PERSISTENCE AND STABILITY OF GENETICALLY MANIPULATED DERIVATIVES OF ENTEROBACTER-AGGLOMERANS IN SOIL MICROCOSMS

Molecular methods and conventional plating were applied to monitor Ent erobacter agglomerans 339 derivatives carrying a Tn5-Mob or an nptI-ca ssette in unsterile soil microcosms. The plate counts of the introduce d bacteria decreased continuously in time until undetectable on select ive media. In contrast, hybridization of the total DNA directly isolat ed from inoculated soil samples showed that the target sequences detec ted corresponded to a much higher number of bacteria than indicated by plating. By PCR-amplification and hybridization of the soil DNA we co uld show that a significant number of target sequences still persisted in the soil microcosms, even when the inoculated bacteria were not ab le to make colonies on selective agar plates. The Tn5 marker caused in stabilities in the genome of the bacteria studied. Some of the clones that grew in the soil samples had rearrangements in their genome. The detection of E. agglomerans 339 derivatives carrying the immobile nptI -cassette was also dependent on its location in the bacterial genome.