Selection of antibiotic hypersusceptible mutants in bacteria

We have designed a new genetic strategy, named Selection for DNA Release (SDR), which can be applied for direct selection of conditional lethal mutants. In the basic form of SDR, a library of random insertional mutants is subjected to experimental conditions. The DNA collected from dying mutants is used to transform wild-type bacteria, selecting for insertion of the marker gene. After several cycles of SDR, the desired mutants, which die under a given experimental condition, begin to dominate in the library. As one of the applications of SDR, this strategy was used to select ampicillin-hypersusceptible mutants of Acinetobacter sp. by treating the library of random insertional mutants with an antibiotic at 1/10 of MIC.; The simple SDR is applicable to only a few experimental conditions that cause cell lysis and the release of DNA. We have broadened its applicability by creating a reporter strain, which lyses upon inhibition of translation. This was achieved by placing the lysis cassette of bacteriophage λ under the control of an rRNA promoter in Acinetobacter sp., which is activated by translational inhibitors such as erythromycin and tetracycline. Application of SDR strategy to this strain allowed for selecting mutants hypersusceptible to erythromycin. Antibiotic hypersusceptibility mutations discovered using SDR can identify bacterial targets for potentiators of antibiotic actions.