A SPECIFIC MARKER, PAT, FOR STUDYING THE FATE OF INTRODUCED BACTERIA AND THEIR DNA IN SOIL USING A COMBINATION OF DETECTION TECHNIQUES

A specific eucaryotic DNA marker from Solanum tuberosum cv Bintje (688 bp patatin cDNA fragment) was cloned into the unique HindIII-site of plasmid RP4. RP4:: pat was transferred from Escherichia coli to Pseudomonas fluorescens R2f by filter mating. Homology to pat was not detected in the microbial population of Ede loamy sand soil, nor in that of the rhizosphere of wheat growing in this soil, as evidenced by colony filter hybridization. More sensitive molecular detection techniques like most-probable-number recovery/hybridization analysis, and analysis of total community DNA from soil by polymerase chain reaction (PCR) amplification did not reveal the presence of the pat sequence either. P. fluorescens R2f (RP4:: pat), introduced into sterile soil extract microcosms, initially showed poor survival and plasmid loss, after which the introduced populations grew and stabilized at a level of about Log10 7 cfu per mL. Between 25 and 50% of the population maintained the plasmid, as evidenced by filter hybridization of colonies from non-selective agar plates using the pat fragment as probe. Introduced R2f (RP4:: pat) could be recovered from soil microcosms using selective plating followed by colony hybridization and MPN recovery/hybridization with the pat probe. The presence of the pat marker always coincided with the presence of the resistance genes on RP4:: pat, indicating pat was an adequate marker of the presence of this plasmid. In addition, it adequately described the population dynamics of the introduced strain in soil, since no loss of the plasmid occurred. Hybridization to pat was also useful to show transfer of plasmid RP4:: pat to a recipient strain in soil; transfer to indigenous bacteria was not detected. Analysis by slot-blot hybridization of total community DNA extracted from inoculated soils indicated about Log10 6 cfu per g of dry soil were still detectable. Application of the PCR on this DNA indicated pat was detectable at least at a level of Log10 4 immunofluorescence-detectable cells per g of dry soil. Thus extraction of total community DNA followed by PCR permitted the detection of genetically engineered microorganisms present in soil as non-culturable cells.