PLASMID MARKER RESCUE TRANSFORMATION IN THERMUS-THERMOPHILUS

The marker rescue transformation method was examined for Thermus therm ophilus. Plasmid pYK134, constructed from a Thermus cryptic plasmid pT T8 with a modified staphylococcal kanamycin resistance (Km(r)) gene an d a heterologous Thermus trpB gene as selection markers was used to tr ansform T. thermophilus HB27 trpB mutant. The transformation efficienc y 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) a nd Trp+ markers. When the pTT8-carrying HB27 trpB mutant was used as a recipient, transformation efficiency with pYK134 increased about 30-f old, ie. about 3 x 10(8) per microgram of DNA or 3% per viable cell co unt. The plasmidless recipient could not be transformed with linearize d pYK134, whereas the pTT8-carrying recipient was transformed by it on ly when linearization was done at the pTT8 region in pYK134. These res ults suggest that flanking homologous regions are necessary for plasmi d marker rescue transformation in T. thermophilus.