Development of engineered genomic DNA to monitor the natural transformation ofPseudomonas stutzeriin soil-like microcosms

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Abstract
The goal of this paper was to demonstrate whether natural transformation could occur in the environment to promote horizontal gene transfer between bacteria. Microcosms consisting of clay, clay and humic acids, or sterile soil were compared with respect to the natural transformation of Pseudomonas stutzeri by mineral-adsorbed DNA. Genes conferring resistance to tetracycline and ampicillin were first inserted in P. stutzeri pp100 chromosome via the pSUP202 suicide plasmid. Then, DNA extracted from the engineered P. stutzeri strain was used for transformation experiments, allowing the new transformed cells to be detected by hybridization with a tet probe. It turned out that DNA adsorbed on clay or soil particles and in presence of humic acids still transformed competent cells with frequencies up to 10−8transformants/viable cell. Finally, natural transformation assays involving two different DNAs were carried out in sterile soil microcosms. The use of nonisogenic DNA extracted from a rifampicin-resistant Pseudomonas fluorescens strain resulted in production of transformants, while isogenic DNA from our engineered strain failed to produce any. These observations confirmed that extracellular DNA adsorbed on a soil matrix composed of minerals and organic matter could still transform competent bacteria under environmental conditions.Key words: transformation, Pseudomonas stutzeri, soil microcosm, DNA, suicide plasmid.