Transposon vectors for stable chromosomal integration of cloned genes in rhizosphere bacteria

A series of Tn5-based transposon-cloning vectors, in which many unique restriction sites lie within the transposon, have been constructed. These transposon vectors can be delivered, by conjugation, using a delivery vehicle containing a pBR322 replicon and the mobilization genes of plasmid RK2. In Pseudomonas sp., this delivery vehicle acts as a suicide plasmid, permitting transposition to the chromosome to be detected. To facilitate cloning into the transposon vector, the delivery vehicle has been simplified so that the useful cloning sites in the transposon are not duplicated. As a model system for the transposition of cloned genes, the xylE (coding for catechol-2,3-dioxygenase) has been transposed to a variety of Pseudomonads. The transposon vectors should be useful when a stable single copy of a cloned gene is desired. They should be particularly advantageous for the genetic engineering of soil bacteria for environmental studies (agriculture or pollution control) where the stability of the engineered strains, in the absence of continuous antibiotic selection, may be important.