High frequency mobilization of the chromosome of Escherichia coli by a mutant of plasmid RP4 temperature-Sensitive for maintenance

Two mutants of plasmid RP4 temperaturesensitive for maintenance were isolated and one of them (pTH 10) was extensively studied. Cells carrying pTH 10 showed temperature-sensitive drug resistance from which we isolated a number of temperature-independent derivatives. Almost all of them were Hfrs donating chromosomal genes to recipient bidirectionally from different points of origin. The Hfrs may be formed in two steps: (1) the transposon (Tn 1) carried by pTH 10 translocates into the host chromosome, and (2) pTH 10 is integrated in the host chromosome by reciprocal recombination between the Tn 1 s, one situated on pTH 10 and another on the host chromosome. That temperature-independent drug resistance selects for this type of derivative, was supported by the following observations: (1) Hfrs thus obtained were usually unstable and segregated at high frequency 'revertants' showing temperature-sensitive drug resistance when they were cultivated at 30° C. (2) The 'revertants', cured of pTH 10 were still ampicillin resistant, indicating existence of Tn 1 inserted in the host chromosome. (3) Tn 1 insertions found in these derivatives mapped in the vicinity of points of origin of the original Hfrs. (4) When new Hfrs were constructed by: (a) transduction with Pl kc of Tn 1 insertions found in derivatives of Hfrs, (b) introduction of pTH 10 into the transductants, and (c) isolation of clones of temperature-independent drug resistance from such pTH 10 carrying strains, they had similar characteristics to the original Hfrs from which Tn 1 insertions were derived. Possibilities for genetic manupulation using pTH 10 in a wide range of Gram-negative bacteria are discussed.