Protoplast transformation of Bacillus stearothermophilus NUB36 by plasmid DNA

Abstract

An efficient protoplast transformation system was established for Bacillus stearothermophilus NUB3621 using thermophilic plasmid pTHT15 Tc(r) (4.5 kb) and mesophilic plasmid pLW05 Cm(r) (3 kb), a spontaneous deletion derivative of pL401 Cm(r) Km(r). The efficiency of transformation of NUB3621 with pLW05 and pTHT15 was 2 x 107 to 4 x 108 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 Bacillus subtilis were transformed with pLW05 [2.5 x 105 transformants (μg DNA)-1] and pTHT15 [1.8 x 105 transformants (μg DNA)-1], respectively. Thus, these plasmids are shuttle vectors for mesophilic and thermophilic bacilli. Plasmid pLW05 Cm(r) was not stably maintained in cultures growing at temperatures between 50 and 65°C but the thermostable chloramphenicol acetyltransferase was active in vivo at temperatures up to 70°C. In contrast, thermophilic plasmid pTHT15 Tc(r) 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.