Bioluminescent most-probable-number monitoring of a genetically engineeredbacterium during a long-term contained field release

Pseudomonas fluorescens HK44 is a lux-based bioluminescent bioreporter capa ble of emitting light upon exposure to naphthalene, salicylate, and other s ubstituted analogs. The bacterium was inoculated into intermediate-scale fi eld lysimeters and population dynamics were monitored with time. Two method s were used to enumerate cell numbers in soil: a standard selective plating technique with colony hybridization verification and a modified flux-based most-probable-number (lux-MPN) assay based on the detection of bioluminesc ence. The lux-MPN assay was developed and evaluated as a possible supplemen t or replacement for the labor-intensive and time-consuming selective plati ng assay. Comparisons between selective plate counts and lux-MPN population estimates showed similar trends over the 2-year study, except that lux-MPN estimates were consistently less than selective plate counts. Verification of P. fluorescens HK44 genotype through colony hybridization techniques re vealed that selective plating was actually overestimating HK44 populations and that lux-MPN values were more closely approximating true HK44 cell dens ities, except within the first few weeks after inoculation, when lux-MPN es timates underrepresented population densities. Thus, utilizing bioluminesce nce as a population monitoring tool for lux-based microorganisms was shown to be more effective and precise than standard selective plating techniques , and provided an accurate ecological analysis of P. fluorescens HK44 popul ation dynamics over an extended period.