Real-time PCR quantification of a green fluorescent protein-labeled, genetically engineered Pseudomonas putida strain during 2-chlorobenzoate degradation in soil

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Abstract

The potential for real-time PCR (RTm-PCR) detection of the genetically engineered strain Pseudomonas putida GN2 was studied during 2-chlorobenzoate (2-CB) degradation in three different soils. The strain contained the constructed plasmid pGN2 which encoded genes for 2-CB oxidation (cbdA) and the green fluorescent protein (gfp). P. putida GN2 numbers were assessed by plating onto 2-CB minimal media and also by RTm-PCR detection of cbdA and gfp. Addition of P. putida GN2 decreased the time required to degrade 2-CB in all tested soils by more than 7 days. The RTm-PCR estimations of P. putida GN2 numbers strongly correlated with those obtained from plate count methods during active 2-CB degradation. However, after 2-CB degradation in the soils had ceased, RTm-PCR estimations of cbdA and gfp genes were generally one order of magnitude lower than those from plate counts. These results indicate the potential for RTm-PCR to rapidly determine degrader numbers in soil following bioaugmentation but also the need to exercise caution when attempting to determine cell numbers of degraders from the RTm-PCR quantification of plasmid encoded genes after substrate is depleted. © 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

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Wang, G., Gentry, T. J., Grass, G., Josephson, K., Rensing, C., & Pepper, I. L. (2004). Real-time PCR quantification of a green fluorescent protein-labeled, genetically engineered Pseudomonas putida strain during 2-chlorobenzoate degradation in soil. FEMS Microbiology Letters, 233(2), 307–314. https://doi.org/10.1016/j.femsle.2004.02.025

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