A double kill gene cassette for the positive selection of transforming non-selective DNA segments in Acinetobacter baylyi BD413.

Natural transformation has been widely used for the monitoring of DNA in biological and environmental samples. These assays depended on selectable traits on the tested DNA. We have now developed a transformation assay system in which recombinational removal of a cassette with two conditional kill genes (hok and sacB) from the recipient genome provides positive selection for non-selective DNA. The cassette was integrated into the Acinetobacter baylyi BD413 chromosome within trpE and was flanked by two segments of non-selective test DNA, which in this study were from a T-DNA construct previously used to generate a transgenic potato. Genes for tetracycline and spectinomycin/streptomycin resistance located at the sides of the cassette allowed to maintain selection pressure against spontaneous loss of the cassette. Plasmid DNA containing the T-DNA gave transformation frequencies ranging linearly from 10(-4) per recipient (at 1 mug DNA ml(-1)) down to 10(-7) (1 ng DNA ml(-1)) by selecting for survivors after activation of both kill functions. Transformation depended on the two flanking homologous segments for recombinational exchange. DNA of the transgenic potato also gave positive scores in spite of the about 10(5)-fold dilution of T-DNA by potato DNA. False positives having a spontaneous deletion of hok and sacB occurred at a frequency of 1.8x10(-9) per cell but could be distinguished by PCR from real transformants. Thus, the system is suitable for detection of transformation frequencies down to about 10(-9). Hok and sacB as well as the regulatory system used (LacI-lac operator and T5 promoter) are known to function in many organisms suggesting wide applicability of the cassette for positive selection.