DEVELOPMENT OF ENGINEERED GENOMIC DNA TO MONITOR THE NATURAL TRANSFORMATION OF PSEUDOMONAS-STUTZERI IN SOIL-LIKE MICROCOSMS

The goal of this paper was to demonstrate whether natural transformati on 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 transformat ion of Pseudomonas stutzeri by mineral-adsorbed DNA. Genes conferring resistance to tetracycline and ampicillin were first inserted in P. st utzeri pp100 chromosome via the pSUP202 suicide plasmid. Then, DNA ext racted from the engineered P. stutzeri strain was used for transformat ion experiments, allowing the new transformed cells to be detected by hybridization with a tet probe. It turned out that DNA adsorbed on cla y or soil particles and in presence of humic acids still transformed c ompetent cells with frequencies up to 10(-8) transformants/viable cell . Finally, natural transformation assays involving two different DNAs were carried out in sterile soil microcosms. The use of nonisogenic DN A 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 envi ronmental conditions.