Establishment of a gene transfer system for Rhodothermus marinus

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 x 10(6) cfu/mug DNA at 22.5 kV/cm, 200 Omega and 25 muF. Transformation failed, however, after chemical preparation of cells according to several protocols. This is the first report of genetic transformation in the genus Rhodothermus.