Triparental conjugation.

Transfer of plasmids from E. coli to P. putida strains was performed by triparental mating using E. coli(pRK600) as a helper, as previously described (55). For each strain, 0.5 ml of overnight cultures grown in LB medium was collected by centrifugation, resuspended in 50 μl of fresh LB medium after several washing steps, and spotted on a nitrocellulose filter (0.22-μm pore diameter) on LB agar plates, which were incubated overnight at 30°C. Then, the cells were scraped off the mating filter and resuspended in 1.5 ml of diluted M9 salts medium. Serial dilutions were plated on selective LB medium supplemented with the appropriate antibiotics to select exconjugants and counterselect donor, helper, and recipient strains.