@article{mbs:/content/journal/micro/10.1099/00221287-135-5-1315, author = "Wu, Lijun and Welker, N. E.", title = "Protoplast Transformation of Bacillus stearothermophilus NUB36 by Plasmid DNA", journal= "Microbiology", year = "1989", volume = "135", number = "5", pages = "1315-1324", doi = "https://doi.org/10.1099/00221287-135-5-1315", url = "https://www.microbiologyresearch.org/content/journal/micro/10.1099/00221287-135-5-1315", publisher = "Microbiology Society", issn = "1465-2080", type = "Journal Article", abstract = "An efficient protoplast transformation system was established for Bacillus stearothermophilus NUB3621 using thermophilic plasmid pTHT15 Tcr (4·5 kb) and mesophilic plasmid pLW05 Cmr (3 kb), a spontaneous deletion derivative of pPL401 Cmr Kmr. 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 Cmr 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 Tcr 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.", }