Analysis of the occurrence of natural transformation of Azospirillum brasilense with DNA from plants containing a kanamycin resistance gene

Abstract

There is a considerable concern about the possibility of a horizontal transfer of antibiotic resistance genes from genetically modified crops to soil bacterial community. One mechanism of gene transfer in bacteria that allows uptake of genetic material from diverged species is the natural transformation. In this work, the recipient strain for natural transformation was Azospirillium brasilense, a nitrogen fixing bacteria. Tobacco transgenic plants containing the nptII gene (kanamycin resistance) were used as donor of the transgenic material. A 12 months period of contact between the transgenic plants and the bacteria was evaluated each two months by PCR analysis of the DNA extracted from the bacterial pellicle grown on selective NFbHP medium. The PCR reactions were performed with oligonucleotides for nptII and nifH genes. No amplification of nptII gene was observed until the end of the experiment. A restriction analysis with TaqI of the purified DNA fragments of nifH PCR was performed to confirm the presence of A. brasilense in the cultures. The bacteria were also spread on NFbHP plates containing kanamycin and no colonies were observed after 72 h of growth. On the contrary, on the plates containing only ampicilin the colonies were similar to those obtained with the control plates. The results obtained could be useful to develop a protocol to evaluate the effects of transgenic plants on the soil microorganisms.