Luminescence based detection of genetically modified Pseudomonas fluorescens in soil
Methods currently available for the detection and enumeration of genetically modified micro-organisms in the environment include culturing methods, direct microscopic detection and nucleic hybridization techniques. The aim of this project was to develop luminescence as a molecular based-marker system in Pseudomonas fluorescens. The lux genes, originally isolated from Vibrio fischeri, were introduced into Ps. fluorescens on plasmid vectors and on the chromosome. The efficiency of these two strategies for the detection of Ps. fluorescens in soil was assessed. Luminometry was used to estimate biomass concentration during growth. The sensitivity of luminescence detection was greater for the plasmid marked Ps. fluorescens in both liquid culture and soil, however, cellular light output was less closely linked to biomass concentration. Enumeration of cells by luminometry was only possible for growing cells as light output is correlated with microbial activity. The lux chromosomal marker was stable in liquid culture for at least 200 generations and in soil for up to 135 days. The plasmid borne lux genes had a half-life of 20 generations in liquid culture. After inoculation in sterile soil, plasmid loss was only observed during cellular growth. The frequency of transfer of the lux genes from Ps. fluorescens, by conjugation and transformation, was assessed in liquid culture. Mobilisation of these genes by three self transmissible plasmids was negligible due to the instability of these vectors in this host. Transformation of Ps. stutzeri with lux genes, by cell contact, was at frequencies below levels of detection. Luminescence provided a valuable marker for tracking pseudomonads in soil. Detection of marked strains by luminometry provided a sensitive, rapid and non-extractive technique for enumeration of growing cells and measurement of microbial activity. As the chromosomally encoded lux genes were stable, regardless of growth conditions, and emitted sufficient levels of light to enable visual enumeration of colonies by eye, this was considered the best system for long term risk assessment studies.