Plasmid carrying the temperature-sensitive mutation in the DNA-methylase gene of the pst1 system: effect on host cells at nonpermissive temperature

摘要

Temperature-sensitive derivatives of plasmid pRMPI, the derivative of PBR322 containing restriction and modification genes of Pst system. were obtained using hydroxylamine mutagenesis. One of the isolated plasmids responsible for the inhibition of Escherichia coli cell growth at 42 deg C, pRMPts, was analyzed in this work. Cells of Rec+ strains carrying this plasmid were unable to divide at 42degC and formed long nonseptated filaments that died upon prolonged cultivation. Cells of the RecA~-strains carrying pRMPts did not form filaments at 42 degC and rapidly disappeared. On agar media with or without ampicillin, Rec~+ and RecA~- strains with this plasmid formed colonies of temperature-resistant derivatives with frequencies ranging from 1.5x10~-4 to 4x10~-6 in independent clones. The structure of plasmids from cells of tr-derivatives of Rec~+ and RecA~- strains carrying plasmid pMRPts was analyzed by the set of restriction enzymes. (1)the insertional inactivation of the PstI restriction enzyme gene in pRMPts;(2)deletions in plasmid DNA fragments that partially or completely cover the restriction enzyme gene; (3) point mutations (4)others. High efficiency loss of the plasmid was detected in pRMPts-carrying Rec~+ cells with the sulA::Tn5 mutation grown in liquid and solid nutrient media at this temperature. Under similar conditions, plasmid loss was not detected in SulA~+ cells. Mutant DNA methylase was unable to methylate all sites in the chromosomal DNA at 42 degC. Some of the unmethyated sites can be digested with the PstI enzyme. Which leads to the induction of SOS response in Rec~+ cells or to total mortality in cells with the recA phenotype.