Genetic manipulation of Rhodothermus marinus has been hampered by the lack of a selection system for gene transfer. We report construction of a Rhodothermus/Escherichia coli shuttle plasmid, containing the R. marinus trpB gene, based on pUC18 and the cryptic R. marinus plasmid pRM21. A plasmid-less R. marinus recipient strain was selected on the basis of growth characteristics and absence of restriction activity. The shuttle plasmid, pRM100, was successfully introduced into a TrpB- mutant of the recipient strain using electroporation and was found to transform it to prototrophy. No loss or rearrangement of pRM100 was observed after growth for 80 generations in non-selective medium. The relative copy numbers of pRM100 and of the parental plasmid, pRM21, were determined as 7±1 and 42±4, respectively. The shuttle plasmid was used to optimize an electroporation protocol, and the maximal number of transformants obtained was 4.3±0.7×106 cfu/μg DNA at 22.5 V/cm, 200 Ω and 25 μF. Transformation failed, however, after chemical preparation of cells according to several protocols. This is the first report of genetic transformation in the genus Rhodothermus.
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Acknowledgements We thank Dr. J. Kristjansson (Prokaria Ltd, Reykjavik, Iceland) for the R. marinus strains and Dr. Ó.S. Andrésson (University of Iceland) and Ó.H. Friðjónsson (Prokaria Ltd) for critical reading of the manuscript. This work was supported by grants from the University of Iceland, the Icelandic Research Council and the Icelandic Research Fund for Graduate Students.