Title

Development of Genetic Tools for Thermotoga SPP.

Date of Award

2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biological Sciences

First Advisor

Zhaohui Xu, Ph.D.

Second Advisor

Lisa Chavers, Ph.D. (Committee Member)

Third Advisor

George Bullerjahn, Ph.D. (Committee Member)

Fourth Advisor

Raymond Larsen, Ph.D. (Committee Member)

Fifth Advisor

Scott Rogers, Ph.D. (Committee Member)

Abstract

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 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.