Molecular methods and conventional plating were applied to monitor Enterobacter agglomerans 339 derivatives carrying a Tn5-Mob or an nptI-cassette in unsterile soil microcosms. The plate counts of the introduced bacteria decreased continuously in time until undetectable on selective media. In contrast, hybridization of the total DNA directly isolated from inoculated soil samples showed that the target sequences detected corresponded to a much higher number of bacteria than indicated by plating. By PCR-amplification and hybridization of the soil DNA we could show that a significant number of target sequences still persisted in the soil microcosms, even when the inoculated bacteria were not able to make colonies on selective agar plates. The Tn5 marker caused instabilities 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.
StrainPlasmid (s) Chromosomal marker(s) References E. coli S17-1 pSUP5011 16 E. coli K-12 JM83 pUC4K 33 E. agglomerans 19-1-1 pEA9::Tn5-Mob Rif r, Nal r, Krnr/Nm r. The Kmr/Nm r was 5, 37 due to two copies of Tn5-Mob Rif r, Str r Rif r, Km r, SaC. The Km r and Sac r were due to an nptI-sacB-sacR cassette Rifr Rifr E. agglomerans 339-E. agglomerans 339CR1