Bacterial Bioluminescence and Gene Probes in Optimizing the Maintenance and Expression of Biodegradative Plasmids in Groundwater

摘要

Reporter gene fusion technology and DNA hybridization gene probing techniques were developed and applied for quantifying the maintenance and activity of biodegradative bacteria of use in groundwater bioremediation and restoration. Transcriptional gene fusion using the bioluminescent genes (lux) of Vibrio fischeri were made with naphthalene catabolic genes (nah) of the NAH7 naphthalene catabolic plasmid harbored in Pseudomonas spp. The nah-lux gene fusions resulted in the development of bioluminescent reporter strains of Pseudomonas that were inducible for light production in the presence of naphthalene or the metabolic intermediate, salicylate. Light production was shown to be a direct response to naphthalene exposure, bioavailability, and biodegradation of naphthalene in contaminated water, subsurface sols, and in soil slurry treatment reactor systems. Using a combination of light response, antibiotic resistance patterns and, a gene probe specific for naphthalene catabolism, both the unmodified naphthalene degrading Pseudomonas strains and the nah-lux reporter strains could be competitively maintained in continuous culture groundwater simulations in excess of 50 days.