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

Temperature-sensitive (ts) derivatives of plasmid pRMP1, the derivative of pBR322 containing restriction and modification (RM) genes of the PstI syste m, were obtained using hydroxylamine mutagenesis. One of the isolated plasm ids responsible for the inhibition of Escherichia coli cell growth at 42 de grees C, pRMPts, was analyzed in this work. Cells of Rec(+) strains carryin g this plasmid were unable to divide at 32 degrees C and formed long, nonse ptated filaments that died upon prolonged cultivation. Cells of the RecA(-) strains carrying pRMPts did not form filaments at 42 degrees C and rapidly disappeared. On agar media with or without ampicillin, Rec(+) and RecA(-) strains with this plasmid formed colonies of temperature-resistant (tr) der ivatives with frequencies ranging from 1.5 x 10(-4) to 4 x 10(-6) in indepe ndent clones. The structure of plasmids from cells of tr-derivatives of Rec (+) and RecA(-) strains carrying plasmid pRMPts was analyzed by the set of restriction endonucleases. Reversions to the temperature-resistant phenotyp e were shown to result from the following events: (1) the insertional inact ivation of the PstI restriction endonuclease gene in pRMPts (the insertion of the ISI element); (2) deletions in plasmid DNA fragments that partially or completely cover the restriction endonuclease gene; (3) point mutations; and (4) others. The effect of the chromosomal sulA mutation on the mainten ance of the ts-plasmid in bacterial cells was studied at 42 degrees C. High efficiency loss of the plasmid was detected in pRMPts-carrying Rec(+) cell s with the sulA: :Tn5 mutation grown in liquid and solid nutrient media at this temperature. Under similar conditions, plasmid loss was not detected i n SulA(+) cells. On the basis of the data obtained, it is concluded that th e ts-mutation is located in the DNA-methylase gene of plasmid pRMPts. Mutan t DNA methylase was unable to methylate all sites in the chromosomal DNA at 42 degrees C. Some of the unmethyated sites can be digested with the PstI endonuclease, which leads to the induction of SOS response in Rec(+) cells or to total mortality of cells with the recA phenotype.