PLASMID MARKER RESCUE TRANSFORMATION IN THERMUS-THERMOPHILUS

The marker rescue transformation method was examined for Thermus thermophilus. Plasmid pYK134, constructed from a Thermus cryptic plasmid pTT8 with a modified staphylococcal kanamycin resistance (Km(r)) gene and a heterologous Thermus trpB gene as selection markers was used to transform T. thermophilus HB27 trpB mutant. The transformation efficiency with pYK134 against a plasmidless recipient was about 1 x 10(7) per microgram of DNA, or about 0.1% per viable cell count for both Km(r) and Trp+ markers. When the pTT8-carrying HB27 trpB mutant was used as a recipient, transformation efficiency with pYK134 increased about 30-fold, ie. about 3 x 10(8) per microgram of DNA or 3% per viable cell count. The plasmidless recipient could not be transformed with linearized pYK134, whereas the pTT8-carrying recipient was transformed by it only when linearization was done at the pTT8 region in pYK134. These results suggest that flanking homologous regions are necessary for plasmid marker rescue transformation in T. thermophilus.