Title

Genetic tools for the study of {\it Thermotoga\/}

Date of Completion

January 1998

Keywords

Biology, Molecular|Biology, Genetics|Biology, Microbiology

Degree

Ph.D.

Abstract

Thermotoga neapolitana is a hyperthermophilic bacterium which occupies one of the deepest phylogenic branches within the bacterial domain. Thermotoga species strains are undeveloped areas for useful genetic systems. The primary goal of this thesis is to establish the genetic tools for the study of Thermotoga strains.^ A T. neapolitana genomic library was constructed in the cosmid conjugal vector pLAFR3. From this library, the 3-phosphoglycerate kinase, triosephosphate isomerase, and enolase were expressed in Escherichia coli strain DH10B, T. neapolitana produces 3-phosphoglycerate kinase both as an individual enzyme and as a fusion protein with triosephosphate isomerase. Triosephosphate isomerase activity is not found without associated 3-phosphoglycerate kinase activity. T. neapolitana expresses both 73 kDa and 81 kDa isozymes of this fusion protein.^ The hyperthermophilic bacterium Thermotoga species strain RQ7 harbors an 846 bp plasmid, pRQ7, with a single open reading frame. Plasmid pRQ7 belongs to the class of plasmids that replicate by single-stranded DNA intermediates--the rolling-circle replicating plasmids. The DNA encoding the open reading frame was cloned and expressed in E. coli and gave a protein with a molecular weight of 26 kDa, similar to that deduced by sequence analysis. This protein binds to a fragment of pRQ7 that contains a putative double stranded replication region in a magnesium-dependent reaction making this fragment sensitive to S1 nuclease activity. This activity on pRQ7 DNA suggests this protein plays a role in plasmid replication.^ Shuttle vector pJY1 has been constructed by combining three fragments: a tac promoter, a chloramphenicol acetyltransferase (cat) gene, and a pRQ7. Another vector, pJY2, replaces the cat gene with a kanamycin nucleotidyltransferase gene. Transformation methods using liposomes and an overlay plating technique have been developed. The transformation frequencies obtained for this liposome-mediated method were $1.1\times10\sp4$ transformants per microgram of DNA per 10$\sp9$ cells. Thermotoga cells could maintain the shuttle vectors up to 3 days without and up to 5 days with antibiotic selection. ^