A mutagenesis system utilizing a Tn1722 derivative containing an Escherichia coli-specific vector plasmid:...

A novel transposon (Tn) mutagenesis system for Gram- non-enteric bacteria was developed which allowed rapid and one-step cloning of the mutated region in Escherichia coli. The Tn constructed was Tn1722-299Km, a Tn1722 derivative containing a KmR gene and the entire sequence of an E. coli-specific plasmid, pACYC184. The hybrid plasmid consisting of Tn1722-299Km and the transfer genes of plasmid R388 was conjugally transferred from E. coli to Pseudomonas putida or P. aeruginosa, and selection of the transconjugants expressing the Tn-specified resistance genes led to isolation of insertion mutants of the recipient strain. The presence of the pACYC184 replicon in the Tn greatly facilitated rapid and easy cloning of the mutated region in E. coli through (i) mini-scale preparation of the genomic DNA from the Tn-inserted mutant, (ii) digestion of the DNA with an appropriate restriction endonuclease, (iii) self-ligation, and (iv) transformation of E. coli to recover the plasmid carrying the Tn-specified resistance marker. This procedure was successfully adapted to clone the Tn-inserted trpBA region of P. putida. Such a cloned region was further employed to isolate the wild-type allele of the trpBA region without construction of a genomic library.