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

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 tomutagenesis,and a mutant plasmid(9.5MDa；penP kan),designated pTRA117,was obtained.A transformant of B.stearothermophilus carrying pTRA117could grow at 63 C in medium containing kanamycin,whereas a transformant carrying pLP11 could not.虽然pTRA117 was detected as covalently closed circular(ccc)DNA when it was extracted fromtransformants cultured at 48c,it was integrated into the host chromosome when the cultureculture temperature was lowered to 48c from 63c,a newplasmid (10.7 MDa;penP kan),designated pTRZ117,could be detected as ccc DNAthe size of this plasmid suggested that it was pTRA117 plus a1.2 MDa DNA fragment of the hostchromosome,and this was confirmed by Southern hybridization.pTRZ90(7.9MDa；was constructed from pTRZ117 by the deletion of a 2.8 MDA fragment that contained penP. Freshtransformants of B.stearothermophilus that carried either pTRZ117 or pTRZ90 could grow at 65 - 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.