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(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 pTRA117could grow at 63°C in medium containing noulin carrying ptra117could grow at 63C in medium containat.Although pTRA117was 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 from63°C，a new plasmid(10.7 MDa；penP kan)，designated pTRZ117，could be detected as ccc DNA；the size this plasmid(Ccc)；Kan)was constructed from pTRZ117 by the deletion of a2.8MDA fragment that contained penP.Fresh transformants of B.stearothermophilus that carried either pTRZ117or pTRZ90could grow at65°C.

A transformant of Bacillus stearothermophilus carrying a recombinant plasmid,pLP11(9.5MDa), on which the penicillinase gene (penP)and kanamycin resistance (Kan)were located was subjected to mutagenesis,and a mutant plasmid(9.5MDa;penP kan),designated pTRA117,was obtained.A transformant of B.stearothermophilus carrying pTRA117 could grow at 63゜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゜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 (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 1.2MDa DNA fragment of the host chromosome,and this was confirmed by Southern hybridization.pTRZ90(7.9MDa; kan) was constructed from pTRZ117 by the deletion of a 2.8 MDA fragment that contained penP. Fresh transformants of B.stearothermophilus that carried either pTRZ117 or pTRZ90 could grow at 65゜C.