Transformation of Bacillus stearothermophilus with plasmid DNA and characterization of shuttle vector plasmids between Bacillus stearothermophilus and Bacillus subtilis.

Publisher Website
Google Scholar
Abstract
A thermophilic bacterium B. stearothermophilus IFO 12550 (ATCC 12980) was transformed by plasmid pUB110 (kanamycin resistance, Kmr), plasmid pTB19 (Kmr and tetracycline resistance [Tcr]), and by the pTB19 derivative, plasmid pTB90 (KmrTcr), by the protoplast procedure in the presence of polyethylene glycol at 48.degree. C. The transformation frequencies per regenerant for pUB110, pTB19 and pTB90 were 5.9 .times. 10-3, 5.5 .times. 10-3 and 2.0 .times. 10-1, respectively. Among these plasmids, pTB90 was newly derived, and the restriction endonuclease cleavage map was constructed. When tetracycline (5 .mu.g/ml) was added into the culture medium, the copy number of pTB90 in B. stearothermophilus was about 4-fold higher than that when kanamycin (5 .mu.g/ml) was added instead of tetracycline. B. subtilis could also be transformed with the plasmids extracted from B. stearothermophilus and vice versa. Accordingly, pUB110, pTB19 and pTB90 served as shuttle vectors between B. stearothermophilus and B. subtilis. The requirements for replication of pTB19 in B. subtilis and B. stearothermophilus appear to be different, because some deletion plasmids (pTB51, pTB52 and pTB53) derived from pTB19 could replicate only in B. subtilis, whereas another deletion plasmid pTB92 could replicate solely in B. stearothermophilus. Plasmids pTB19 and pTB90 could be maintained and expressed in B. stearothermophilus up to 65.degree. C, whereas the expression of pUB110 in the same strain was up to 55.degree. C.