Temperature-dependent plasmid integration into and excision from the chromosome of Bacillus stearothermophilus

A transformant of Bacillus stearothermophilus carrying a recombinant plasmid, pLP11 (9.5 MDa), on which the penicillinase gene (penP) and kanamycin resistance gene (kan) were located was subjected to mutagenesis, and a mutant plasmid (9.5 MDa; penP kan), designated pTRA117, was obtained. A transformant of B. stearothermophilus carrying pTRA117 could grow at 63 degrees C in medium containing kanamycin, whereas a transformant carrying pLP11 could not. Although pTRA117 was detected as covalently closed circular (ccc) DNA when it was extracted from transformants cultured at 48 degrees C, it was integrated into the host chromosome when the culture temperature was shifted up to 63 degrees C. If the culture temperature was lowered to 48 degrees C from 63 degrees C, a new plasmid (10.7 MDa; penP kan), designated pTRZ117, could be detected as ccc DNA; the size of this plasmid suggested that it was pTRA117 plus a 1.2 MDa DNA fragment of the host chromosome, and this was confirmed by Southern hybridization. pTRZ90 (7.9 MDa; kan) was constructed from pTRZ117 by the deletion of a 2.8 MDa DNA fragment that contained penP. Fresh transformants of B. stearothermophilus that carried either pTRZ117 or pTRZ90 could grow at 65 degrees C.