Doctoral Dissertations

Date of Award

5-1998

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Gary S. Sayler

Committee Members

Paul Bienkowski, Dwayne Savage, David White

Abstract

This study describes the construction and application of chromosomally-based bioluminescent reporter strains to monitor monocyclic and polycyclic aromatic hydrocarbon (PAH) bioavailability and biodegradation. 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-/i«: fusions were constructed and inserted into their respective hosts to create ah-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. Since the complete lux cassette was used, bioluminescence was measured in whole cells without added substrate, aldehyde. Mid-exponential phase cells were exposed to test compounds and bioluminescence was measured over time.

The nah-lux fusion was inserted into Pseudomonas fluorescens 5R yielding strain 135R75. The bioluminescence response of this strain to naphthalene was compared with that of other nah-lux reporter strains, P. fluorescens HK44 (nahG-lux) and P. putida RBI 351 (pUTK9, P &subnah;-lux). 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 using 135R75. 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, approximately 9 to 110 mg/L.

Reporter strains showed the utility to determine the range of substrates that would induce the specific pathway. The tod-lux reporter P. putida TVA8 responded to BTEX compounds (benzene, toluene, ethylbenzene, xylenes), phenol and water-soluble JP-4 jet fuel components which 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 <50 μg/L compared to <5 mg/L. The response for TVA8 at 50 μg/L toluene was 80-fold above background.

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. Experiments with control strains and addition of aldehyde (to saturate the bioluminescence reaction) showed that solvents and surfactants were having additional positive effects on bioluminescence production independent of gene induction. The control strain showed the utility to account for non-specific influences on bioluminescence. Other applications of transposon constructs generated in this study included nahR promoter and ahR-P&subsal; expression transposon vectors for creating transcriptional fusions with genes for which constitutive or salicylate-inducible expression, respectively, is desired.

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