Construction, characterization, and application of the bioluminescent bioreporter Pseudomonas fluorescens M3A
Abstract
Whole-cell bioluminescent bioreporters have been used in a wide-variety of assays and studies, including monitoring of cellular toxicity, bioavailability of pollutants, and DNA-damaging agents. These systems are attractive in that they are noninvasive, highly sensitive, can quantify/relate gene expression, can be measured in real-time, and are a measure of enzymatic activity. Accordingly, this study aimed at constructing a bioluminescent bioreporter to assess the physiological effects of environmental conditions on cells. A bioluminescent reporter, Pseudomonas fluorescens M3A, was constructed from P. fluorescens strain Migula and examined in a variety of systems, which include: environmental toxicity from pollutants and contaminants, host/bacteriophage kinetics, and food-matrix interactions. Commonly associated toxic compounds, such as toluene and phenol, were demonstrated to have a "solvent effect" on bioluminescent reporters, resulting from sublethal effects on the cells, such that 9-fold increases in bioluminescence were observed with toluene, and phenol elicited as much as a 4 fold increase. These effects were a result of substrate availability for the bioluminescent reaction, rather than increases in expression of the bioluminescent genes. This was also observed in milk matrices, where milk fat content caused a significant increase in bioluminescence. Bacteriophage are commonly studied in environmental microbiology as environmental control agents or as prophylactics. Utilizing Pseudomonas fluorescens M3A, host/phage interactions were detected in situ at low concentrations (1.93 x 104 pfu/mL; MOI of 0.0001). The bioreporter was also used as a surrogate to facilitate food-safety applications. The constructed bioreporter demonstrated the sensitivity and real-time assessment of physiological effects from environmental perturbations.
Degree
Ph.D.
Advisors
Applegate, Purdue University.
Subject Area
Microbiology
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