Tn 1 insertion mutagenesis in Escherichia coli K-12 using a temperature-sensitive mutant of plasmid RP4

A method for Tn 1 insertion mutagenesis in Escherichia coli has been developed using pTH10, a mutant plasmid of RP4 temperature-sensitive for maintenance. The mutagenesis involves three steps. Firstly, from strains carrying pTH10 showing resistance to the antibiotics kanamycin, tetracycline, and ampicillin at 30° C but not at 42° C, clones are isolated resistant to kanamycin at 42° C. Such temperature-independent, drug resistant clones probably carry pTH10 integrated into the host chromosome. Secondly, they are cultivated at 30° C. At this temperature segregants carrying pTH10, which has been excised from the host chromosome, accumulate. Thirdly, to cure such segregants of autonomous pTH10, they are cultivated at 42° C. By these procedures, clones free of pTH10, but carrying Tn 1 insertions on the host chromosome, were obtained. About 3% of the clones carrying Tn 1 insertions were auxotrophic. Distribution of auxotrophic mutations was not random, indicating the existence of preferential integration sites of Tn 1 on the host chromosome. The frequency of precise excision of Tn 1 was less than 10^-10. The pTH10 plasmid has a wide host range among Gram-negative bacteria and thus may serve as a excellent vector for insertion mutagenesis of Tn₁ in many Gram-negative bacterial species.