Effect of Temperature, pH, and Initial Cell Number on luxCDABE and nah Gene Expression during Naphthalene and Salicylate Catabolism in the Bioreporter Organism Pseudomonas putida RB1353

Abstract

One limitation of employing lux bioreporters to monitor in situ microbial gene expression in dynamic, laboratory-scale systems is the confounding variability in the luminescent responses. For example, despite careful control of oxygen tension, growth stage, and cell number, luminescence from Pseudomonas putida RB1353, a naphthalene-degrading lux bioreporter, varied by more than sevenfold during saturated flow column experiments in our laboratory. Therefore, this study was conducted to determine what additional factors influence the luminescent response. Specifically, this study investigated the impact of temperature, pH, and initial cell number (variations within an order of magnitude) on the peak luminescence of P. putida RB1353 and the maximum degradation rate (V max ) during salicylate and naphthalene catabolism. Statistical analyses based on general linear models indicated that under constant oxygen tension, temperature and pH accounted for 98.1% of the variability in luminescence during salicylate catabolism and 94.2 and 49.5% of the variability in V max during salicylate and naphthalene catabolism, respectively. Temperature, pH, and initial substrate concentration accounted for 99.9% of the variability in luminescence during naphthalene catabolism. Initial cell number, within an order of magnitude, did not have a significant influence on either peak luminescence or V max during salicylate and naphthalene catabolism. Over the ranges of temperature and pH evaluated, peak luminescence varied by more than 4 orders of magnitude. The minimum parameter deviation required to alter lux gene expression during salicylate and naphthalene catabolism was a change in temperature of 1°C, a change in pH of 0.2, or a change in initial cell number of 1 order of magnitude. Results from this study indicate that there is a need for careful characterization of the impact of environmental conditions on both the expression of the reporter and catabolic genes and the activities of the gene products. For example, even though lux gene expression was occurring at ϳ35°C, the luciferase enzyme was inactive. Furthermore, this study demonstrates that with careful characterization and standardization of measurement conditions, the attainment of a reproducible luminescent response and an understanding of the response are feasible.One of the major constraints on implementing in situ bioremediation is the lack of understanding of how physical, biological, and chemical factors affect microbial activity (7). In an attempt to understand how these factors impact bioremediation, reporter organisms have been developed that allow monitoring of microbial interaction with organic compounds in dynamic systems (6,20). Reporter organisms are genetically engineered organisms in which a reporter gene(s), such as lux, luc, or gfp, that encodes a detectable gene product is under regulatory control of an inducible catabolic operon (12). In general, due to a relatively short half-life, luciferase (lux or luc) is preferable in applications where dynamic measure...

Measurement of Biologically Available Naphthalene in Gas and Aqueous Phases by Use of a Pseudomonas putida Biosensor

“…Bioluminescent biosensors have even been coupled to a chip that was designed to detect, process, and report low levels of bioluminescence (30). Unfortunately, luciferase activity is energetically costly for the cell, and physiological differences or compounds inhibitory to cells' energy metabolism can reduce light output (7,12). Compounds interfering with fatty acid metabolism or cell membrane synthesis may even increase nonspecific light output from some biosensors, probably by providing a larger pool of substrates for the light reaction (12).…”

“…For our model system, we chose a bacterial biosensor for the detection of naphthalene, which is relatively poorly water soluble and hence a good model for the behavior of a large class of hydrophobic compounds with moderate gas phase partition constants (Henry constant, 0.02) (25). Bacterial biosensors for naphthalene have been described and used before (3,7,13,14,20), but they have not been systematically tested for accurate measurement of naphthalene through the gas phase, although the principle has been applied previously (23). Here we show that bacterial biosensors can be optimally maintained in controlled chemostat cultivation, react very reliably to naphthalene exposure with a proportional bioluminescence signal, and can be applied in gas phase measurements to increase the bioavailable amount of naphthalene to the cells, thereby effectively lowering the detectable naphthalene concentration range.…”

Alternative Luciferase for Monitoring Bacterial Cells under Adverse Conditions

“…The reaction is catalyzed by bacterial luciferase, a heterodimeric 77-kDa enzyme composed of an alpha subunit and a beta subunit encoded by the luxA and luxB genes, respectively (22,25). Bioluminescence is an excellent reporter system (recently reviewed in reference 56), a sensitive marker for microbial detection (13,15,27,28,49,57,58,70), a real-time, noninvasive reporter for measuring gene expression (11,12,19,33,36,48,51,53,54,55,69), and a way to measure intracellular biochemical function (cell viability) (1,4,5,16,17,27,28,29,30,34).…”

Characterization of Bioluminescent Derivatives of Assimilable Organic Carbon Test Bacteria

“…Expression of bioluminescence is known to be closely related to cellular physiology (2,4,10,11). We therefore reasoned that peak luminescence, just prior to the onset of the stationary phase after exhaustion of AOC in a water sample, would be an early physiological indicator of full cell yield.…”