| Using transcriptional fusions between degradative pathway promoters and the luxCDABE cassette from Vibrio fischeri, bioluminescence was a measure of degradative gene expression in response to specific chemical inducers. A specialized cloning plasmid, pLJS in conjunction with a modified mini-Tn5 transposon delivery system allowed for sequential assembly of genetic constructs for introduction into the bacterial chromosome. Three different promoter-lux fusions were constructed and inserted into their respective hosts to create nah-lux, tod-lux and xyl-lux reporter strains. Control strains were used to distinguish between a specific effect of the genetic sensing system and other non-specific influences such as factors affecting the supply of aldehyde substrate for the luciferase reaction. Mid-exponential phase cells were exposed to test compounds and bioluminescence was measured over time in whole cells without added substrate, aldehyde.; The bioluminescence response of chromosomally-based nah-lux reporter Pseudomonas fluorescens 135R75 to naphthalene was compared with that of other nah-lux reporter strains P. fluorescens HK44 and P. putida RB1351. In addition, the effect of two alkyl ethoxylate surfactants, Witconol SN-70 and Poly-Tergent SL-62, and a rhamnolipid biosurfactant R1 to solubilize naphthalene from artificially and naturally contaminated soil was evaluated. Bioluminescence was shown to be a good indicator of naphthalene bioavailability in surfactant/ or biosurfactant/soil wash solutions. The light response was correlated with solubilized naphthalene concentration over a large range, to 110 mg/L. Chromosomally-based reporter systems were genetically more stable, had lower background bioluminescence levels and responded more consistently to inducer concentration than their plasmid-based or pathway-insertion counterparts.; Reporter strains were used to determine the range of substrates that would induce the specific pathway. The tod-lux reporter P. putida TVA8 responded to BTEX compounds, phenol and water-soluble JP-4 jet fuel components that are mainly BTEX compounds. Response to toluene or JP-4 components was proportional to concentration, showing saturation-type behavior at higher concentrations. The chromosomally-based reporter, TVA8 showed higher bioluminescence responses than the plasmid-based tod-lux reporter, P. putida B2. The tod-lux reporter TVA8 was more sensitive to toluene than the xyl-lux reporter in growing cell assays showing a detection limit for toluene of {dollar}<{dollar}50 {dollar}mu{dollar}g/L that was 80-fold above background. |
