An efficient protoplast transformation system was established for NUB3621 using thermophilic plasmid pTHT15 Tc (4·5 kb) and mesophilic plasmid pLW05 Cm (3 kb), a spontaneous deletion derivative of pPL401 Cm Km. The efficiency of transformation of NUB3621 with pLW05 and pTHT15 was 2 x 10 to 4 x 10 transformants per µg DNA. The transformation frequency (transformants per regenerant) was 0·5 to 1·0. Chloramphenicol-resistant and tetracycline-resistant transformants were obtained when competent cells of were transformed with pLW05 [2·5 x 10 transformants (µg DNA)] and pTHT15 [1·8 x 10 transformants (µg DNA)], respectively. Thus, these plasmids are shuttle vectors for mesophilic and thermophilic bacilli. Plasmid pLW05 Cm was not stably maintained in cultures growing at temperatures between 50 and 65 °C but the thermostable chloramphenicol acetyltransferase was active at temperatures up to 70 °C. In contrast, thermophilic plasmid pTHT15 Tc was stable in cultures growing at temperatures up to 60 °C but the tetracycline resistance protein was relatively thermolabile at higher temperatures. The estimated copy number of pLW05 in cells of NUB3621 growing at 50,60, and 65 °C was 69,18, and 1 per chromosome equivalent, respectively. The estimated copy number of pTHT15 in cells of NUB3621 growing at 50 or 60 °C was about 41 to 45 per chromosome equivalent and 12 in cells growing at 65 °C.


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