil auxotrophs of T. sp. RQ7 and a new thermostable selective marker for Thermotoga ................................................................................. 63 Introduction .......................................................................................................... 63 Materials and Methods ......................................................................................... 64 Strains and growth conditions ....................................................................... 64 DNA analysis ................................................................................................. 65 Construction of vectors ................................................................................. 66 The inhibition
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