Factors Influencing Expression of luxCDABE and nah Genes in Pseudomonas putida RB1353(NAH7, pUTK9) in Dynamic Systems

Abstract

ABSTRACT Bioluminescent reporter organisms have been successfully exploited as analytical tools for in situ determination of bioavailable levels of contaminants in static environmental samples. Continued characterization and development of such reporter systems is needed to extend the application of these bioreporters to in situ monitoring of degradation in dynamic environmental systems. In this study, the naphthalene-degrading, lux bioreporter bacterium Pseudomonas putida RB1353 was used to evaluate the relative influences of cell growth stage, cell density, substrate concentration, oxygen tension, and background carbon substrates on both the magnitude of the light response and the rate of salicylate disappearance. The effect of these variables on the lag time required to obtain maximum luminescence and degradation was also monitored. Strong correlations were observed between the first three factors and both the magnitude and induction time of luminescence and degradation rate. The maximum luminescence response to nonspecific background carbon substrates (soil extract broth or Luria broth) was 50% lower than that generated in response to 1 mg of sodium salicylate liter −1 . Oxygen tension was evaluated over the range of 0.5 to 40 mg liter −1 , with parallel inhibition to luminescence and degradation rate (20 mg of sodium salicylate liter −1 ) observed at 1.5 mg liter −1 and below and no effect observed above 5 mg liter −1 . Oxygen tensions from 2 to 4 mg liter −1 influenced the magnitude of luminescence but not the salicylate degradation rate. The results suggest that factors causing parallel shifts in the magnitude of both luminescence and degradation rate were influencing regulation of the nah operon promoters. For factors that cause nonparallel shifts, other regulatory mechanisms are explored. This study demonstrates that lux reporter bacteria can be used to monitor both substrate concentration and metabolic response in dynamic systems. However, each lux reporter system and application will require characterization and calibration.