Temperature-dependent Plasmid Integration into and Excision from the Chromosome of Bacillus stearothermophilus

A transformant of Bacillus stearothermophilus carrying a recombinant plasmid, pLP 1 1 (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 pTRA 1 17, was obtained. A transformant of B. stearothermophilus carrying pTRA 1 17 could grow at 63 "C in medium containing kanamycin, whereas a transformant carrying pLP 1 1 could not. Although pTRAll7 was detected as covalently closed circular (ccc) DNA when it was extracted from transformants cultured at 48 "C, it was integrated into the host chromosome when the culture temperature was shifted up to 63 "C. If the culture temperature was lowered to 48 "C from 63 "C, a new plasmid (1 0.7 MDa; penP kan), designated pTRZ 1 17, 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 pTRZll7 by the deletion of a 2.8 MDa DNA fragment that contained penP. Fresh transformants of B. stearothermophilus that carried either pTRZll7 or pTRZ90 could grow at 65 "C