Transformation of Acinetobacter sp. strain BD413(pFG4DeltanptII) with transgenic plant DNA in soil microcosms and effects of kanamycin on selection of transformants

Abstract

Here we show that horizontal transfer of DNA, extracted from transgenic sugar beets, to bacteria, based on homologous recombination, can occur in soil. Restoration of a 317-bp-deleted nptII gene in Acinetobacter sp. strain BD413(pFG4) cells incubated in sterile soil microcosms was detected after addition of nutrients and transgenic plant DNA encoding a functional nptII gene conferring bacterial kanamycin resistance. Selective effects of the addition of kanamycin on the population dynamics of Acinetobacter sp. cells in soil were found, and high concentrations of kanamycin reduced the CFU of Acinetobacter sp. cells from 10(9) CFU/g of soil to below detection. In contrast to a chromosomal nptII-encoded kanamycin resistance, the pFG4-generated resistance was found to be unstable over a 31-day incubation period in vitro.

FIG. 1

Natural transformation and restoration of a 317-bp internal deletion in the nptII gene in Acinetobacter sp. strain BD413(pFG4) cells incubated on nitrocellulose filters with increasing concentrations of transgenic sugar beet DNA. T bars, standard deviations.

FIG. 2

PCR amplification of the Tn5 region 1236 to 2929, which includes the nptII promoter, the complete nptII gene, and the bleo gene of pFG4, demonstrating the presence of the restored nptII gene construct in the transformant colonies and the 317-bp partially deleted gene in the recipient bacterium. Lane 1 from the left side, 1-kb ladder (Gibco-BRL); lane 2, Acinetobacter sp. strain BD413(pFG4) Km^s; lane 3, Acinetobacter sp. strain BD413(pFG4) Km^r; lane 4, Acinetobacter sp. strain BD413(pFG4) Km^s recipient; lane 5 to 16, Acinetobacter sp. strain BD413(pFG4) Km^r transformants obtained with transgenic nptII-containing sugar beet DNA.

FIG. 3

Stability of KM-resistant phenotypes of Acinetobacter sp. strain BD413(pFG4::nptII) (circles) and Acinetobacter sp. strain BD413 chr::nptII (triangles) in liquid LB medium measured after plating on KM-free (open symbols) and KM-containing (filled symbols) LB medium. T bars, standard deviations. CFU are per 5 ml.

FIG. 4

CFU of KM-sensitive recipients (open symbols) and KM-resistant transformants (filled symbols) of Acinetobacter sp. strain BD413(pFG4) isolated from soil after addition of increasing amounts of kanamycin to soil microcosms (a) or liquid soil suspensions (b). T bars, standard deviations. CFU are per microcosm of 1.2 g (dry weight) of soil.