Liposome-mediated DNA uptake and transient expression in Thermotoga

We report here the successful application of a PCR-based method to detect genetic transformation of Thermotoga neapolitana and Thermotoga maritima. Plasmid vectors were constructed using pRQ7, an 846-bp plasmid found in Thermotoga species strain RQ7, which replicates by a rolling circle mechanism. The vector pJY1 was constructed by placing a gene encoding a thermostable chloramphenicol acetyltransferase from Stacphylococcus aureus under the control of the tac promoter and joining this with pRQ7 in a pBluescript vector. A second vector, pJY2, was similarly constructed using a gene encoding a kanamycin nucleotidyltransferase previously engineered for thermostability. Genetic transformation of T. neapolitana and T. maritima spheroplasts was achieved using cationic liposomes. The transforming DNA was detected in cells grown in liquid cultures using polymerase chain reaction amplification of the cat or kan genes. T. neapolitana could maintain pJY1 for at least 25 generations in liquid medium containing chloramphenicol. The pJY2 vector conferred kanamycin resistance to T. maritima cells grown in liquid culture. Isolation of stable transformants on solid media after 2-3 days of incubation at 77 degrees C was not possible with either vector, probably because of the instability of both vectors and antibiotics under these conditions. However, this transformation procedure provides, for the first time, a method to introduce DNA into this hyperthermophilic bacterium for potential applications such as targeted gene disruption analyses.