Construction and transformation of a Thermotoga-E. coli shuttle vector

Methods for preparing and handling Thermotoga solid cultures under aerobic conditions were optimized. A plating efficiency of ~50% was achieved when the bacterial cells were embedded in 0.3% Gelrite. A Thermotoga-E. coli shuttle vector pDH10 was constructed using pRQ7, a cryptic mini-plasmid found in T. sp. RQ7. Plasmid pDH10 was introduced to T. maritima and T. sp. RQ7 by electroporation and liposome-mediated transformation. Transformants were isolated, and the transformed kanamycin resistance gene (kan) was detected from the plasmid DNA extracts of the recombinant strains by PCR and was confirmed by restriction digestions. The transformed DNA was stably maintained in both Thermotoga and E. coli even without the selective pressure.Thermotoga are transformable by multiple means. Recombinant Thermotoga strains have been isolated for the first time. A heterologous kan gene is functionally expressed and stably maintained in Thermotoga.Besides Aquifex, Thermotoga are the only group of bacteria that can grow up to 90°C. Isolates of Thermotoga have been discovered from heated sea floors [1], continental hot springs [2], and oil fields [3]. Analysis of their 16S rRNA sequences have positioned Thermotoga spp. to a deep branch of the tree of life, suggesting that these strict anaerobes emerged at an early stage of evolution, when the surface of the Earth was hot and its atmosphere contained little oxygen. Study of the molecular genetics of Thermotoga is expected to shed light on the fundamental questions related to the origin of life as well as the mechanisms of the thermostability of macromolecules under extreme conditions. Most importantly, Thermotoga hydrolyze a number of polysaccharides through fermentative catabolism and produce hydrogen gas as one of the final products [4]. This has stimulated tremendous interest in utilizing these bacteria to produce biomass-based clean energy, especially through metabolic engineering approaches. However, due to the lack of genetic to