CHROMOSOMAL INSERTION OF THE ENTIRE ESCHERICHIA-COLI LACTOSE OPERON, INTO 2 STRAINS OF PSEUDOMONAS, USING A MODIFIED MINI-TN5 DELIVERY SYSTEM

A 12-kb PstI fragment including the entire E. coli lactose operon (lac IPOZYA) was inserted in one copy into the chromosome of Pseudomonas pu tida, Pseudomonas fluorescens and an E. coli strain with lac(-) phenot ype. This was made possible by improvements of an already existing min i-Tn5 transposon delivery system (de Lorenzo et al., 1990; Herrero et al., 1990), which integrates cloned DNA fragments at random sites on t he chromosome of the recipient bacteria in single copies. This has res ulted in: (a) the making of two useful low copy-number cloning vectors both with extensive multi-cloning regions flanked by NotI sites neede d in the mini-Tn5 delivery system; (b) the generation of E. coli nonly sogenic strains expressing the pi protein thus being capable of mainta ining and delivering R6K-based mini-Tn5 vectors to other E. coli strai ns; (c) the successful insertion of the E. coli lactose operon into th e P. fluorescens chromosome giving P. fluorescens the ability to grow on lactose; (d) evidence from Southern blotting that contradicts the a ssumption that the mini-Tn5 delivery system always creates one-copy in serts. These improvements allow insertion of large DNA fragments encod ing highly expressed proteins into the chromosome of a large variety o f Gram-negative bacteria including E. coli.