FIG 3.

Curing pTT27 from T. thermophilus HB27. (A) Strategy to cure the pTT27 megaplasmid from HB27. When HB27 was transformed by pUC27H11, the intact pTT27 was excluded due to plasmid incompatibility. The transformant lacking pTT27 but containing pUC27H11 was subsequently transformed by pUC27K11. After both plasmid-possessing transformants obtained in the presence of both Hm and Km were cultivated without drugs, their colonies on a drug-free plate were checked for drug sensitivity. One drug-sensitive strain without any plasmid was selected as the PFW substrain. (B) Confirmation of the absence of plasmids in the PFW strain. Intact genomic DNAs of the WT (lane 1) and PFW (lane 2) strains were prepared in gel plugs and analyzed by CHEF electrophoresis after in-gel digestion by both EcoT22I and MunI. Lane M, size marker of concatemeric λ DNA and a HindIII-digested one. Running conditions were 5 V/cm with a 30-s pulse time and 20-h running time at 15°C. The absence of a linearized megaplasmid in lane 2 is indicated by the arrowhead.