FIG. 4.
Insertion of the upp TOL catabolic segment into the chromosome of P. putida KT2442 and deletion of selection markers. The organization of pCK05, the delivery plasmid for the mobile element mini-Tn5 [upp TOL], is shown at the top (the vector part is not to scale). It includes all of the features of pJMS11 (Fig. 3) plus the NotI insert of pCK04AxylR (upp TOL segment) within the boundaries of the I and O ends of Tn5. This plasmid was mobilized to P. putida KT2442, and the insertion of the mini-Tn5 [upp TOL] transposon was selected with kanamycin and verified upon spraying of the exconjugants with catechol. In a subsequent step, the Kmr/xylE markers were deleted from P. putida KT2442::mini-Tn5 [upp TOL] by mobilization of the suicide plasmid pJMSB8 (which bears the parA sequence downstream of lacI/Plac). The final result is stable inheritance of the remainder of the hybrid transposon, i.e., mini-Tn5 [upp TOL] Δ[Kmr/xylE].