Degradation of plasmid and plant DNA in water microcosms monitored by natural transformation and real-time polymerase chain reaction (PCR)

Abstract

Extracellular DNA exists in the environment and can be taken up by competent bacterial cells, leading to horizontal gene transfer. The persistence of extracellular plasmid and plant DNA in water microcosms was monitored in this study. Water samples were two groundwater (GW1 and GW2) and one river water (RW) samples. Three treatments included: (1) intact, (2) 0.22 μm filter-sterilized, and (3) autoclaved water. DNA from a plasmid (pNS1) and a transgenic Bt ( Bacillus thuringiensis) corn line, both carrying a neomycin phosphotransferase gene ( nptII gene) conferring kanamycin and neomycin resistance, was inoculated into the microcosms at 0.4 and 0.8 μg/ml, respectively. By monitoring its ability to transform a competent Pseudomonas stutzeri strain harboring plasmid pMR7 ( P. stutzeri pMR7), plasmid DNA was degraded to undetectable levels in the intact and/or filter-sterilized water treatments within 48–96 h in GW1, GW2, and RW. Meanwhile, plasmid DNA persisted in the autoclaved treatment throughout the entire incubation period. For plant DNA, a highly sensitive real-time PCR method using SYBR Green I was developed to monitor the degradation dynamics of the nptII gene carried by the transgenic corn line in the microcosms. The results showed that the concentration of plant DNA was reduced by two orders of magnitude (from 0.8–0.008 μg/ml) within 96 h in the intact and filter-sterilized treatments of GW1, GW2, and RW, in contrast to its persistence in the autoclaved treatment. In addition, no kanamycin resistant (Km R) transformants were detected from in situ transformation of P. stutzeri pMR7 with plasmid pNS1 DNA.